A Review on Microfluidic Paper-Based Analytical Devices for Glucose Detection
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paper_title: Paper-based nanobiosensors for diagnostics

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In this review we discuss how nanomaterials can be integrated in diagnostic paper-based biosensors for the detection of proteins, nucleic acids and cells. In particular first the different types and properties of paper-based nanobiosensors and nanomaterials are briefly explained. Then several examples of their application in diagnostics of several biomarkers are reported. Finally our opinions regarding future trends in this field are discussed.
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paper_title: Laser-treated hydrophobic paper: an inexpensive microfluidic platform

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We report a method for fabricating inexpensive microfluidic platforms on paper using laser treatment. Any paper with a hydrophobic surface coating (e.g., parchment paper, wax paper, palette paper) can be used for this purpose. We were able to selectively modify the surface structure and property (hydrophobic to hydrophilic) of several such papers using a CO(2) laser. We created patterns down to a minimum feature size of 62±1 µm. The modified surface exhibited a highly porous structure which helped to trap/localize chemical and biological aqueous reagents for analysis. The treated surfaces were stable over time and were used to self-assemble arrays of aqueous droplets. Furthermore, we selectively deposited silica microparticles on patterned areas to allow lateral diffusion from one end of a channel to the other. Finally, we demonstrated the applicability of this platform to perform chemical reactions using luminol-based hemoglobin detection.
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paper_title: Microfluidic Paper-Based Analytical Device for Aerosol Oxidative Activity

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Human exposure to particulate matter (PM) air pollution has been linked with respiratory, cardiovascular, and neurodegenerative diseases, in addition to various cancers. Consistent among all of these associations is the hypothesis that PM induces inflammation and oxidative stress in the affected tissue. Consequently, a variety of assays have been developed to quantify the oxidative activity of PM as a means to characterize its ability to induced oxidative stress. The vast majority of these assays rely on high-volume, fixed-location sampling methods due to limitations in assay sensitivity and detection limit. As a result, our understanding of how personal exposure contributes to the intake of oxidative air pollution is limited. To further this understanding, we present a microfluidic paper-based analytical device (μPAD) for measuring PM oxidative activity on filters collected by personal sampling. The μPAD is inexpensive to fabricate and provides fast and sensitive analysis of aerosol oxidative activity. T...
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paper_title: A novel chemiluminescence paper microfluidic biosensor based on enzymatic reaction for uric acid determination.

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In this work, chemiluminescence (CL) method was combined with microfluidic paper-based analytical device (μPAD) to establish a novel CL μPAD biosensor for the first time. This novel CL μPAD biosensor was based on enzyme reaction which produced H(2)O(2) while decomposing the substrate and the CL reaction between rhodanine derivative and generated H(2)O(2) in acid medium. Microchannels in μPAD were fabricated by cutting method. And the possible CL assay principle of this CL μPAD biosensor was explained. Rhodanine derivative system was used to reach the purpose of high sensitivity and well-defined signal for this CL μPAD biosensor. And the optimum reaction conditions were investigated. The quantitative determination of uric acid could be achieved by this CL μPAD biosensor with accurate and satisfactory result. And this biosensor could provide good reproducible results upon storage at 4°C for at least 10 weeks. The successful integration of μPAD and CL reaction made the final biosensor inexpensive, easy-to-use, low-volume, and portable for uric acid determination, which also greatly reduces the cost and increases the efficiency required for an analysis. We believe this simple, practical CL μPAD biosensor will be of interest for use in areas such as disease diagnosis.
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paper_title: Paper-fluidic electrochemical biosensing platform with enzyme paper and enzymeless electrodes

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Abstract A miniaturized paper-based microfluidic electrochemical enzymatic biosensing platform was developed and the effects of fluidic behaviors in paper substrate on electrochemical sensing were systemically investigated. The biosensor is composed of an enzyme-immobilized pure cellulose paper pad, an enzymeless screen-printed electrode (SPE) modified with platinum nanoparticles (PtNPs), and a pair of clamped acrylonitrile butadiene styrene (ABS) plastic holders to provide good alignment for stable signal sensing. The wicking rate of liquid sample in paper was predicted, using a two-dimensional Fickian-diffusion model, to be 1.0 × 10 −2 cm 2 /s, and was verified experimentally. Dip-coating was used to prepare the enzyme-modified paper pad (EPP), which is amenable for mass manufacturing. The EPP retained excellent hydrophilicity and mechanical properties, with even slightly improved tensile strength and break strain. No significant difference in voltammetric behaviors was observed between measurements made in bulk buffer solution and with different sample volumes applied to EPP beyond its saturation wicking volume. Glucose oxidase (GO x ), an enzyme specific for glucose (Glc) substrate, was used as a model enzyme and its enzymatic reaction product H 2 O 2 was detected by the enzymeless PtNPs-SPE in the presence of ambient electron mediator O 2 . Consequently, Glc was detected with its concentration linearly depending on H 2 O 2 oxidation current with sensitivity of 10.5 μA mM -1 cm -2 and detection limit of 9.3 μM (at S / N = 3). The biosensor can be quickly regenerated with memory effects removed by buffer additions for continuous real-time detection of multiple samples in one run for point-of-care purposes. This integrated platform is also inexpensive since the EPP is easily stored, and enzymeless PtNPs-SPEs can be used multiple times with different EPPs. The green and facile preparation in bulk, excellent mechanical strength, well-maintained enzyme activity, disposability, and good reproducibility and stability make our paper-fluidic biosensor platform suitable for various real-time electrochemical bioassays without any external power for mixing, especially in resource-limited conditions.
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paper_title: A fast and low-cost spray method for prototyping and depositing surface-enhanced Raman scattering arrays on microfluidic paper based device

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In this study, a fast, low-cost, and facile spray method was proposed. This method deposits highly sensitive surface-enhanced Raman scattering (SERS) silver nanoparticles (AgNPs) on the paper-microfluidic scheme. The procedures for substrate preparation were studied including different strategies to synthesize AgNPs and the optimization of spray cycles. In addition, the morphologies of the different kinds of paper substrates were characterized by SEM and investigated by their SERS signals. The established method was found to be favorable for obtaining good sensitivity and reproducible results. The RSDs of Raman intensity of randomly analyzing 20 spots on the same paper or different filter papers depositing AgNPs are both below 15%. The SERS enhancement factor is approximately 2 × 10(7) . The whole fabrication is very rapid, robust, and does not require specific instruments. Furthermore, the total cost for 1000 pieces of chip is less than $20. These advantages demonstrated the potential for growing SERS applications in the area of environmental monitoring, food safety, and bioanalysis in the future.
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paper_title: Method for fabrication of paper-based microfluidic devices by alkylsilane self-assembling and UV/O3-patterning.

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This work presents a novel and facile method for fabricating paper-based microfluidic devices by means of coupling of hydrophobic silane to paper fibers followed by deep UV-lithography. After filter paper being simply immersed in an octadecyltrichlorosilane (OTS) solution in n-hexane for 5 min, the hydrophilic paper became highly hydrophobic (water contact angle of about 125°) due to the hydrophobic OTS molecules were coupled to paper's cellulose fibers. The hydrophobized paper was then exposed to deep UV-lights through a quartz mask that had the pattern of the to-be-prepared channel network. Thus, the UV-exposed regions turned highly hydrophilic whereas the masked regions remained highly hydrophobic, generating hydrophilic channels, reservoirs and reaction zones that were well-defined by the hydrophobic regions. The resolution for hydrophilic channels was 233 ± 30 μm and that for between-channel hydrophobic barrier was 137 ± 21 μm. Contact angle measurement, X-ray photoelectron spectroscopy (XPS) and attenuated total reflectance Fourier transform-infrared (ATR-FT-IR) spectroscopy were employed to characterize the surface chemistry of the OTS-coated and UV/O(3)-treated paper, and the related mechanism was discussed. Colorimetric assays of nitrite are demonstrated with the developed paper-based microfluidic devices.
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paper_title: A paper-based skin patch for the diagnostic screening of cystic fibrosis

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A thin and flexible paper-based skin patch was developed for the diagnostic screening of cystic fibrosis. It utilized a unique combination of both anion exchange and pH test papers to enable the quantitative, colorimetric and on-skin detection of sweat anions.
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paper_title: Electrogenerated Chemiluminescence Detection in Paper-Based Microfluidic Sensors

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This paper describes the first approach at combining paper microfluidics with electrochemiluminescent (ECL) detection. Inkjet printing is used to produce paper microfluidic substrates which are combined with screen-printed electrodes (SPEs) to create simple, cheap, disposable sensors which can be read without a traditional photodetector. The sensing mechanism is based on the orange luminescence due to the ECL reaction of tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+) with certain analytes. Using a conventional photodetector, 2-(dibutylamino)ethanol (DBAE) and nicotinamide adenine dinucleotide (NADH) could be detected to levels of 0.9 μM and 72 μM, respectively. Significantly, a mobile camera phone can also be used to detect the luminescence from the sensors. By analyzing the red pixel intensity in digital images of the ECL emission, a calibration curve was constructed demonstrating that DBAE could be detected to levels of 250 μM using the phone.
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paper_title: Determination of glucose and uric acid with bienzyme colorimetry on microfluidic paper-based analysis devices.

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In this work, we first employ a drying method combining with the bienzyme colorimetric detection of glucose and uric acid on microfluidic paper-based analysis devices (μPADs). The channels of 3D μPADs are also designed by us to get better results. The color results are recorded by both Gel Documentation systems and a common camera. By using Gel Documentation systems, the limits of detection (LOD) of glucose and uric acid are 3.81 × 10(-5)M and 4.31 × 10(-5)M, respectively one order of magnitude lower than that of the reported methods on μPADs. By using a common camera, the limits of detection (LOD) of glucose and uric acid are 2.13 × 10(-4)M and 2.87 × 10(-4)M, respectively. Furthermore, the effects of detection conditions have been investigated and discussed comprehensively. Human serum samples are detected with satisfactory results, which are comparable with the clinical testing results. A low-cost, simple and rapid colorimetric method for the simultaneous detection of glucose and uric acid on the μPADs has been developed with enhanced sensitivity.
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paper_title: Automating Quantum Dot Barcode Assays Using Microfluidics and Magnetism for the Development of a Point-of-Care Device

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The impact of detecting multiple infectious diseases simultaneously at point-of-care with good sensitivity, specificity, and reproducibility would be enormous for containing the spread of diseases in both resource-limited and rich countries. Many barcoding technologies have been introduced for addressing this need as barcodes can be applied to detecting thousands of genetic and protein biomarkers simultaneously. However, the assay process is not automated and is tedious and requires skilled technicians. Barcoding technology is currently limited to use in resource-rich settings. Here we used magnetism and microfluidics technology to automate the multiple steps in a quantum dot barcode assay. The quantum dot-barcoded microbeads are sequentially (a) introduced into the chip, (b) magnetically moved to a stream containing target molecules, (c) moved back to the original stream containing secondary probes, (d) washed, and (e) finally aligned for detection. The assay requires 20 min, has a limit of detection of ...
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paper_title: Microfluidic paper-based chemiluminescence biosensor for simultaneous determination of glucose and uric acid.

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In this study, a novel microfluidic paper-based chemiluminescence analytical device (μPCAD) with a simultaneous, rapid, sensitive and quantitative response for glucose and uric acid was designed. This novel lab-on-paper biosensor is based on oxidase enzyme reactions (glucose oxidase and urate oxidase, respectively) and the chemiluminescence reaction between a rhodanine derivative and generated hydrogen peroxide in an acid medium. The possible chemiluminescence assay principle of this μPCAD is explained. We found that the simultaneous determination of glucose and uric acid could be achieved by differing the distances that the glucose and uric acid samples traveled. This lab-on-paper biosensor could provide reproducible results upon storage at 4 °C for at least 10 weeks. The application test of our μPCAD was then successfully performed with the simultaneous determination of glucose and uric acid in artificial urine. This study shows the successful integration of the μPCAD and the chemiluminescence method will be an easy-to-use, inexpensive, and portable alternative for point-of-care monitoring.
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paper_title: Paper-Based Microfluidic Devices by Plasma Treatment

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Paper-based microfluidic patterns have been demonstrated in recent literature to have a significant potential in developing low-cost analytical devices for telemedicine and general health monitoring. This study reports a new method for making microfluidic patterns on a paper surface using plasma treatment. Paper was first hydrophobized and then treated using plasma in conjunction with a mask. This formed well defined hydrophilic channels on the paper. Paper-based microfluidic systems produced in this way retained the flexibility of paper and a variety of patterns could be formed. A major advantage of this system is that simple functional elements such as switches and filters can be built into the patterns. Examples of these elements are given in this study.
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paper_title: Point-of-Care Multiplexed Assays of Nucleic Acids Using Microcapillary-based Loop-Mediated Isothermal Amplification

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This report demonstrates a straightforward, robust, multiplexed and point-of-care microcapillary-based loop-mediated isothermal amplification (cLAMP) for assaying nucleic acids. This assay integrates capillaries (glass or plastic) to introduce and house sample/reagents, segments of water droplets to prevent contamination, pocket warmers to provide heat, and a hand-held flashlight for a visual readout of the fluorescent signal. The cLAMP system allows the simultaneous detection of two RNA targets of human immunodeficiency virus (HIV) from multiple plasma samples, and achieves a high sensitivity of two copies of standard plasmid. As few nucleic acid detection methods can be wholly independent of external power supply and equipment, our cLAMP holds great promise for point-of-care applications in resource-poor settings.
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paper_title: Integrated electrochemical microsystems for genetic detection of pathogens at the point of care.

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The capacity to achieve rapid, sensitive, specific, quantitative, and multiplexed genetic detection of pathogens via a robust, portable, point-of-care platform could transform many diagnostic applications. And while contemporary technologies have yet to effectively achieve this goal, the advent of microfluidics provides a potentially viable approach to this end by enabling the integration of sophisticated multistep biochemical assays (e.g., sample preparation, genetic amplification, and quantitative detection) in a monolithic, portable device from relatively small biological samples. Integrated electrochemical sensors offer a particularly promising solution to genetic detection because they do not require optical instrumentation and are readily compatible with both integrated circuit and microfluidic technologies. Nevertheless, the development of generalizable microfluidic electrochemical platforms that integrate sample preparation and amplification as well as quantitative and multiplexed detection remains a challenging and unsolved technical problem. Recognizing this unmet need, we have developed a series of microfluidic electrochemical DNA sensors that have progressively evolved to encompass each of these critical functionalities. For DNA detection, our platforms employ label-free, single-step, and sequence-specific electrochemical DNA (E-DNA) sensors, in which an electrode-bound, redox-reporter-modified DNA "probe" generates a current change after undergoing a hybridization-induced conformational change. After successfully integrating E-DNA sensors into a microfluidic chip format, we subsequently incorporated on-chip genetic amplification techniques including polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) to enable genetic detection at clinically relevant target concentrations. To maximize the potential point-of-care utility of our platforms, we have further integrated sample preparation via immunomagnetic separation, which allowed the detection of influenza virus directly from throat swabs and developed strategies for the multiplexed detection of related bacterial strains from the blood of septic mice. Finally, we developed an alternative electrochemical detection platform based on real-time LAMP, which not is only capable of detecting across a broad dynamic range of target concentrations, but also greatly simplifies quantitative measurement of nucleic acids. These efforts represent considerable progress toward the development of a true sample-in-answer-out platform for genetic detection of pathogens at the point of care. Given the many advantages of these systems, and the growing interest and innovative contributions from researchers in this field, we are optimistic that iterations of these systems will arrive in clinical settings in the foreseeable future.
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paper_title: A low-cost paper-based inkjet-printed platform for electrochemical analyses

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An electrode platform printed on a recyclable low-cost paper substrate was characterized using cyclic voltammetry. The working and counter electrodes were directly printed gold-stripes, while the reference electrode was a printed silver stripe onto which an AgCl layer was deposited electrochemically. The novel paper-based chips showed comparable performance to conventional electrochemical cells. Different types of electrode modifications were carried out to demonstrate that the printed electrodes behave similarly with conventional electrodes. Firstly, a self-assembled monolayer (SAM) of alkanethiols was successfully formed on the Au electrode surface. As a consequence, the peak currents were suppressed and no longer showed clear increase as a function of the scan rate. Such modified electrodes have potential in various sensor applications when terminally substituted thiols are used. Secondly, a polyaniline film was electropolymerized on the working electrode by cyclic voltammetry and used for potentiometric pH sensing. The calibration curve showed close to Nerstian response. Thirdly, a poly(3,4-ethylenedioxythiophene) (PEDOT) layer was electropolymerized both by galvanostatic and cyclic potential sweep method on the working electrode using two different dopants; Cl− to study ion-to-electron transduction on paper-Au/PEDOT system and glucose oxidase in order to fabricate a glucose biosensor. The planar paper-based electrochemical cell is a user-friendly platform that functions with low sample volume and allows the sample to be applied and changed by e.g. pipetting. Low unit cost is achieved with mask- and mesh-free inkjet-printing technology.
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paper_title: Three-dimensional microfluidic devices fabricated in layered paper and tape

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This article describes a method for fabricating 3D microfluidic devices by stacking layers of patterned paper and double-sided adhesive tape. Paper-based 3D microfluidic devices have capabilities in microfluidics that are difficult to achieve using conventional open-channel microsystems made from glass or polymers. In particular, 3D paper-based devices wick fluids and distribute microliter volumes of samples from single inlet points into arrays of detection zones (with numbers up to thousands). This capability makes it possible to carry out a range of new analytical protocols simply and inexpensively (all on a piece of paper) without external pumps. We demonstrate a prototype 3D device that tests 4 different samples for up to 4 different analytes and displays the results of the assays in a side-by-side configuration for easy comparison. Three-dimensional paper-based microfluidic devices are especially appropriate for use in distributed healthcare in the developing world and in environmental monitoring and water analysis.
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paper_title: Agarose-Based Microfluidic Device for Point-of-Care Concentration and Detection of Pathogen

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Preconcentration of pathogens from patient samples represents a great challenge in point-of-care (POC) diagnostics. Here, a low-cost, rapid, and portable agarose-based microfluidic device was developed to concentrate biological fluid from micro- to picoliter volume. The microfluidic concentrator consisted of a glass slide simply covered by an agarose layer with a binary tree-shaped microchannel, in which pathogens could be concentrated at the end of the microchannel due to the capillary effect and the strong water permeability of the agarose gel. The fluorescent Escherichia coli strain OP50 was used to demonstrate the capacity of the agarose-based device. Results showed that 90% recovery efficiency could be achieved with a million-fold volume reduction from 400 μL to 400 pL. For concentration of 1 × 10(3) cells mL(-1) bacteria, approximately ten million-fold enrichment in cell density was realized with volume reduction from 100 μL to 1.6 pL. Urine and blood plasma samples were further tested to validate the developed method. In conjugation with fluorescence immunoassay, we successfully applied the method to the concentration and detection of infectious Staphylococcus aureus in clinics. The agarose-based microfluidic concentrator provided an efficient approach for POC detection of pathogens.
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paper_title: Paper based colorimetric biosensing platform utilizing cross-linked siloxane as probe

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Abstract Paper based colorimetric biosensing platform utilizing cross-linked siloxane 3-aminopropyltriethoxysilane (APTMS) as probe was developed for the detection of a broad range of targets including H 2 O 2 , glucose and protein biomarker. APTMS was extensively used for the modification of filter papers to develop paper based analytical devices. We discovered when APTMS was cross-linked with glutaraldehyde (GA), the resulting complex (APTMS–GA) displays brick-red color, and a visual color change was observed when the complex reacted with H 2 O 2 . By integrating the APTMS–GA complex with filter paper, the modified paper enables quantitative detection of H 2 O 2 through the monitoring of the color intensity change of the paper via software Image J. Then, with the immobilization of glucose oxidase (GOx) onto the modified paper, glucose can be detected through the detection of enzymatically generated H 2 O 2 . For protein biomarker prostate specific antigen (PSA) assay, we immobilized capture, not captured anti-PSA antibody (Ab 1 ) onto the paper surface and using GOx modified gold nanorod (GNR) as detection anti-PSA antibody (Ab 2 ) label. The detection of PSA was also achieved via the liberated H 2 O 2 when the GOx label reacted with glucose. The results demonstrated the possibility of this paper based sensor for the detection of different analytes with wide linear range. The low cost and simplicity of this paper based sensor could be developed for “point-of-care” analysis and find wide application in different areas.
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paper_title: Flexographically Printed Fluidic Structures in Paper

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This Technical Note demonstrates a simple method based on flexographic printing of polystyrene to form liquid guiding boundaries and layers on paper substrates. The method allows formation of hydrophobic barrier structures that partially or completely penetrate through the substrate. This unique property enables one to form very thin fluidic channels on paper, leading to reduced sample volumes required in point-of-care diagnostic devices. The described method is compatible with roll-to-roll flexography units found in many printing houses, making it an ideal method for large-scale production of paper-based fluidic structures.
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paper_title: One-step polymer screen-printing for microfluidic paper-based analytical device (μPAD) fabrication

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We report a simple, low-cost, one-step fabrication method for microfluidic paper-based analytical devices (μPAD) using only polystyrene and a patterned screen. The polystyrene solution applied through the screen penetrates through the paper, forming a three-dimensional hydrophobic barrier, defining a hydrophilic analysis zone. The optimal polystyrene concentration and paper types were first investigated. Adjusting polystyrene concentration allows for various types of paper to be used for successful device fabrication. Using an optimized polystyrene concentration with Whatman#4 filter paper, a linear relationship was found to exist between the design width and the printed width. The smallest hydrophilic channel and hydrophobic barrier that can be obtained are 670 ± 50 μm and 380 ± 40 μm, respectively. High device-to-device fabrication reproducibility was achieved yielding a relative standard deviation (%RSD) in the range of 1.12–2.54% (n = 64) of the measured diameter of the well-shaped fabricated test zones with a designed diameter of 5 and 7 mm. To demonstrate the significance of the fabricated μPAD, distance-based and well-based paper devices were constructed for the analysis of H2O2 and antioxidant activity, respectively. The analysis of H2O2 in real samples using distance-based measurement with CeO2 nanoparticles as the colorimetric agent produced the same results at 95% confidence level, as those obtained using KMnO4 titration. A proof-of-concept antioxidant activity determination based on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was also demonstrated. The results verify that the polymer screen-printing method can be used as an alternative method for μPAD fabrication.
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paper_title: Highly stable and sensitive glucose biosensor based on covalently assembled high density Au nanostructures

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Abstract We describe the development of a highly stable and sensitive glucose biosensor based on the nanohybrid materials derived from gold nanoparticles (AuNPs) and multi-walled carbon nanotubes (MWCNT). The biosensing platform was developed by using layer-by-layer (LBL) self-assembly of the nanohybrid materials and the enzyme glucose oxidase (GOx). A high density of AuNPs and MWCNT nanocomposite materials were constructed by alternate self assembly of thiol functionalized MWCNTs and AuNPs, followed by chemisoption of GOx. The surface morphology of multilayered AuNPs/MWCNT structure was characterized by field emission-scanning electron microscope (FE-SEM), and the surface coverage of AuNPs was investigated by cyclic voltammetry (CV), showing that 5 layers of assembly achieves the maximum particle density on electrode. The immobilization of GOx was monitored by electrochemical impedance spectroscopy (EIS). CV and amperometry methods were used to study the electrochemical oxidation of glucose at physiological pH 7.4. The Au electrode modified with five layers of AuNPs/MWCNT composites and GOx exhibited an excellent electrocatalytic activity towards oxidation of glucose, which presents a wide liner range from 20 μM to 10 mM, with a sensitivity of 19.27 μA mM−1 cm−2. The detection limit of present modified electrode was found to be 2.3 μM (S/N = 3). In addition, the resulting biosensor showed a faster amperometric current response (within 3 s) and low apparent Michaelis–Menten constant ( K m app ) . Our present study shows that the high density of AuNPs decorated MWCNT is a promising nanohybrid material for the construction of enzyme based electrochemical biosensors.
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paper_title: Self-monitoring of blood glucose in non-insulin treated patients with type 2 diabetes: a systematic review and meta-analysis.

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OBJECTIVE ::: To assess the effect of self-monitoring of blood glucose (SMBG) on glycaemic control in non-insulin treated patients with type 2 diabetes by means of a systematic review and meta-analysis. ::: ::: ::: RESEARCH DESIGN AND METHODS ::: MEDLINE and the Cochrane Controlled Trials Register were searched from inception to January 2009 for randomised controlled trials comparing SMBG with non-SMBG or more frequent SMBG with less intensive SMBG. Electronic searches were supplemented by manual searching of reference lists and reviews. The comparison of SMBG with non-SMBG was the primary, the comparison of more frequent SMBG with less intensive SMBG the secondary analysis. Stratified analyses were performed to evaluate modifying factors. ::: ::: ::: MAIN OUTCOME MEASURES ::: The primary endpoint was glycated haemoglobin A(1c) (HbA(1c)), secondary outcomes included fasting glucose and the occurrence of hypoglycaemia. Using random effects models a weighted mean difference (WMD) was calculated for HbA(1c) and a risk ratio (RR) was calculated for hypoglycaemia. Due to considerable heterogeneity, no combined estimate was computed for fasting glucose. ::: ::: ::: RESULTS ::: Fifteen trials (3270 patients) were included in the analyses. SMBG was associated with a larger reduction in HbA(1c) compared with non-SMBG (WMD -0.31%, 95% confidence interval -0.44 to -0.17). The beneficial effect associated with SMBG was not attenuated over longer follow-up. SMBG significantly increased the probability of detecting a hypoglycaemia (RR 2.10, 1.37 to 3.22). More frequent SMBG did not result in significant changes of HbA(1c) compared with less intensive SMBG (WMD -0.21%, 95% CI -0.57 to 0.15). ::: ::: ::: CONCLUSIONS ::: SMBG compared with non-SMBG is associated with a significantly improved glycaemic control in non-insulin treated patients with type 2 diabetes. The added value of more frequent SMBG compared with less intensive SMBG remains uncertain.
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paper_title: 3D origami-based multifunction-integrated immunodevice: low-cost and multiplexed sandwich chemiluminescence immunoassay on microfluidic paper-based analytical device.

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A novel 3D microfluidic paper-based immunodevice, integrated with blood plasma separation from whole blood samples, automation of rinse steps, and multiplexed CL detections, was developed for the first time based on the principle of origami (denoted as origami-based device). This 3D origami-based device, comprised of one test pad surrounded by four folding tabs, could be patterned and fabricated by wax-printing on paper in bulk. In this work, a sandwich-type chemiluminescence (CL) immunoassay was introduced into this 3D origami-based immunodevice, which could separate the operational procedures into several steps including (i) folding pads above/below and (ii) addition of reagent/buffer under a specific sequence. The CL behavior, blood plasma separation, washing protocol, and incubation time were investigated in this work. The developed 3D origami-based CL immunodevice, combined with a typical luminuol-H(2)O(2) CL system and catalyzed by Ag nanoparticles, showed excellent analytical performance for the simultaneous detection of four tumor markers. The whole blood samples were assayed and the results obtained were in agreement with the reference values from the parallel single-analyte test. This paper-based microfluidic origami CL detection system provides a new strategy for a low-cost, sensitive, simultaneous multiplex immunoassay and point-of-care diagnostics.
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paper_title: Oral pharmacologic management of type 2 diabetes.

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Epidemiologic and interventional studies have led to lower treatment targets for type 2 diabetes (formerly known as non-insulin-dependent diabetes), including a glycosylated hemoglobin level of 7 percent or less and a before-meal blood glucose level of 80 to 120 mg per dL (4.4 to 6.7 mmol per L). New oral medications make these targets easier to achieve, especially in patients with recently diagnosed diabetes. Acarbose, metformin, miglitol, pioglitazone, rosiglitazone and troglitazone help the patient's own insulin control glucose levels and allow early treatment with little risk of hypoglycemia. Two new long-acting sulfonylureas (glimepiride and extended-release glipizide) and a short-acting sulfonylurea-like agent (repaglinide) simply and reliably augment the patient's insulin supply. Combinations of agents have additive therapeutic effects and can restore glucose control when a single agent is no longer successful. Oral therapy for early type 2 diabetes can be relatively inexpensive, and evidence of its cost-effectiveness is accumulating.
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paper_title: Quantitative biomarker assay with microfluidic paper-based analytical devices

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This article describes the use of microfluidic paper-based analytical devices (muPADs) to perform quantitative chemical assays with internal standards. MicroPADs are well-suited for colorimetric biochemical assays; however, errors can be introduced from the background color of the paper due to batch difference and age, and from color measurement devices. To reduce errors from these sources, a series of standard analyte solutions and the sample solution are assayed on a single device with multiple detection zones simultaneously; an analyte concentration calibration curve can thus be established from the standards. Since the muPAD design allows the colorimetric measurements of the standards and the sample to be conducted simultaneously and under the same condition, errors from the above sources can be minimized. The analytical approach reported in this work shows that muPADs can perform quantitative chemical analysis at very low cost.
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paper_title: Low-cost bioanalysis on paper-based and its hybrid microfluidic platforms.

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Low-cost assays have broad applications ranging from human health diagnostics and food safety inspection to environmental analysis. Hence, low-cost assays are especially attractive for rural areas and developing countries, where financial resources are limited. Recently, paper-based microfluidic devices have emerged as a low-cost platform which greatly accelerates the point of care (POC) analysis in low-resource settings. This paper reviews recent advances of low-cost bioanalysis on paper-based microfluidic platforms, including fully paper-based and paper hybrid microfluidic platforms. In this review paper, we first summarized the fabrication techniques of fully paper-based microfluidic platforms, followed with their applications in human health diagnostics and food safety analysis. Then we highlighted paper hybrid microfluidic platforms and their applications, because hybrid platforms could draw benefits from multiple device substrates. Finally, we discussed the current limitations and perspective trends of paper-based microfluidic platforms for low-cost assays.
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paper_title: A low-cost, simple, and rapid fabrication method for paper-based microfluidics using wax screen-printing

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Wax screen-printing as a low-cost, simple, and rapid method for fabricating paper-based microfluidic devices (µPADs) is reported here. Solid wax was rubbed through a screen onto paper filters. The printed wax was then melted into the paper to form hydrophobic barriers using only a hot plate. We first studied the relationship between the width of a hydrophobic barrier and the width of the original design line. We also optimized the heating temperature and time and determined the resolution of structures fabricated using this technique. The minimum width of hydrophilic channel and hydrophobic barrier is 650 and 1300 µm, respectively. Next, our fabrication method was compared to a photolithographic method using the reaction between bicinchoninic acid (BCA) and Cu1+ to demonstrate differences in background reactivity. Photolithographically defined channels exhibited a high background while wax printed channels showed a very low background. Finally, the utility of wax screen-printing was demonstrated for the simultaneous determination of glucose and total iron in control human serum samples using an electrochemical method with glucose oxidase and a colorimetric method with 1,10-phenanthroline. This study demonstrates that wax screen-printing is an easy-to-use and inexpensive alternative fabrication method for µPAD, which will be especially useful in developing countries.
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paper_title: Paper-based microfluidic devices for analysis of clinically relevant analytes present in urine and saliva.

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We report the use of paper-based microfluidic devices fabricated from a novel polymer blend for the monitoring of urinary ketones, glucose, and salivary nitrite. Paper-based devices were fabricated via photolithography in less than 3 min and were immediately ready for use for these diagnostically relevant assays. Patterned channels on filter paper as small as 90 microm wide with barriers as narrow as 250 microm could be reliably patterned to permit and block fluid wicking, respectively. Colorimetric assays for ketones and nitrite were adapted from the dipstick format to this paper microfluidic chip for the quantification of acetoacetate in artificial urine, as well as nitrite in artificial saliva. Glucose assays were based on those previously demonstrated (Martinez et al., Angew Chem Int Ed 8:1318-1320, 1; Martinez et al., Anal Chem 10:3699-3707, 2; Martinez et al., Proc Nat Acad Sci USA 50:19606-19611, 3; Lu et al., Electrophoresis 9:1497-1500, 4; Abe et al., Anal Chem 18:6928-6934, 5). Reagents were spotted on the detection pad of the paper device and allowed to dry prior to spotting of samples. The ketone test was a two-step reaction requiring a derivitization step between the sample spotting pad and the detection pad, thus for the first time, confirming the ability of these paper devices to perform online multi-step chemical reactions. Following the spotting of the reagents and sample solution onto the paper device and subsequent drying, color images of the paper chips were recorded using a flatbed scanner, and images were converted to CMYK format in Adobe Photoshop CS4 where the intensity of the color change was quantified using the same software. The limit of detection (LOD) for acetoacetate in artificial urine was 0.5 mM, while the LOD for salivary nitrite was 5 microM, placing both of these analytes within the clinically relevant range for these assays. Calibration curves for urinary ketone (5 to 16 mM) and salivary nitrite (5 to 2,000 microM) were generated. The time of device fabrication to the time of test results was about 25 min.
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paper_title: Electrochemical sensing in paper-based microfluidic devices

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This paper describes the fabrication and the performance of microfluidic paper-based electrochemical sensing devices (we call the microfluidic paper-based electrochemical devices, µPEDs). The µPEDs comprise paper-based microfluidic channels patterned by photolithography or wax printing, and electrodes screen-printed from conducting inks (e.g., carbon or Ag/AgCl). We demonstrated that the µPEDs are capable of quantifying the concentrations of various analytes (e.g., heavy-metal ions and glucose) in aqueous solutions. This low-cost analytical device should be useful for applications in public health, environmental monitoring, and the developing world.
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paper_title: Multivalent Nanoparticle Networks Enable Point-of-Care Detection of Human Phospholipase-A2 in Serum

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A rapid and highly sensitive point-of-care (PoC) lateral flow assay for phospholipase A2 (PLA2) is demonstrated in serum through the enzyme-triggered release of a new class of biotinylated multiarmed polymers from a liposome substrate. Signal from the enzyme activity is generated by the adhesion of polystreptavidin-coated gold nanoparticle networks to the lateral flow device, which leads to the appearance of a red test line due to the localized surface plasmon resonance effect of the gold. The use of a liposome as the enzyme substrate and multivalent linkers to link the nanoparticles leads to amplification of the signal, as the cleavage of a small amount of lipids is able to release a large amount of polymer linker and adhesion of an even larger amount of gold nanoparticles. By optimizing the molecular weight and multivalency of these biotinylated polymer linkers, the sensitivity of the device can be tuned to enable naked-eye detection of 1 nM human PLA2 in serum within 10 min. This high sensitivity enabled the correct diagnosis of pancreatitis in diseased clinical samples against a set of healthy controls using PLA2 activity in a point-of-care device for the first time.
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paper_title: Robust and Highly Sensitive Fluorescence Approach for Point-of-Care Virus Detection Based on Immunomagnetic Separation

paper_content:

In this work, robust approach for a highly sensitive point-of-care virus detection was established based on immunomagnetic nanobeads and fluorescent quantum dots (QDs). Taking advantage of immunomagnetic nanobeads functionalized with the monoclonal antibody (mAb) to the surface protein hemagglutinin (HA) of avian influenza virus (AIV) H9N2 subtype, H9N2 viruses were efficiently captured through antibody affinity binding, without pretreatment of samples. The capture kinetics could be fitted well with a first-order bimolecular reaction with a high capturing rate constant kf of 4.25 × 109 (mol/L)−1 s–1, which suggested that the viruses could be quickly captured by the well-dispersed and comparable-size immunomagnetic nanobeads. In order to improve the sensitivity, high-luminance QDs conjugated with streptavidin (QDs-SA) were introduced to this assay through the high affinity biotin-streptavidin system by using the biotinylated mAb in an immuno sandwich mode. We ensured the selective binding of QDs-SA to the ...
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paper_title: Biomarker detection for disease diagnosis using cost-effective microfluidic platforms.

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Early and timely detection of disease biomarkers can prevent the spread of infectious diseases, and drastically decrease the death rate of people suffering from different diseases such as cancer and infectious diseases. Because conventional diagnostic methods have limited application in low-resource settings due to the use of bulky and expensive instrumentation, simple and low-cost point-of-care diagnostic devices for timely and early biomarker diagnosis is the need of the hour, especially in rural areas and developing nations. The microfluidics technology possesses remarkable features for simple, low-cost, and rapid disease diagnosis. There have been significant advances in the development of microfluidic platforms for biomarker detection of diseases. This article reviews recent advances in biomarker detection using cost-effective microfluidic devices for disease diagnosis, with the emphasis on infectious disease and cancer diagnosis in low-resource settings. This review first introduces different microfluidic platforms (e.g. polymer and paper-based microfluidics) used for disease diagnosis, with a brief description of their common fabrication techniques. Then, it highlights various detection strategies for disease biomarker detection using microfluidic platforms, including colorimetric, fluorescence, chemiluminescence, electrochemiluminescence (ECL), and electrochemical detection. Finally, it discusses the current limitations of microfluidic devices for disease biomarker detection and future prospects.
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paper_title: Paper-based planar reaction arrays for printed diagnostics

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Abstract A reaction plate mimicking the working principle of a conventional three-dimensional microplate was printed on various hydrophilic paper substrates. Planar well patterns with high wetting/non-wetting contrast were formed using hydrophobic polydimethylsiloxane (PDMS) based ink with fast curing time, which enables truly low cost roll-to-roll fabrication. The formation and functionality of the printed reaction arrays were verified by two proof-of-concept demonstrations. Firstly a colorimetric glucose sensor, based on an enzymatic reaction sequence involving glucose oxidase, was screen-printed on the reaction plate. A detection limit of 0.1 mg/mL and a fairly linear sensor response was obtained on a logarithmic scale. Secondly, the employment of the reaction plate for electrical applications was demonstrated by modulating the resistance of a drop-casted polyaniline film as a function of pH.
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paper_title: Development, characterization, and application of paper spray ionization.

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Paper spray is developed as a direct sampling ionization method for mass spectrometric analysis of complex mixtures. Ions of analyte are generated by applying a high voltage to a paper triangle wetted with a small volume (<10 microL) of solution. Samples can be preloaded onto the paper, added with the wetting solution, or transferred from surfaces using the paper as a wipe. It is demonstrated that paper spray is applicable to the analysis of a wide variety of compounds, including small organic compounds, peptides, and proteins. Procedures are developed for analysis of dried biofluid spots and applied to therapeutic drug monitoring with whole blood samples and to illicit drug detection in raw urine samples. Limits of detection of 50 ng/mL (or 20 pg absolute) are achieved for atenolol in bovine blood. The combination of sample collection from surfaces and paper spray ionization also enables fast chemical screening at high sensitivity, for example 100 pg of heroin distributed on a surface and agrochemicals on fruit peels are detectable. Online derivatization with a preloaded reagent is demonstrated for analysis of cholesterol in human serum. The combination of paper spray with miniature mass spectrometers offers a powerful impetus to wide application of mass spectrometry in nonlaboratory environments.
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paper_title: Highly selective and sensitive paper-based colorimetric sensor using thiosulfate catalytic etching of silver nanoplates for trace determination of copper ions.

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A novel, highly selective and sensitive paper-based colorimetric sensor for trace determination of copper (Cu(2+)) ions was developed. The measurement is based on the catalytic etching of silver nanoplates (AgNPls) by thiosulfate (S2O3(2-)). Upon the addition of Cu(2+) to the ammonium buffer at pH 11, the absorption peak intensity of AuNPls/S2O3(2-) at 522 nm decreased and the pinkish violet AuNPls became clear in color as visible to the naked eye. This assay provides highly sensitive and selective detection of Cu(2+) over other metal ions (K(+), Cr(3+), Cd(2+), Zn(2+), As(3+), Mn(2+), Co(2+), Pb(2+), Al(3+), Ni(2+), Fe(3+), Mg(2+), Hg(2+) and Bi(3+)). A paper-based colorimetric sensor was then developed for the simple and rapid determination of Cu(2+) using the catalytic etching of AgNPls. Under optimized conditions, the modified AgNPls coated at the test zone of the devices immediately changes in color in the presence of Cu(2+). The limit of detection (LOD) was found to be 1.0 ng mL(-1) by visual detection. For semi-quantitative measurement with image processing, the method detected Cu(2+) in the range of 0.5-200 ng mL(-1)(R(2)=0.9974) with an LOD of 0.3 ng mL(-1). The proposed method was successfully applied to detect Cu(2+) in the wide range of real samples including water, food, and blood. The results were in good agreement according to a paired t-test with results from inductively coupled plasma-optical emission spectrometry (ICP-OES).
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paper_title: Microfluidic paper-based analytical devices fabricated by low-cost photolithography and embossing of Parafilm®

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Microfluidic paper-based analytical devices (μPADs) attract tremendous attention as an economical tool for in-field diagnosis, food safety and environmental monitoring. We innovatively fabricated 2D and 3D μPADs by photolithography-patterning microchannels on a Parafilm® and subsequently embossing them to paper. This truly low-cost, wax printer and cutter plotter independent approach offers the opportunity for researchers from resource-limited laboratories to work on paper-based analytical devices.
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paper_title: A Simple Point-of-Care Microfluidic Immunomagnetic Fluorescence Assay for Pathogens

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In this work, we reported a simple rapid and point-of-care magnetic immunofluorescence assay for avian influenza virus (AIV) and developed a portable experimental setup equipped with an optical fiber spectrometer and a microfluidic device. We achieved the integration of immunomagnetic target capture, concentration, and fluorescence detection in the microfluidic chip. By optimizing flow rate and incubation time, we could get a limit of detection low up to 3.7 × 104 copy/μL with a sample consumption of 2 μL and a total assay time of less than 55 min. This approach had proved to possess high portability, fast analysis, high specificity, high precision, and reproducibility with an intra-assay variability of 2.87% and an interassay variability of 4.36%. As a whole, this microfluidic system may provide a powerful platform for the rapid detection of AIV and may be extended for detection of other viral pathogens; in addition, this portable experimental setup enables the development of point-of-care diagnostic sys...
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paper_title: Quantitative fluorescence assays using a self-powered paper-based microfluidic device and a camera-equipped cellular phone

paper_content:

Fluorescence assays often require specialized equipment and, therefore, are not easily implemented in resource-limited environments. Herein we describe a point-of-care assay strategy in which fluorescence in the visible region is used as a readout, while a camera-equipped cellular phone is used to capture the fluorescent response and quantify the assay. The fluorescence assay is made possible using a paper-based microfluidic device that contains an internal fluidic battery, a surface-mount LED, a 2 mm section of a clear straw as a cuvette, and an appropriately designed small molecule reagent that transforms from weakly fluorescent to highly fluorescent when exposed to a specific enzyme biomarker. The resulting visible fluorescence is digitized by photographing the assay region using a camera-equipped cellular phone. The digital images are then quantified using image processing software to provide sensitive as well as quantitative results. In a model 30 min assay, the enzyme β-D-galactosidase was measured quantitatively down to 700 pM levels. This communication describes the design of these types of assays in paper-based microfluidic devices and characterizes the key parameters that affect the sensitivity and reproducibility of the technique.
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paper_title: Paper-Based Microfluidic Surface Acoustic Wave Sample Delivery and Ionization Source for Rapid and Sensitive Ambient Mass Spectrometry

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A surface acoustic wave-based sample delivery and ionization method that requires minimal to no sample pretreatment and that can operate under ambient conditions is described. This miniaturized technology enables real-time, rapid, and high-throughput analysis of trace compounds in complex mixtures, especially high ionic strength and viscous samples that can be challenging for conventional ionization techniques such as electrospray ionization. This technique takes advantage of high order surface acoustic wave (SAW) vibrations that both manipulate small volumes of liquid mixtures containing trace analyte compounds and seamlessly transfers analytes from the liquid sample into gas phase ions for mass spectrometry (MS) analysis. Drugs in human whole blood and plasma and heavy metals in tap water have been successfully detected at nanomolar concentrations by coupling a SAW atomization and ionization device with an inexpensive, paper-based sample delivery system and mass spectrometer. The miniaturized SAW ioniza...
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paper_title: Microfluidic Distance Readout Sweet Hydrogel Integrated Paper-Based Analytical Device (μDiSH-PAD) for Visual Quantitative Point-of-Care Testing

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A disposable, equipment-free, versatile point-of-care testing platform, microfluidic distance readout sweet hydrogel integrated paper-based analytical device (μDiSH-PAD), was developed for portable quantitative detection of different types of targets. The platform relies on a target-responsive aptamer cross-linked hydrogel for target recognition, cascade enzymatic reactions for signal amplification, and microfluidic paper-based analytic devices (μPADs) for visual distance-based quantitative readout. A “sweet” hydrogel with trapped glucoamylase (GA) was synthesized using an aptamer as a cross-linker. When target is present in the sample, the “sweet” hydrogel collapses and releases enzyme GA into the sample, generating glucose by amylolysis. A hydrophilic channel on the μPADs is modified with glucose oxidase (GOx) and colorless 3,3′-diaminobenzidine (DAB) as the substrate. When glucose travels along the channel by capillary action, it is converted to H2O2 by GOx. In addition, DAB is converted into brown ins...
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paper_title: Simple, distance-based measurement for paper analytical devices

paper_content:

Paper-based analytical devices (PADs) represent a growing class of elegant, yet inexpensive chemical sensor technologies designed for point-of-use applications. Most PADs, however, still utilize some form of instrumentation such as a camera for quantitative detection. We describe here a simple technique to render PAD measurements more quantitative and straightforward using the distance of colour development as a detection motif. The so-called distance-based detection enables PAD chemistries that are more portable and less resource intensive compared to classical approaches that rely on the use of peripheral equipment for quantitative measurement. We demonstrate the utility and broad applicability of this technique with measurements of glucose, nickel, and glutathione using three different detection chemistries: enzymatic reactions, metal complexation, and nanoparticle aggregation, respectively. The results show excellent quantitative agreement with certified standards in complex sample matrices. This work provides the first demonstration of distance-based PAD detection with broad application as a class of new, inexpensive sensor technologies designed for point-of-use applications.
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paper_title: Microfluidic diagnostic technologies for global public health

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The developing world does not have access to many of the best medical diagnostic technologies; they were designed for air-conditioned laboratories, refrigerated storage of chemicals, a constant supply of calibrators and reagents, stable electrical power, highly trained personnel and rapid transportation of samples. Microfluidic systems allow miniaturization and integration of complex functions, which could move sophisticated diagnostic tools out of the developed-world laboratory. These systems must be inexpensive, but also accurate, reliable, rugged and well suited to the medical and social contexts of the developing world.
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paper_title: Synthesis of triphenylamine-containing conjugated polyelectrolyte and fabrication of fluorescence color-changeable, paper-based sensor strips for biothiol detection

paper_content:

A new water-soluble conjugated polyelectrolyte containing triphenylamine groups with aldehyde pendants was synthesized, which featured distinctly different emission colors according to its states, in aqueous solution and in the solid. Paper-based strips containing the polymer were prepared by simple immersion of filter paper in the polyelectrolyte solution for practical and efficient detection of biothiols including cysteine and homocysteine. The presence of aldehyde groups enables us to demonstrate noticeable fluorescence emission color changes (green-to-blue) because of the alterations in electron push–pull structure in the polymer via a reaction between the aldehyde group of the polymer and the aminothiol moiety in biothiol compounds. The presence of an aldehyde group and a sulfonate side chain was found to be indispensable for the cysteine reaction site and for a hydrophilic environment allowing the easy approach of cysteine, respectively, resulting in a simple and easy detection protocol for biothiol compounds.
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paper_title: FLASH: A rapid method for prototyping paper-based microfluidic devices

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This article describes FLASH (Fast Lithographic Activation of Sheets), a rapid method for laboratory prototyping of microfluidic devices in paper. Paper-based microfluidic devices are emerging as a new technology for applications in diagnostics for the developing world, where low cost and simplicity are essential. FLASH is based on photolithography, but requires only a UV lamp and a hotplate; no clean-room or special facilities are required (FLASH patterning can even be performed in sunlight if a UV lamp and hotplate are unavailable). The method provides channels in paper with dimensions as small as 200 µm in width and 70 µm in height; the height is defined by the thickness of the paper. Photomasks for patterning paper-based microfluidic devices can be printed using an ink-jet printer or photocopier, or drawn by hand using a waterproof black pen. FLASH provides a straightforward method for prototyping paper-based microfluidic devices in regions where the technological support for conventional photolithography is not available.
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paper_title: Fully-drawn origami paper analytical device for electrochemical detection of glucose

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Abstract In this work, an origami paper-based analytical device for glucose biosensor by employing fully-drawn pencil electrodes has been reported. The three-electrode system was prepared on paper directly by drawing with nothing more than pencils. By simple printing, two separated zones on paper were designed for the immobilization of the mediator and glucose oxidase (GOx), respectively. The used paper provides a favorable and biocompatible support for maintaining the bioactivities of GOx. With a sandwich-type scheme, the origami biosensor exhibited great analytical performance for glucose sensing including acceptable reproducibility and favorable selectivity against common interferents in physiological fluids. The limit of detection and linear range achieved with the approach was 0.05 mM and 1–12 mM, respectively. Its analytical performance was also demonstrated in the analysis of human blood samples. Such fully-drawn paper-based device is cheap, flexible, portable, disposable, and environmentally friendly, affording great convenience for practical use under resource-limited conditions. We therefore envision that this approach can be extended to generate other functional paper-based devices.
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paper_title: Chemiluminescence diminishment on a paper-based analytical device: high throughput determination of β-agonists in swine hair

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beta-Agonists are a group of illegal but widely used feed additives in the stockbreeding industry. In order to achieve simple-to-use, fast and high-throughput testing of this banned chemical, herein we suggest a paper-based analytical device on which a chemiluminescence diminishment method was performed. In this approach, extracts from swine hair samples as well as luminescent reagents, such as luminol and potassium periodate solution, in a low volume were applied to our device. It was found that the light emission was diminished by the beta-agonists extracted from the swine hair samples. The degree of diminishment is proportional to the concentration of the beta-agonists from 1.0 x 10(-5) to 1.0 x 10(-8) mol L-1. Also, the concentrations of solutions for chemiluminescence were optimized. The mechanism and reaction kinetics of chemiluminescence were discussed as well. The detection limit was obtained as 1.0 x 10(-9) mol L-1, and recoveries from 96% to 110% were achieved, both of which suggested that our method will be favourable in field applications for swine hair samples.
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paper_title: A handheld stamping process to fabricate microfluidic paper-based analytical devices with chemically modified surface for clinical assays

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This paper describes the development and use of a handheld and lightweight stamp for the production of microfluidic paper-based analytical devices (μPADs). We also chemically modified the paper surface for improved colorimetric measurements. The design of the microfluidic structure has been patterned in a stamp, machined in stainless steel. Prior to stamping, the paper surface was oxidized to promote the conversion of hydroxyl into aldehyde groups, which were then chemically activated for covalent coupling of enzymes. Then, a filter paper sheet was impregnated with paraffin and sandwiched with a native paper (n-paper) sheet, previously oxidized. The metal stamp was preheated at 150 °C and then brought in contact with the paraffined paper (p-paper) to enable the thermal transfer of the paraffin to the n-paper, thus forming the hydrophobic barriers under the application of a pressure of ca. 0.1 MPa for 2 s. The channel and barrier widths measured in 50 independent μPADs exhibited values of 2.6 ± 0.1 and 1.4 ± 0.1 mm, respectively. The chemical modification for covalent coupling of enzymes on the paper surface also led to improvements in the colour uniformity generated inside the sensing area, a known bottleneck in this technology. The relative standard deviation (RSD) values for glucose and uric acid (UA) assays decreased from 40 to 10% and from 20 to 8%, respectively. Bioassays related to the detection of glucose, UA, bovine serum albumin (BSA), and nitrite were successfully performed in concentration ranges useful for clinical assays. The semi-quantitative analysis of all four analytes in artificial urine samples revealed an error smaller than 4%. The disposability of μPADs, the low instrumental requirements of the stamp-based fabrication, and the improved colour uniformity enable the use of the proposed devices for the point-of-care diagnostics or in limited resources settlements.
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paper_title: Post-prandial hypoglycemia results from a non-glucose-dependent inappropriate insulin secretion in Roux-en-Y gastric bypassed patients

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Abstract Background After Roux-en-Y gastric bypass (RYGB), hypoglycemia can occur and be associated with adverse events such as intense malaise and impaired quality of life. Objective To compare insulin secretion, sensitivity, and clearance between two groups of patients, with or without hypoglycemia, after an oral glucose tolerance test (OGTT 75-g), and also to compare real-life glucose profiles within these two groups. Setting Bariatric surgery referral center. Methods This study involves a prospective cohort of 46 consecutive patients who complained of malaise compatible with hypoglycemia after RYGB, in whom an OGTT 75-g was performed. A plasma glucose value of lower than 2.8 mmol/L (50 mg/dl) between 90 and 120 min after the load was considered to be a significant hypoglycemia. The main outcome measures were insulin sensitivity, beta-cell function, and glycemic profiles during the test. Glucose parameters were also evaluated by continuous glucose monitoring (CGM) in a real-life setting in 43 patients. Results Twenty-five patients had plasma glucose that was lower than 2.8 mmol/L between 90 and 120 from the load (HYPO group). Twenty-one had plasma glucose that was higher than 2.8 mmol/L (NONHYPO group). The HYPO patients were younger, had lost more weight after RYGB, were less frequently diabetic before surgery, and displayed higher early insulin secretion rates compared with the NONHYPO patients after the 75-g OGTT, and they had lower late insulin secretion rates. The HYPO patients had lower interstitial glucose values in real life, which suggests that a continuum exists between observations with an oral glucose load and real-life interstitial glucose concentrations. Conclusions This study suggests that HYPO patients after RYGB display an early increased insulin secretion rate when tested with an OGTT. CGM shows that HYPO patients spend more time below 3.3 mmol/L when compared with NONHYPO patients. This phenotype of patients should be monitored carefully after RYGB.
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paper_title: Toner and paper-based fabrication techniques for microfluidic applications.

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The interest in low-cost microfluidic platforms as well as emerging microfabrication techniques has increased considerably over the last years. Toner- and paper-based techniques have appeared as two of the most promising platforms for the production of disposable devices for on-chip applications. This review focuses on recent advances in the fabrication techniques and in the analytical/bioanalytical applications of toner and paper-based devices. The discussion is divided in two parts dealing with (i) toner and (ii) paper devices. Examples of miniaturized devices fabricated by using direct-printing or toner transfer masking in polyester-toner, glass, PDMS as well as conductive platforms as recordable compact disks and printed circuit board are presented. The construction and the use of paper-based devices for off-site diagnosis and bioassays are also described to cover this emerging platform for low-cost diagnostics.
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paper_title: Paper microfluidics for red wine tasting

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A paper microfluidic chip was designed and fabricated to evaluate the taste of 10 different red wines using a set of chemical dyes. The digital camera of a smartphone captured the images, and its red-green-blue (RGB) pixel intensities were analyzed by principal component analysis (PCA). Using 8 dyes and 2 principal components (PCs), we were able to distinguish each wine by the grape variety and the oxidation status. Through comparing with the flavor map by human evaluation, PC1 seemed to represent the sweetness and PC2 the bodyness of red wine. This superior performance is attributed to: (1) careful selection of commercially available dyes through a series of linear correlation studies with the taste chemicals in red wines, (2) minimization of sample-to-sample variation by splitting a single sample into multiple wells on the paper microfluidics, and (3) filtration of particulate matter through paper fibers. The image processing and PCA procedure can eventually be implemented as a stand-alone smartphone application and can be adopted as an extremely low-cost, disposable, fully handheld, easy-to-use, yet sensitive and specific quality control method for appraising red wine or similar beverage products in resource-limited environments.
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paper_title: Determination of human blood glucose levels using microchip electrophoresis

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A high-performance monitoring system for human blood glucose levels was developed using microchip electrophoresis with a plastic chip. The combination of reductive amination as glucose labeling with fluorescent 2-aminoacridone (AMAC) and glucose-borate complex formation realized the highly selective detection of glucose even in a complex matrix such as a blood sample. The migration time of a single peak, observed on an electropherogram of AMAC-labeled plasma, closely resembled that of glucose standard solution. The treatment of plasma with hexokinase or glucokinase for glucose phosphorylation resulted in a peak shift from approximately 145 to 70 s, corresponding to glucose and glucose-6-phosphate, respectively. A double-logarithm plot revealed a linear relationship between glucose concentration and fluorescence intensity in the range of 1-300 microM of glucose (r(2) = 0.9963; p <0.01), and the detection limit was 0.92 microM. Furthermore, blood glucose concentrations estimated from the standard curves of three subjects were compared with results obtained by conventional colorimetric analysis using glucose dehydrogenase. Good correlation was observed between methods according to simple linear regression analysis (p <0.05). The reproducibility of the assay was about 6.3-9.1% (RSD) and the within-days and between-days reproducibility were 1.6-8.4 and 5.2-7.2%, respectively. This system enables us to determine blood glucose with high sensitivity and accuracy, and will be applicable to clinical diagnosis.
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paper_title: Polyelectrolyte-Coated Gold Magnetic Nanoparticles for Immunoassay Development: Toward Point of Care Diagnostics for Syphilis Screening

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Immediate response for disease control relies on simple, inexpensive, and sensitive diagnostic tests, highly sought after for timely and accurate test of various diseases, including infectious diseases. Composite Fe3O4/Au nanoparticles have attracted considerable interest in diagnostic applications due to their unique physical and chemical properties. Here, we developed a simple coating procedure for gold magnetic nanoparticles (GMNs) with poly(acrylic acid) (PAA). PAA-coated GMNs (PGMNs) were stable and monodispersed and characterized by Fourier transform-infrared spectroscopy (FT-IR), transmission electron microscopy, UV-visible scanning spectrophotometry, thermogravimetric analysis, and Zetasizer methodologies. For diagnostic application, we established a novel lateral flow immunoassay (LFIA) strip test system where recombinant Treponema pallidum antigens (r-Tp) were conjugated with PGMNs to construct a particle probe for detection of anti-Tp antibodies. Intriguingly, the particle probes specifically identified Tp antibodies with a detection limitation as low as 1 national clinical unit/mL (NCU/mL). An ample pool of 1020 sera samples from three independent hospitals were obtained to assess our PGMNs-based LFIA strips, which exhibited substantially high values of sensitivity and specificity for all clinical tests (higher than 97%) and, therefore, proved to be a suitable approach for syphilis screening at a point-of-care test manner.
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paper_title: A portable explosive detector based on fluorescence quenching of pyrene deposited on coloured wax-printed μPADs

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A new technique for the detection of explosives has been developed based on fluorescence quenching of pyrene on paper-based analytical devices (μPADs). Wax barriers were generated (150 °C, 5 min) using ten different colours. Magenta was found as the most suitable wax colour for the generation of the hydrophobic barriers with a nominal width of 120 μm resulting in fully functioning hydrophobic barriers. One microliter of 0.5 mg mL(-1) pyrene dissolved in an 80:20 methanol-water solution was deposited on the hydrophobic circle (5 mm diameter) to produce the active microchip device. Under ultra-violet (UV) illumination, ten different organic explosives were detected using the μPAD, with limits of detection ranging from 100-600 ppm. A prototype of a portable battery operated instrument using a 3 W power UV light-emitting-diode (LED) (365 nm) and a photodiode sensor was also built and evaluated for the successful automatic detection of explosives and potential application for field-based screening.
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paper_title: Two general designs for fluidic batteries in paper-based microfluidic devices that provide predictable and tunable sources of power for on-chip assays

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Microfluidic devices fabricated out of paper (and paper and tape) have emerged as promising platforms for conducting multiple diagnostic assays simultaneously in resource-limited settings. Certain types of assays in these devices, however, require a source of power to function. Lithium ion, nickel-cadmium, and other types of batteries have been used to power these devices, but these traditional batteries are too expensive and pose too much of a disposal hazard for diagnostic applications in resource-limited settings. To circumvent this problem, we previously designed a “fluidic battery” that is composed of multiple galvanic cells, incorporated directly into a multilayer paper-based microfluidic device. We now show that multiple cells of these fluidic batteries can be connected in series and/or in parallel in a predictable way to obtain desired values of current and potential, and that the batteries can be optimized to last for a short period of time (<1 min) or for up to 10–15 min. This paper also (i) outlines and quantifies the parameters that can be adjusted to maximize the current and potential of fluidic batteries, (ii) describes two general configurations for fluidic batteries, and (iii) provides equations that enable prediction of the current and potential that can be obtained when these two general designs are varied. This work provides the foundation upon which future applications of fluidic batteries will be based.
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paper_title: Fabrication of a microfluidic paper-based analytical device by silanization of filter cellulose using a paper mask for glucose assay

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We developed a novel, low-cost and simple method for the fabrication of microfluidic paper-based analytical devices (μPADs) by silanization of filter cellulose using a paper mask having a specific pattern. The paper mask was penetrated with trimethoxyoctadecylsilane (TMOS) by immersing into TMOS-heptane solution. By heating the filter paper sandwiched between the paper mask and glass slides, TMOS was immobilized onto the filter cellulose via the reaction between cellulose OH and TMOS, while the hydrophilic area was not silanized because it was not in contact with the paper mask penetrated with TMOS. The effects of some factors including TMOS concentration, heating temperature and time on the fabrication of μPADs were studied. This method is free of any expensive equipment and metal masks, and could be performed by untrained personnel. These features are very attractive for the fabrication and applications of μPADs in developing countries or resource-limited settings. A flower-shaped μPAD was fabricated and used to determine glucose in human serum samples. The contents determined by this method agreed well with those determined by a standard method.
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paper_title: Preparation of paper micro-fluidic devices used in bio-assay based on drop-on-demand wax droplet generation

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Paper microfluidic devices are a promising technology in developing analytical devices for point-of-care diagnosis in the developing world. This article describes a simple method for paper microfluidic devices based on a PZT drop-on-demand droplet generator. Wax was jetted in the form of droplet, linked with each other and formed into wax pattern on filter paper with a PZT actuator and a glass nozzle. The heated wax pattern became a hydrophobic barrier for reagent used in bio-assay. The glass nozzle fabricated by a home-made micronozzle puller without complicated fabrication technology was low cost, simple and easily made. Coefficient of variation of the jetted wax droplet diameter was 4.0% which showed good reproducibility. The width of wax line was experimentally studied by changing the driving voltage, nozzle diameters and degree of overlapping. The wax line with width of 700–1700 μm was prepared for paper based microfluidic devices. Multi-assay of glucose, protein and pH and 3 × 3 arrays of glucose, protein and pH assay were realized with the prepared paper microfluidic devices. The wax droplet generating system supplied a low-cost, simple, easy-to-use and fast fabrication method for paper microfluidic devices.
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paper_title: Self monitoring of blood glucose in type 2 diabetes mellitus: Systematic review of economic evidence

paper_content:

BACKGROUND ::: Self-monitoring of Blood Glucose (SMBG) is purported to improve glycaemic control, measured by glycosylated haemoglobin (HbA1c). The effectiveness of SMBG in type 2 diabetes mellitus (T2DM) is well-documented though no systematic review of the economic evidence surrounding the use of SMBG in T2DM has been performed. ::: ::: ::: OBJECTIVES ::: To perform a systematic review of economic evaluations of SMBG in T2DM patients. ::: ::: ::: INCLUSION CRITERIA ::: All adult patients suffering from T2DM were included. Outcomes of differing treatment groups, where specified, were also recorded. Studies which examined SMBG as an intervention to control blood glucose were considered. To be included, studies must have made a formal attempt to relate cost to outcome data in a cost-effectiveness or cost utility analysis.The main outcomes were in terms of cost-effectiveness and cost-utility. ::: ::: ::: SEARCH STRATEGY ::: Extensive electronic searches were conducted. Searching was carried out, for the time period 1990 to January 2009, for full text papers and conference abstracts. ::: ::: ::: METHODOLOGICAL QUALITY ::: Methodological quality of included studies was assessed by two reviewers using the standard critical appraisal tools from the JBI-Actuari (Joanna Briggs Institute-Analysis of Cost, Technology and Utilisation Assessment and Review Instrument). Included modelling studies were also assessed using the review criteria of economic models set out by Phillips and colleagues. ::: ::: ::: DATA COLLECTION ::: Data from included studies were extracted using the JBI-Actuari extraction tool. ::: ::: ::: DATA SYNTHESIS ::: Studies were grouped by outcome measure and summarised using tabular and narrative formats. ::: ::: ::: RESULTS ::: Five studies met the review criteria. Three were model-based analyses assessing long-term cost-effectiveness of SMBG, all of which concluded that SMBG was cost-effective. Two further primary economic evaluations assessed short-term cost-effectiveness. Their results found SMBG to be associated with increased cost and no significant reduction in HbA1c. The studies examined subgroups in terms of their treatment protocols and SMBG was considered more likely to be cost-effective in drug and insulin treated groups compared to diet and exercise groups. ::: ::: ::: CONCLUSIONS ::: Economic evidence surrounding SMBG in T2DM remains unclear. For the most part, included studies found SMBG to be cost-effective though analyses are extremely sensitive to relative effects, time-frame of analyses and model assumptions. Whilst large uncertainty exists, SMBG may be cost-effective in certain subgroups e.g. drug and insulin-treated patients. ::: ::: ::: IMPLICATION FOR PRACTICE ::: No strong evidence to recommend the regular use of SMBG in well-controlled diabetes patients, treated only with diet and exercise programmes, exists. The evidence does offer support for SMBG in drug and insulin treated T2DM. It is recommended that clinicians select appropriate patients for SMBG, from these groups, based on their domain expertise. ::: ::: ::: IMPLICATIONS FOR RESEARCH ::: Large-scale prospective RCTs of SMBG, particularly in drug and insulin treated patients, with well-conducted economic evaluations performed alongside them, will enable a more accurate estimation of the cost-effectiveness of SMBG. The optimal frequency and administration of SMBG is still unknown and is another area that warrants further research.
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paper_title: Bottom-Up Fabrication of Paper-Based Microchips by Blade Coating of Cellulose Microfibers on a Patterned Surface

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We report a method for the bottom-up fabrication of paper-based capillary microchips by the blade coating of cellulose microfibers on a patterned surface. The fabrication process is similar to the paper-making process in which an aqueous suspension of cellulose microfibers is used as the starting material and is blade-coated onto a polypropylene substrate patterned using an inkjet printer. After water evaporation, the cellulose microfibers form a porous, hydrophilic, paperlike pattern that wicks aqueous solution by capillary action. This method enables simple, fast, inexpensive fabrication of paper-based capillary channels with both width and height down to about 10 μm. When this method is used, the capillary microfluidic chip for the colorimetric detection of glucose and total protein is fabricated, and the assay requires only 0.30 μL of sample, which is 240 times smaller than for paper devices fabricated using photolithography.
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paper_title: Translating bacterial detection by DNAzymes into a litmus test.

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Microbial pathogens pose serious threats to public health and safety, and results in millions of illnesses and deaths as well as huge economic losses annually. Laborious and expensive pathogen tests often represent a significant hindrance to implementing effective front-line preventative care, particularly in resource-limited regions. Thus, there is a significant need to develop low-cost and easy-to-use methods for pathogen detection. Herein, we present a simple and inexpensive litmus test for bacterial detection. The method takes advantage of a bacteria-specific RNA-cleaving DNAzyme probe as the molecular recognition element and the ability of urease to hydrolyze urea and elevate the pH value of the test solution. By coupling urease to the DNAzyme on magnetic beads, the detection of bacteria is translated into a pH increase, which can be readily detected using a litmus dye or pH paper. The simplicity, low cost, and broad adaptability make this litmus test attractive for field applications, particularly in the developing world.
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paper_title: Inkjet Printed Surface Enhanced Raman Spectroscopy Array on Cellulose Paper

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A novel, ultra low-cost surface enhanced Raman spectroscopy (SERS) substrate has been developed by modifying the surface chemistry of cellulose paper and patterning nanoparticle arrays, all with a consumer inkjet printer. Micro/nanofabrication of SERS substrates for on-chip chemical and biomolecular analysis has been under intense investigation. However, the high cost of producing these substrates and the limited shelf life severely limit their use, especially for routine laboratory analysis and for point-of-sample analysis in the field. Paper-based microfluidic biosensing systems have shown great potential as low-cost disposable analysis tools. In this work, this concept is extended to SERS-based detection. Using an inexpensive consumer inkjet printer, cellulose paper substrates are modified to be hydrophobic in the sensing regions. Synthesized silver nanoparticles are printed onto this hydrophobic paper substrate with microscale precision to form sensing arrays. The hydrophobic surface prevents the aque...
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paper_title: Comparable efficacy of self-monitoring of quantitative urine glucose with self-monitoring of blood glucose on glycaemic control in non-insulin-treated type 2 diabetes☆☆☆

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Abstract Aim To assess whether self-monitoring of quantitative urine glucose or blood glucose is effective, convenient and safe for glycaemic control in non-insulin treated type 2 diabetes. Methods Adults with non-insulin treated type 2 diabetes were recruited and randomized into three groups: Group A, self-monitoring with a quantitative urine glucose meter (n = 38); Group B, selfmonitoring with a blood glucose meter (n = 35); Group C, the control group without selfmonitoring (n = 35). All patients were followed up for six months, during which identical diabetes care was provided. Results There was a significant decrease in HbA1c within each group (p Conclusions This study suggests that self-monitoring of urine glucose has comparable efficacy on glycaemic control, and facilitates better compliance than blood self monitoring, without influencing the quality of life or risk of hypoglycaemia.
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paper_title: Patterned Paper as a Platform for Inexpensive, Low‐Volume, Portable Bioassays

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This communication describes a simple method for patterning paper to create well-defined, millimeter-sized channels, comprising hydrophilic paper bounded by hydrophobic polymer. We believe that this type of patterned paper will become the basis for low-cost, portable, and technically simple multiplexed bioassays. We demonstrate this capability by the simultaneous detection of glucose and protein in 5 μL of urine. The assay system is small, disposable, easy to use (and carry), and requires no external equipment, reagents, or power sources. We believe this kind of system is attractive for uses in less-industrialized countries, in the field, or as an inexpensive alternative to more advanced technologies already used in clinical settings.[1-4] ::: ::: The analysis of biological fluids is necessary for monitoring the health of populations,[2] but these measurements are difficult to implement in remote regions such as those found in less-industrialized countries, in emergency situations, or in home health-care settings.[3] Conventional laboratory instruments provide quantitative measurements of biological samples, but they are unsuitable for these situations since they are large, expensive, and require trained personnel and considerable volumes of biological samples.[2] Other bioassay platforms provide alternatives to more expensive instruments,[5-7] but the need remains for a platform that uses small volumes of sample and that is sufficiently inexpensive to be used widely for measuring samples from large populations. ::: ::: We believe that paper may serve as a particularly convenient platform for running bioassays in the remote situations locations. As a prototype for a mthod we believe to be particularly promosing, we patterned photoresist onto chromatography paper to form defined areas of hydrophilic paper separated by hydrophobic lines or “walls”; these patterns provide spatial control of biological fluids and enable fluid transport, without pumping, due to capillary action in the millimeter-sized channels produced. This method for patterning paper makes it possible to run multiple diagnostic assays on one strip of paper, while still using only small volumes of a single sample. In a fully developed technology, patterned photoresist would be replaced by an appropriate printing technology, but patterning paper with photoresist is: i) convenient for prototyping these devices, and ii) a useful new micropatterning technology in its own right. ::: ::: We patterned chromatography paper with SU-8 2010 photoresist as shown in Figure 1a and as described below: we soaked a 7.5-cm diameter piece of chromatography paper in 2 mL of SU-8 2010 for 30 s, spun it at 2000 rpm for 30 s, and then baked it at 95 °C for 5 min to remove the cyclopentanone in the SU-8 formula. We then exposed the photoresist and paper to 405 nm UV light (50 mW/cm2) for 10 s through a photo-mask (CAD/Art Services, Inc.) that was aligned using a mask aligner (OL-2 Mask Aligner, AB-M, Inc). After exposure, we baked the paper a second time at 95 °C for 5 min to cross-link the exposed portions of the resist. The unpolymerized photoresist was removed by soaking the paper in propylene glycol monomethyl ether acetate (PGMEA) (5 min), and by washing the pattern with propan-2-ol (3 × 10 mL). The paper was more hydrophobic after it was patterned, presumably due to residual resist bound to the paper, so we exposed the entire surface to an oxygen plasma for 10 s at 600 millitorr (SPI Plasma-Prep II, Structure Probe, Inc) to increase the hydrophilicity of the paper (Figures 2a and 2b). ::: ::: ::: ::: Figure 1 ::: ::: Chromatography paper patterned with photoresist. The darker lines are cured photoresist; the lighter areas are unexposed paper. (a) Patterned paper after absorbing 5 μL of Waterman red ink by capillary action. The central channel absorbs the sample ... ::: ::: ::: ::: ::: ::: Figure 2 ::: ::: Assays contaminated with (a) dirt, (b) plant pollen, and (c) graphite powder. The pictures were taken before and after running an artificial urine solution that contained 550 mM glucose and 75 μM BSA. The particulates do not move up the channels ... ::: ::: ::: ::: The patterned paper can be derivatized for biological assays by adding appropriate reagents to the test areas (Figures 1b and ​and2b).2b). In this communication, we demonstrate the method by detecting glucose and protein,[8] but the surface should be suitable for measuring many other analytes as well.[7] The glucose assay is based on the enzymatic oxidation of iodide to iodine,[9] where a color change from clear to brown is associated with the presence of glucose.[10] The protein assay is based on the color change of tetrabromophenol blue (TBPB) when it ionizes and binds to proteins;[11] a positive result in this case is indicated by a color change from yellow to blue. ::: ::: For the glucose assay, we spotted 0.3 μL of a 0.6 M solution of potassium iodide, followed by 0.3 μL of a 1:5 horseradish peroxidase/glucose oxidase solution (15 units of protein per mL of solution). For the protein assay, we spotted 0.3 μL of a 250-mM citrate buffer (pH 1.8) in a well separate from the glucose assay, and then layered 0.3 μL of a 3.3 mM solution of tetrabromophenol blue (TBPB) in 95% ethanol over the citrate buffer. The spotted reagents were allowed to air dry at room temperature. This pre-loaded paper gave consistent results for the protein assay regardless of storage temperature and time (when stored for 15 d both at 0 °C and at 23 °C, wrapped in aluminum foil). The glucose assay was sensitive to storage conditions, and showed decreased signal for assays run 24 h after spotting the reagents (when stored at 23 °C); when stored at 0 °C, however, the glucose assay was as sensitive after day 15 as it was on day 1. ::: ::: We measured artificial samples of glucose and protein in clinically relevant ranges (2.5-50 mM for glucose and 0.38-7.5 μM for bovine serum albumin (BSA))[12, 13] by dipping the bottom of each test strip in 5 μL of a pre-made test solution (Figure 2d). The fluid filled the entire pattern within ca. one minute, but the assays required 10-11 min for the paper to dry and for the color to fully develop.[14] In all cases, we observed color changes corresponding roughly in intensity to the amount of glucose and protein in the test samples, where the lowest concentrations define the lower limits to which these assays can be used (Figure 2e). For comparison, commercially-available dipsticks detect glucose at concentrations as low as 5 mM[7, 9] and protein as low as 0.75 μM;[6, 15] these limits indicate that these paper-based assays are comparable in sensitivity to commercial dipstick assays. Our assay format also allows for the measurement of multiple analytes. ::: ::: This paper-based assay is suitable for measuring multiple samples in parallel and in a relatively short period of time. For example, in one trial, one researcher was able to run 20 different samples (all with 550 mM glucose and 75 μM BSA) within 7.5 min (followed by another 10.5 min for the color to fully develop). An 18-min assay of this type—one capable of measuring two analytes in 20 different sample—may be efficient enough to use in high-throughput screens of larger sample pools. ::: ::: In the field, samples will not be measured under sterile conditions, and dust and dirt may contaminate the assays. The combination of paper and capillary action provides a mechanism for separating particulates from a biological fluid. As a demonstration, we purposely contaminated the artificial urine samples with quantities of dirt, plant pollen, and graphite powder at levels higher than we might expect to see in the samples in the field. These particulates do not move up the channels under the action of capillary wicking, and do not interfere with the assay (Figure 3). ::: ::: Paper strips have been used in biomedical assays for decades because they offer an inexpensive platform for colorimetric chemical testing.[1] Patterned paper has characteristics that lead to miniaturized assays that run by capillary action (e.g., without external pumping), with small volumes of fluids. These methods suggest a path for the development of simple, inexpensive, and portable diagnostic assays that may be useful in remote settings, and in particular, in less-industrialized countries where simple assays are becoming increasingly important for detecting disease and monitoring health,[16, 17], for environmental monitoring, in veterinary and agricultural practice and for other applications.
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paper_title: Understanding Wax Printing: A Simple Micropatterning Process for Paper-Based Microfluidics

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This technical note describes a detailed study on wax printing, a simple and inexpensive method for fabricating microfluidic devices in paper using a commercially available printer and hot plate. The printer prints patterns of solid wax on the surface of the paper, and the hot plate melts the wax so that it penetrates the full thickness of the paper. This process creates complete hydrophobic barriers in paper that define hydrophilic channels, fluid reservoirs, and reaction zones. The design of each device was based on a simple equation that accounts for the spreading of molten wax in paper.
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paper_title: Three-Dimensional Wax Patterning of Paper Fluidic Devices

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In this paper we describe a method for three-dimensional wax patterning of microfluidic paper-based analytical devices (μPADs). The method is rooted in the fundamental details of wax transport in paper and provides a simple way to fabricate complex channel architectures such as hemichannels and fully enclosed channels. We show that three-dimensional μPADs can be fabricated with half as much paper by using hemichannels rather than ordinary open channels. We also provide evidence that fully enclosed channels are efficiently isolated from the exterior environment, decreasing contamination risks, simplifying the handling of the device, and slowing evaporation of solvents.
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paper_title: Paper-based microfluidic point-of-care diagnostic devices

paper_content:

Dipstick and lateral-flow formats have dominated rapid diagnostics over the last three decades. These formats gained popularity in the consumer markets due to their compactness, portability and facile interpretation without external instrumentation. However, lack of quantitation in measurements has challenged the demand of existing assay formats in consumer markets. Recently, paper-based microfluidics has emerged as a multiplexable point-of-care platform which might transcend the capabilities of existing assays in resource-limited settings. However, paper-based microfluidics can enable fluid handling and quantitative analysis for potential applications in healthcare, veterinary medicine, environmental monitoring and food safety. Currently, in its early development stages, paper-based microfluidics is considered a low-cost, lightweight, and disposable technology. The aim of this review is to discuss: (1) fabrication of paper-based microfluidic devices, (2) functionalisation of microfluidic components to increase the capabilities and the performance, (3) introduction of existing detection techniques to the paper platform and (4) exploration of extracting quantitative readouts via handheld devices and camera phones. Additionally, this review includes challenges to scaling up, commercialisation and regulatory issues. The factors which limit paper-based microfluidic devices to become real world products and future directions are also identified.
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paper_title: Point-of-care biochemical assays using gold nanoparticle-implemented microfluidics.

paper_content:

One of the goals of point-of-care (POC) is a chip-based, miniaturized, portable, self-containing system that allows the assay of proteins, nucleic acids, and cells in complex samples. The integration of nanomaterials and microfluidics can help achieve this goal. This tutorial review outlines the mechanism of assaying biomarkers by gold nanoparticles (AuNPs), and the implementation of AuNPs for microfluidic POC devices. In line with this, we discuss some recent advances in AuNP-coupled microfluidic sensors with enhanced performance. Portable and automated instruments for device operation and signal readout are also included for practical applications of these AuNP-combined microfluidic chips.
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paper_title: Rapid prototyping of paper-based microfluidics with wax for low-cost, portable bioassay

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Here we present a simple and low-cost production method to generate paper-based microfluidic devices with wax for portable bioassay. The wax patterning method we introduced here included three different ways: (i) painting with a wax pen, (ii) printing with an inkjet printer followed by painting with a wax pen, (iii) printing by a wax printer directly. The whole process was easy to operate and could be finished within 5-10 min without the use of a clean room, UV lamp, organic solvent, etc. Horse radish peroxidase, BSA and glucose assays were conducted to verify the performance of wax-patterned paper.
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paper_title: Association between ischemia modified albumin, inflammation and hyperglycemia in type 2 diabetes mellitus.

paper_content:

Abstract Objectives The aim of this study was assess serum ischemia modified albumin (IMA) in type 2 diabetes patients and determine its correlation with other risk factors for chronic complications such as inflammation and hyperglycemia. Design and methods Fasting glucose, glycated albumin, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, creatinine, uric acid, albumin, lactic acid, high-sensitivity C-reactive protein (hs-CRP) and IMA were measured in 80 patients with type 2 diabetes and 26 controls. Results Fasting glucose, glycated albumin, triglycerides, creatinine, IMA and hs-CRP were significantly higher in patients with type 2 diabetes. Correlations were weak but significant between IMA and fasting glucose, IMA and hs-CRP, hs-CRP and HDL cholesterol and hs-CRP and fasting glucose were observed. Conclusions We have shown higher levels of IMA and hs-CRP in type 2 diabetes. Hyperglycemia and inflammation reduces the capacity of albumin to bind cobalt, resulting in higher IMA levels.
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paper_title: An instrument-free, screen-printed paper microfluidic device that enables bio and chemical sensing

paper_content:

This paper describes a simple and instrument-free screen-printing method to fabricate hydrophilic channels by patterning polydimethylsiloxane (PDMS) onto chromatography paper. Clearly recognizable border lines were formed between hydrophilic and hydrophobic areas. The minimum width of the printed channel to deliver an aqueous sample was 600 μm, as obtained by this method. Fabricated microfluidic paper-based analytical devices (μPADs) were tested for several colorimetric assays of pH, glucose, and protein in both buffer and artificial urine samples and results were obtained in less than 30 min. The limits of detection (LODs) for glucose and bovine serum albumin (BSA) were 5 mM and 8 μM, respectively. Furthermore, the pH values of different solutions were visually recognised with the naked eye by using a sensitive ink. Ultimately, it is expected that this PDMS-screen-printing (PSP) methodology for μPADs can be readily translated to other colorimetric detection and hydrophilic channels surrounded by a hydrophobic polymer can be formed to transport fluids toward target zones.
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paper_title: Screen Printed Paper-based Diagnostic Devices with Polymeric Inks

paper_content:

A simple and low-cost fabrication method for paper-based diagnostic devices (PBDDs) is described in this study. Street-available polymer solutions were screen printed onto filter papers to create hydrophobic patterns for fluidic channels. In order to obtain fully functional hydrophobic patterns for fluids, the original polymer solutions were diluted with butyl acetate to yield a suitable viscosity range between 30-200 cP for complete patterning on paper. Typical pH and glucose tests with color indicators were performed on the screen printed PBDDs. Images of the PBDDs were analyzed by computers to obtain calibration curves for pH between 2 and 12 and glucose concentration ranging from 10-1000 mmol dm(-3). Detection of formaldehyde in acetone was also carried out to show the possibility of using this PBBD for analytical detection with organic solvents. An exemplar PBDD with simultaneous pH and glucose detection was also used to demonstrate the feasibility of applying this technique for realistic diagnostic applications.
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paper_title: Colorimetric detection and chromatographic analyses of designer drugs in biological materials: a comprehensive review

paper_content:

A number of analogues of phenethylamine and tryptamine, which are prepared by modification of the chemical structures, are being developed for circulation on the black market. Often called “designer drugs,” they are abused in many countries, and cause serious social problems in many parts of the world. Acute deaths have been reported after overdoses of designer drugs. Various methods are required for screening and routine analysis of designer drugs in biological materials for forensic and clinical purposes. Many sample preparation and chromatographic methods for analysis of these drugs in biological materials and seized items have been published. This review presents various colorimetric detections, gas chromatographic (GC)–mass spectrometric, and liquid chromatographic (LC)–mass spectrometric methods proposed for designer drug analyses. Basic information on extractions, derivatizations, GC columns, LC columns, detection limits, and linear ranges is also summarized.
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paper_title: High-Throughput Metabolic Engineering: Advances in Small-Molecule Screening and Selection

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Metabolic engineering for the overproduction of high-value small molecules is dependent upon techniques in directed evolution to improve production titers. The majority of small molecules targeted for overproduction are inconspicuous and cannot be readily obtained by screening. We provide a review on the development of high-throughput colorimetric, fluorescent, and growth-coupled screening techniques, enabling inconspicuous small-molecule detection. We first outline constraints on throughput imposed during the standard directed evolution workflow (library construction, transformation, and screening) and establish a screening and selection ladder on the basis of small-molecule assay throughput and sensitivity. An in-depth analysis of demonstrated screening and selection approaches for small-molecule detection is provided. Particular focus is placed on in vivo biosensor-based detection methods that reduce or eliminate in vitro assay manipulations and increase throughput. We conclude by providing our prospec...
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paper_title: Laser-induced photo-polymerisation for creation of paper-based fluidic devices

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Paper-based microfluidics is a rapidly progressing inter-disciplinary technology driven by the need for low-cost alternatives to conventional point-of-care diagnostic tools. For transport of reagents/analytes, such devices often consist of interconnected hydrophilic fluid-flow channels that are demarcated by hydrophobic barrier walls that extend through the thickness of the paper. Here, we present a laser-based fabrication procedure that uses polymerisation of a photopolymer to produce the required fluidic channels in paper. Experimental results showed that the structures successfully guide the flow of fluids and allow containment of fluids in wells, and hence the technique is suitable for fabrication of paper-based microfluidic devices. The minimum width for the hydrophobic barriers that successfully prevented fluid leakage was ~120 μm and the minimum width for the fluidic channels that can be formed was ~80 μm, the smallest reported so far for paper-based fluidic patterns.
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paper_title: Fabrication of paper-based microfluidic device using printed circuit technology

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This paper describes the use of a printed circuit technology to generate hydrophilic channels in a filter paper. Patterns of channels were designed using Protel soft, and printed on a blank paper. Then, the patterns were transferred to a sheet copper using a thermal transfer printer. The sheet copper with patterns was dipped into ferric chloride solution to etch the whole patterns of the sheet copper. At last, the etched sheet copper was coated with a film of paraffin and then a filter paper. An electric iron was used to heat the other side of the sheet copper. The melting paraffin penetrated full thickness of the filter paper and formed a hydrophobic “wall”. Colorimetric assays for the presence of protein and glucose were demonstrated using the paper-based device. The work is helpful to researchers to fabricate paper-based microfluidic devices for monitoring health and detecting disease.
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paper_title: A handheld stamping process to fabricate microfluidic paper-based analytical devices with chemically modified surface for clinical assays

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This paper describes the development and use of a handheld and lightweight stamp for the production of microfluidic paper-based analytical devices (μPADs). We also chemically modified the paper surface for improved colorimetric measurements. The design of the microfluidic structure has been patterned in a stamp, machined in stainless steel. Prior to stamping, the paper surface was oxidized to promote the conversion of hydroxyl into aldehyde groups, which were then chemically activated for covalent coupling of enzymes. Then, a filter paper sheet was impregnated with paraffin and sandwiched with a native paper (n-paper) sheet, previously oxidized. The metal stamp was preheated at 150 °C and then brought in contact with the paraffined paper (p-paper) to enable the thermal transfer of the paraffin to the n-paper, thus forming the hydrophobic barriers under the application of a pressure of ca. 0.1 MPa for 2 s. The channel and barrier widths measured in 50 independent μPADs exhibited values of 2.6 ± 0.1 and 1.4 ± 0.1 mm, respectively. The chemical modification for covalent coupling of enzymes on the paper surface also led to improvements in the colour uniformity generated inside the sensing area, a known bottleneck in this technology. The relative standard deviation (RSD) values for glucose and uric acid (UA) assays decreased from 40 to 10% and from 20 to 8%, respectively. Bioassays related to the detection of glucose, UA, bovine serum albumin (BSA), and nitrite were successfully performed in concentration ranges useful for clinical assays. The semi-quantitative analysis of all four analytes in artificial urine samples revealed an error smaller than 4%. The disposability of μPADs, the low instrumental requirements of the stamp-based fabrication, and the improved colour uniformity enable the use of the proposed devices for the point-of-care diagnostics or in limited resources settlements.
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paper_title: Fabrication of a microfluidic paper-based analytical device by silanization of filter cellulose using a paper mask for glucose assay

paper_content:

We developed a novel, low-cost and simple method for the fabrication of microfluidic paper-based analytical devices (μPADs) by silanization of filter cellulose using a paper mask having a specific pattern. The paper mask was penetrated with trimethoxyoctadecylsilane (TMOS) by immersing into TMOS-heptane solution. By heating the filter paper sandwiched between the paper mask and glass slides, TMOS was immobilized onto the filter cellulose via the reaction between cellulose OH and TMOS, while the hydrophilic area was not silanized because it was not in contact with the paper mask penetrated with TMOS. The effects of some factors including TMOS concentration, heating temperature and time on the fabrication of μPADs were studied. This method is free of any expensive equipment and metal masks, and could be performed by untrained personnel. These features are very attractive for the fabrication and applications of μPADs in developing countries or resource-limited settings. A flower-shaped μPAD was fabricated and used to determine glucose in human serum samples. The contents determined by this method agreed well with those determined by a standard method.
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paper_title: Preparation of paper micro-fluidic devices used in bio-assay based on drop-on-demand wax droplet generation

paper_content:

Paper microfluidic devices are a promising technology in developing analytical devices for point-of-care diagnosis in the developing world. This article describes a simple method for paper microfluidic devices based on a PZT drop-on-demand droplet generator. Wax was jetted in the form of droplet, linked with each other and formed into wax pattern on filter paper with a PZT actuator and a glass nozzle. The heated wax pattern became a hydrophobic barrier for reagent used in bio-assay. The glass nozzle fabricated by a home-made micronozzle puller without complicated fabrication technology was low cost, simple and easily made. Coefficient of variation of the jetted wax droplet diameter was 4.0% which showed good reproducibility. The width of wax line was experimentally studied by changing the driving voltage, nozzle diameters and degree of overlapping. The wax line with width of 700–1700 μm was prepared for paper based microfluidic devices. Multi-assay of glucose, protein and pH and 3 × 3 arrays of glucose, protein and pH assay were realized with the prepared paper microfluidic devices. The wax droplet generating system supplied a low-cost, simple, easy-to-use and fast fabrication method for paper microfluidic devices.
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paper_title: Bottom-Up Fabrication of Paper-Based Microchips by Blade Coating of Cellulose Microfibers on a Patterned Surface

paper_content:

We report a method for the bottom-up fabrication of paper-based capillary microchips by the blade coating of cellulose microfibers on a patterned surface. The fabrication process is similar to the paper-making process in which an aqueous suspension of cellulose microfibers is used as the starting material and is blade-coated onto a polypropylene substrate patterned using an inkjet printer. After water evaporation, the cellulose microfibers form a porous, hydrophilic, paperlike pattern that wicks aqueous solution by capillary action. This method enables simple, fast, inexpensive fabrication of paper-based capillary channels with both width and height down to about 10 μm. When this method is used, the capillary microfluidic chip for the colorimetric detection of glucose and total protein is fabricated, and the assay requires only 0.30 μL of sample, which is 240 times smaller than for paper devices fabricated using photolithography.
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paper_title: Integrated separation of blood plasma from whole blood for microfluidic paper-based analytical devices.

paper_content:

Many diagnostic tests in a conventional clinical laboratory are performed on blood plasma because changes in its composition often reflect the current status of pathological processes throughout the body. Recently, a significant research effort has been invested into the development of microfluidic paper-based analytical devices (μPADs) implementing these conventional laboratory tests for point-of-care diagnostics in resource-limited settings. This paper describes the use of red blood cell (RBC) agglutination for separating plasma from finger-prick volumes of whole blood directly in paper, and demonstrates the utility of this approach by integrating plasma separation and a colorimetric assay in a single μPAD. The μPAD was fabricated by printing its pattern onto chromatography paper with a solid ink (wax) printer and melting the ink to create hydrophobic barriers spanning through the entire thickness of the paper substrate. The μPAD was functionalized by spotting agglutinating antibodies onto the plasma separation zone in the center and the reagents of the colorimetric assay onto the test readout zones on the periphery of the device. To operate the μPAD, a drop of whole blood was placed directly onto the plasma separation zone of the device. RBCs in the whole blood sample agglutinated and remained in the central zone, while separated plasma wicked through the paper substrate into the test readout zones where analyte in plasma reacted with the reagents of the colorimetric assay to produce a visible color change. The color change was digitized with a portable scanner and converted to concentration values using a calibration curve. The purity and yield of separated plasma was sufficient for successful operation of the μPAD. This approach to plasma separation based on RBC agglutination will be particularly useful for designing fully integrated μPADs operating directly on small samples of whole blood.
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paper_title: Fabrication of paper micro-devices with wax jetting

paper_content:

Paper microfluidic devices are a promising technology in developing analytical devices for point-of-care diagnosis in the developing world. This article describes a novel method of wax jetting with a PZT (piezoelectric ceramic transducer) actuator and glass nozzle for the fabrication of paper microfluidic devices. The hydrophobic fluid pattern was formed by the permeation of filter paper with wax droplets. Results showed that the size of the wax droplet, which was determined by the voltage of the driving signal and nozzle diameter, ranged from 150 μm to 380 μm, and the coefficient of variation of the droplet diameter was under 4.0%. The smallest width of the fluid channel was 600 μm frontside and 750 μm backside. The patterned filter paper was without any leakage, and multi-assay of glucose, protein, and pH on the paper microfluidic device, and laminar diffusion flow with blue and yellow dye were realized. The wax jetting system supplied a low-cost, simple, easy-to-use and fast fabrication method for paper microfluidic devices.
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paper_title: Draw your assay: Fabrication of low-cost paper-based diagnostic and multi-well test zones by drawing on a paper.

paper_content:

Interest in low-cost diagnostic devices has recently gained attention, in part due to the rising cost of healthcare and the need to serve populations in resource-limited settings. A major challenge in the development of such devices is the need for hydrophobic barriers to contain polar bio-fluid analytes. Key approaches in lowering the cost in diagnostics have centered on (i) development of low-cost fabrication techniques/processes, (ii) use of affordable materials, or, (iii) minimizing the need for high-tech tools. This communication describes a simple, low-cost, adaptable, and portable method for patterning paper and subsequent use of the patterned paper in diagnostic tests. Our approach generates hydrophobic regions using a ball-point pen filled with a hydrophobizing molecule suspended in a solvent carrier. An empty ball-point pen was filled with a solution of trichloro perfluoroalkyl silane in hexanes (or hexadecane), and the pen used to draw lines on Whatman® chromatography 1 paper. The drawn regions defined the test zones since the trichloro silane reacts with the paper to give a hydrophobic barrier. The formation of the hydrophobic barriers is reaction kinetic and diffusion-limited, ensuring well defined narrow barriers. We performed colorimetric glucose assays and enzyme-linked immuno-sorbent assay (ELISA) using the created test zones. To demonstrate the versatility of this approach, we fabricated multiple devices on a single piece of paper and demonstrated the reproducibility of assays on these devices. The overall cost of devices fabricated by drawing are relatively lower (
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paper_title: Portable paper-based device for quantitative colorimetric assays relying on light reflectance principle

paper_content:

This paper presents a novel paper-based analytical device based on the colorimetric paper assays through its light reflectance. The device is portable, low cost (<20 dollars), and lightweight (only 176 g) that is available to assess the cost-effectiveness and appropriateness of the original health care or on-site detection information. Based on the light reflectance principle, the signal can be obtained directly, stably and user-friendly in our device. We demonstrated the utility and broad applicability of this technique with measurements of different biological and pollution target samples (BSA, glucose, Fe, and nitrite). Moreover, the real samples of Fe (II) and nitrite in the local tap water were successfully analyzed, and compared with the standard UV absorption method, the quantitative results showed good performance, reproducibility, and reliability. This device could provide quantitative information very conveniently and show great potential to broad fields of resource-limited analysis, medical diagnostics, and on-site environmental detection.
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paper_title: Spatial and spectral detection of protein monolayers with deterministic aperiodic arrays of metal nanoparticles

paper_content:

Light scattering phenomena in periodic systems have been investigated for decades in optics and photonics. Their classical description relies on Bragg scattering, which gives rise to constructive interference at specific wavelengths along well defined propagation directions, depending on illumination conditions, structural periodicity, and the refractive index of the surrounding medium. In this paper, by engineering multifrequency colorimetric responses in deterministic aperiodic arrays of nanoparticles, we demonstrate significantly enhanced sensitivity to the presence of a single protein monolayer. These structures, which can be readily fabricated by conventional Electron Beam Lithography, sustain highly complex structural resonances that enable a unique optical sensing approach beyond the traditional Bragg scattering with periodic structures. By combining conventional dark-field scattering micro-spectroscopy and simple image correlation analysis, we experimentally demonstrate that deterministic aperiodic surfaces with engineered structural color are capable of detecting, in the visible spectral range, protein layers with thickness of a few tens of Angstroms.
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paper_title: Determination of glucose and uric acid with bienzyme colorimetry on microfluidic paper-based analysis devices.

paper_content:

In this work, we first employ a drying method combining with the bienzyme colorimetric detection of glucose and uric acid on microfluidic paper-based analysis devices (μPADs). The channels of 3D μPADs are also designed by us to get better results. The color results are recorded by both Gel Documentation systems and a common camera. By using Gel Documentation systems, the limits of detection (LOD) of glucose and uric acid are 3.81 × 10(-5)M and 4.31 × 10(-5)M, respectively one order of magnitude lower than that of the reported methods on μPADs. By using a common camera, the limits of detection (LOD) of glucose and uric acid are 2.13 × 10(-4)M and 2.87 × 10(-4)M, respectively. Furthermore, the effects of detection conditions have been investigated and discussed comprehensively. Human serum samples are detected with satisfactory results, which are comparable with the clinical testing results. A low-cost, simple and rapid colorimetric method for the simultaneous detection of glucose and uric acid on the μPADs has been developed with enhanced sensitivity.
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paper_title: Development of cellulose paper testing strips for quick measurement of glucose using chromogen agent

paper_content:

Abstract Cellulose paper based glucose test strips were successfully prepared using 2,4,6-tribromo-3-hydroxy benzoic acid (TBHBA) as the chromogen agent. Cellulose paper is a good substrate for carrying chromogen agents and other chemicals so that the quantitative analysis can be done based on the colorimetric chemistry. The color intensity of the developed compounds, which was measured as the differential diffusive reflectance of the test strip at 510 nm, was correlated to the glucose concentration of the sample solutions in the range of 0.18–9.91 mg/ml. These colorimetric test strips could be conveniently used, do not have to use an electronic device, and would have potential applications in the home monitoring of blood glucose for people with diabetes.
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paper_title: Bienzyme colorimetric detection of glucose with self-calibration based on tree-shaped paper strip

paper_content:

Abstract In this contribution, we first developed a semiquantitative method for the detection of glucose with self-calibration based on bienzyme colorimetry by using tree-shaped paper strip. The GOD/HRP bienzyme system was utilized to amplify the color signal in the aqueous phase. Moreover, we employed a paper as microfluidic media for running colorimetric assay, while tree-shaped paper strip was designed to ensure uniform microfluidic flow for multiple branches. Our proposed method gives direct outcomes which can be observed by the naked eye or recorded by a simple camera. The linear range is from 1.0 × 10 −3 to 11.0 × 10 −3 M, with a detection limit of 3 × 10 −4 M. Furthermore, the effect of detection condition has been investigated and discussed comprehensively. The result of determining glucose in human serum is consistent with that of detecting standard glucose solution by using our developed approach. A low-cost, simple, and rapid colorimetric method for the simultaneous detection of glucose with self-calibration on the tree-shaped paper has been proposed.
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paper_title: Fabrication of a microfluidic paper-based analytical device by silanization of filter cellulose using a paper mask for glucose assay

paper_content:

We developed a novel, low-cost and simple method for the fabrication of microfluidic paper-based analytical devices (μPADs) by silanization of filter cellulose using a paper mask having a specific pattern. The paper mask was penetrated with trimethoxyoctadecylsilane (TMOS) by immersing into TMOS-heptane solution. By heating the filter paper sandwiched between the paper mask and glass slides, TMOS was immobilized onto the filter cellulose via the reaction between cellulose OH and TMOS, while the hydrophilic area was not silanized because it was not in contact with the paper mask penetrated with TMOS. The effects of some factors including TMOS concentration, heating temperature and time on the fabrication of μPADs were studied. This method is free of any expensive equipment and metal masks, and could be performed by untrained personnel. These features are very attractive for the fabrication and applications of μPADs in developing countries or resource-limited settings. A flower-shaped μPAD was fabricated and used to determine glucose in human serum samples. The contents determined by this method agreed well with those determined by a standard method.
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paper_title: Integrated separation of blood plasma from whole blood for microfluidic paper-based analytical devices.

paper_content:

Many diagnostic tests in a conventional clinical laboratory are performed on blood plasma because changes in its composition often reflect the current status of pathological processes throughout the body. Recently, a significant research effort has been invested into the development of microfluidic paper-based analytical devices (μPADs) implementing these conventional laboratory tests for point-of-care diagnostics in resource-limited settings. This paper describes the use of red blood cell (RBC) agglutination for separating plasma from finger-prick volumes of whole blood directly in paper, and demonstrates the utility of this approach by integrating plasma separation and a colorimetric assay in a single μPAD. The μPAD was fabricated by printing its pattern onto chromatography paper with a solid ink (wax) printer and melting the ink to create hydrophobic barriers spanning through the entire thickness of the paper substrate. The μPAD was functionalized by spotting agglutinating antibodies onto the plasma separation zone in the center and the reagents of the colorimetric assay onto the test readout zones on the periphery of the device. To operate the μPAD, a drop of whole blood was placed directly onto the plasma separation zone of the device. RBCs in the whole blood sample agglutinated and remained in the central zone, while separated plasma wicked through the paper substrate into the test readout zones where analyte in plasma reacted with the reagents of the colorimetric assay to produce a visible color change. The color change was digitized with a portable scanner and converted to concentration values using a calibration curve. The purity and yield of separated plasma was sufficient for successful operation of the μPAD. This approach to plasma separation based on RBC agglutination will be particularly useful for designing fully integrated μPADs operating directly on small samples of whole blood.
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paper_title: Graphene Oxide: Intrinsic Peroxidase Catalytic Activity and Its Application to Glucose Detection

paper_content:

Carboxyl-modified graphene oxide (GO-COOH) possesses intrinsic peroxidase-like activity that can catalyze the reaction of peroxidase substrate 3,3,5,5-tetramethyl-benzidine (TMB) in the presence of H2O2 to produce a blue color reaction. A simple, cheap, and highly sensitive and selective colorimetric method for glucose detection has been developed and will facilitate the utilization of GO-COOH intrinsic peroxidase activity in medical diagnostics and biotechnology.
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paper_title: Determination of glucose and uric acid with bienzyme colorimetry on microfluidic paper-based analysis devices.

paper_content:

In this work, we first employ a drying method combining with the bienzyme colorimetric detection of glucose and uric acid on microfluidic paper-based analysis devices (μPADs). The channels of 3D μPADs are also designed by us to get better results. The color results are recorded by both Gel Documentation systems and a common camera. By using Gel Documentation systems, the limits of detection (LOD) of glucose and uric acid are 3.81 × 10(-5)M and 4.31 × 10(-5)M, respectively one order of magnitude lower than that of the reported methods on μPADs. By using a common camera, the limits of detection (LOD) of glucose and uric acid are 2.13 × 10(-4)M and 2.87 × 10(-4)M, respectively. Furthermore, the effects of detection conditions have been investigated and discussed comprehensively. Human serum samples are detected with satisfactory results, which are comparable with the clinical testing results. A low-cost, simple and rapid colorimetric method for the simultaneous detection of glucose and uric acid on the μPADs has been developed with enhanced sensitivity.
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paper_title: Development of cellulose paper testing strips for quick measurement of glucose using chromogen agent

paper_content:

Abstract Cellulose paper based glucose test strips were successfully prepared using 2,4,6-tribromo-3-hydroxy benzoic acid (TBHBA) as the chromogen agent. Cellulose paper is a good substrate for carrying chromogen agents and other chemicals so that the quantitative analysis can be done based on the colorimetric chemistry. The color intensity of the developed compounds, which was measured as the differential diffusive reflectance of the test strip at 510 nm, was correlated to the glucose concentration of the sample solutions in the range of 0.18–9.91 mg/ml. These colorimetric test strips could be conveniently used, do not have to use an electronic device, and would have potential applications in the home monitoring of blood glucose for people with diabetes.
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paper_title: Paper based colorimetric biosensing platform utilizing cross-linked siloxane as probe

paper_content:

Abstract Paper based colorimetric biosensing platform utilizing cross-linked siloxane 3-aminopropyltriethoxysilane (APTMS) as probe was developed for the detection of a broad range of targets including H 2 O 2 , glucose and protein biomarker. APTMS was extensively used for the modification of filter papers to develop paper based analytical devices. We discovered when APTMS was cross-linked with glutaraldehyde (GA), the resulting complex (APTMS–GA) displays brick-red color, and a visual color change was observed when the complex reacted with H 2 O 2 . By integrating the APTMS–GA complex with filter paper, the modified paper enables quantitative detection of H 2 O 2 through the monitoring of the color intensity change of the paper via software Image J. Then, with the immobilization of glucose oxidase (GOx) onto the modified paper, glucose can be detected through the detection of enzymatically generated H 2 O 2 . For protein biomarker prostate specific antigen (PSA) assay, we immobilized capture, not captured anti-PSA antibody (Ab 1 ) onto the paper surface and using GOx modified gold nanorod (GNR) as detection anti-PSA antibody (Ab 2 ) label. The detection of PSA was also achieved via the liberated H 2 O 2 when the GOx label reacted with glucose. The results demonstrated the possibility of this paper based sensor for the detection of different analytes with wide linear range. The low cost and simplicity of this paper based sensor could be developed for “point-of-care” analysis and find wide application in different areas.
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paper_title: Modification of microfluidic paper-based devices with silica nanoparticles.

paper_content:

This paper describes a silica nanoparticle-modified microfluidic paper-based analytical device (μPAD) with improved color intensity and uniformity for three different enzymatic reactions with clinical relevance (lactate, glucose, and glutamate). The μPADs were produced on a Whatman grade 1 filter paper and using a CO2 laser engraver. Silica nanoparticles modified with 3-aminopropyltriethoxysilane were then added to the paper devices to facilitate the adsorption of selected enzymes and prevent the washing away effect that creates color gradients in the colorimetric measurements. According to the results herein described, the addition of silica nanoparticles yielded significant improvements in color intensity and uniformity. The resulting μPADs allowed for the detection of the three analytes in clinically relevant concentration ranges with limits of detection (LODs) of 0.63 mM, 0.50 mM, and 0.25 mM for lactate, glucose, and glutamate, respectively. An example of an analytical application has been demonstrated for the semi-quantitative detection of all three analytes in artificial urine. The results demonstrate the potential of silica nanoparticles to avoid the washing away effect and improve the color uniformity and intensity in colorimetric bioassays performed on μPADs.
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paper_title: Paper Bioassay Based on Ceria Nanoparticles as Colorimetric Probes

paper_content:

We report the first use of redox nanoparticles of cerium oxide as colorimetric probes in bioanalysis. The method is based on changes in the physicochemical properties of ceria nanoparticles, used here as chromogenic indicators, in response to the analyte. We show that these particles can be fully integrated in a paper-based bioassay. To construct the sensor, ceria nanoparticles and glucose oxidase were coimmobilized onto filter paper using a silanization procedure. In the presence of glucose, the enzymatically generated hydrogen peroxide induces a visual color change of the ceria nanoparticles immobilized onto the bioactive sensing paper, from white-yellowish to dark orange, in a concentration-dependent manner. A detection limit of 0.5 mM glucose with a linear range up to 100 mM and a reproducibility of 4.3% for n = 11 ceria paper strips were obtained. The assay is fully reversible and can be reused for at least 10 consecutive measurement cycles, without significant loss of activity. Another unique featur...
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paper_title: Controlled fabrication of gold nanoparticles biomediated by glucose oxidase immobilized on chitosan layer-by-layer films

paper_content:

Abstract The control of size and shape of metallic nanoparticles is a fundamental goal in nanochemistry, and crucial for applications exploiting nanoscale properties of materials. We present here an approach to the synthesis of gold nanoparticles mediated by glucose oxidase (GOD) immobilized on solid substrates using the Layer-by-Layer (LbL) technique. The LbL films contained four alternated layers of chitosan and poly(styrene sulfonate) (PSS), with GOD in the uppermost bilayer adsorbed on a fifth chitosan layer: (chitosan/PSS)4/(chitosan/GOD). The films were inserted into a solution containing gold salt and glucose, at various pHs. Optimum conditions were achieved at pH 9, producing gold nanoparticles of ca. 30 nm according to transmission electron microscopy. A comparative study with the enzyme in solution demonstrated that the synthesis of gold nanoparticles is more efficient using immobilized GOD.
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paper_title: Bienzyme colorimetric detection of glucose with self-calibration based on tree-shaped paper strip

paper_content:

Abstract In this contribution, we first developed a semiquantitative method for the detection of glucose with self-calibration based on bienzyme colorimetry by using tree-shaped paper strip. The GOD/HRP bienzyme system was utilized to amplify the color signal in the aqueous phase. Moreover, we employed a paper as microfluidic media for running colorimetric assay, while tree-shaped paper strip was designed to ensure uniform microfluidic flow for multiple branches. Our proposed method gives direct outcomes which can be observed by the naked eye or recorded by a simple camera. The linear range is from 1.0 × 10 −3 to 11.0 × 10 −3 M, with a detection limit of 3 × 10 −4 M. Furthermore, the effect of detection condition has been investigated and discussed comprehensively. The result of determining glucose in human serum is consistent with that of detecting standard glucose solution by using our developed approach. A low-cost, simple, and rapid colorimetric method for the simultaneous detection of glucose with self-calibration on the tree-shaped paper has been proposed.
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paper_title: Microfluidic Distance Readout Sweet Hydrogel Integrated Paper-Based Analytical Device (μDiSH-PAD) for Visual Quantitative Point-of-Care Testing

paper_content:

A disposable, equipment-free, versatile point-of-care testing platform, microfluidic distance readout sweet hydrogel integrated paper-based analytical device (μDiSH-PAD), was developed for portable quantitative detection of different types of targets. The platform relies on a target-responsive aptamer cross-linked hydrogel for target recognition, cascade enzymatic reactions for signal amplification, and microfluidic paper-based analytic devices (μPADs) for visual distance-based quantitative readout. A “sweet” hydrogel with trapped glucoamylase (GA) was synthesized using an aptamer as a cross-linker. When target is present in the sample, the “sweet” hydrogel collapses and releases enzyme GA into the sample, generating glucose by amylolysis. A hydrophilic channel on the μPADs is modified with glucose oxidase (GOx) and colorless 3,3′-diaminobenzidine (DAB) as the substrate. When glucose travels along the channel by capillary action, it is converted to H2O2 by GOx. In addition, DAB is converted into brown ins...
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paper_title: Simple, distance-based measurement for paper analytical devices

paper_content:

Paper-based analytical devices (PADs) represent a growing class of elegant, yet inexpensive chemical sensor technologies designed for point-of-use applications. Most PADs, however, still utilize some form of instrumentation such as a camera for quantitative detection. We describe here a simple technique to render PAD measurements more quantitative and straightforward using the distance of colour development as a detection motif. The so-called distance-based detection enables PAD chemistries that are more portable and less resource intensive compared to classical approaches that rely on the use of peripheral equipment for quantitative measurement. We demonstrate the utility and broad applicability of this technique with measurements of glucose, nickel, and glutathione using three different detection chemistries: enzymatic reactions, metal complexation, and nanoparticle aggregation, respectively. The results show excellent quantitative agreement with certified standards in complex sample matrices. This work provides the first demonstration of distance-based PAD detection with broad application as a class of new, inexpensive sensor technologies designed for point-of-use applications.
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paper_title: A paper strip based non-invasive glucose biosensor for salivary analysis.

paper_content:

In our present study, we developed an optical biosensor for direct determination of salivary glucose by using immobilized glucose oxidase enzyme on filter paper strip (specific activity 1.4 U/strip) and then reacting it with synthetic glucose samples in presence of co-immobilized color pH indicator. The filter paper changed color based on concentration of glucose in reaction media and hence, by scanning this color change (using RGB profiling) through an office scanner and open source image processing software (GIMP) the concentration of glucose in the reaction medium could be deduced. Once the biosensor was standardized, the synthetic glucose sample was replaced with human saliva from donors. The individual's blood glucose level at the time of obtaining saliva was also measured using an Accuchek(™) active glucometer (Roche Inc.). In this preliminary study, a correlation of nearly 0.64 was found between glucose levels in saliva and blood of healthy individuals and in diabetic patients it was nearly in the order of 0.95, thereby validating the importance of salivary analysis. The RGB profiling method obtained a detection range of 9-1350 mg/dL glucose at a response time of 45 s and LOD of 22.2 mg/dL.
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paper_title: Reagent pencils: a new technique for solvent-free deposition of reagents onto paper-based microfluidic devices

paper_content:

Custom-made pencils containing reagents dispersed in a solid matrix were developed to enable rapid and solvent-free deposition of reagents onto membrane-based fluidic devices. The technique is as simple as drawing with the reagent pencils on a device. When aqueous samples are added to the device, the reagents dissolve from the pencil matrix and become available to react with analytes in the sample. Colorimetric glucose assays conducted on devices prepared using reagent pencils had comparable accuracy and precision to assays conducted on conventional devices prepared with reagents deposited from solution. Most importantly, sensitive reagents, such as enzymes, are stable in the pencils under ambient conditions, and no significant decrease in the activity of the enzyme horseradish peroxidase stored in a pencil was observed after 63 days. Reagent pencils offer a new option for preparing and customizing diagnostic tests at the point of care without the need for specialized equipment.
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paper_title: Enzymatic synthesis of gold nanoparticles wrapped by glucose oxidase

paper_content:

An enzymatic synthesis route to protein-wrapped gold nanoparticles is developed. Glucose oxidase (GOD) reduces Au(III) ion in the presence of β-D-glucose, and stable gold nanoparticles with average diameter of 14.5 nm areformed. FT-IR spectra, zeta potential and CD spectra of purified nanoparticles indicate that they are stabilized by the adsorbed protein layer.
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paper_title: Multiplexed bioactive paper based on GO@SiO2@CeO2 nanosheets for a low-cost diagnostics platform

paper_content:

Abstract In this paper, Graphene oxide@SiO 2 @CeO 2 hybrid nanosheets (GSCs) have been successfully synthesized by the wet-chemical strategy. TEM, FITR and XPS were applied to characterize the morphology and composition of the nanosheets. The colorimetric assay of these nanosheets indicated that they possessed high intrinsic peroxidase activity, which should be ascribed to the combination of graphene oxide and CeO 2 . A fully integrated reagentless bioactive paper based on GSCs was fabricated, which were able to simultaneously detect glucose, lactate, uric acid and cholesterol. The results demonstrated that GSCs have great potential as an alternative to the commonly employed peroxidase in daily nursing and general physical examination.
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paper_title: Highly sensitive colorimetric detection of glucose and uric acid in biological fluids using chitosan-modified paper microfluidic devices

paper_content:

This paper describes the modification of microfluidic paper-based analytical devices (μPADs) with chitosan to improve the analytical performance of colorimetric measurements associated with enzymatic bioassays. Chitosan is a natural biopolymer extensively used to modify biosensing surfaces due to its capability of providing a suitable microenvironment for the direct electron transfer between an enzyme and a reactive surface. This hypothesis was investigated using glucose and uric acid (UA) colorimetric assays as model systems. The best colorimetric sensitivity for glucose and UA was achieved using a chromogenic solution composed of 4-aminoantipyrine and sodium 3,5-dichloro-2-hydroxy-benzenesulfonate (4-AAP/DHBS), which provided a linear response for a concentration range between 0.1 and 1.0 mM. Glucose and UA were successfully determined in artificial serum samples with accuracies between 87 and 114%. The limits of detection (LODs) found for glucose and UA assays were 23 and 37 μM, respectively. The enhanced analytical performance of chitosan-modified μPADs allowed the colorimetric detection of glucose in tear samples from four nondiabetic patients. The achieved concentration levels ranged from 130 to 380 μM. The modified μPADs offered analytical reliability and accuracy as well as no statistical difference from the values achieved through a reference method. Based on the presented results, the proposed μPAD can be a powerful alternative tool for non-invasive glucose analysis.
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paper_title: Colorimetric detection of sugars based on gold nanoparticle formation

paper_content:

Abstract In this paper gold nanoparticles (Au-NPs) have been used as colorimetric reporters for the detection of sugars. The synthesis of Au-NPs has been obtained in presence of glucose as reducing agent in different conditions, allowing the formation of pink or blue coloured NPs, and has been employed in the design of two colorimetric assays. Both assays rely on the analyte induced intensity increase (without any shift) of the NPs plasmon band absorption. The “pink assay” is based on the sugar assisted chemical synthesis of NPs and it represents a simple one-step colorimetric approach to the quantification of all potentially reducing sugars (sucrose included) with a LOD of 10 μM. The “blue assay” is based on the Au-NP synthesis catalysed by the enzyme glucose oxidase and it is specific for glucose, with a LOD of 5 μM. Compared to the classical bi-enzymatic (glucose oxidase/peroxidase) optical assay, it uses only one enzyme and does not suffer of the bleaching of the final colour because the reporter Au-NPs are very stable.
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paper_title: Paper-based glucose biosensing system utilizing a smartphone as a signal reader

paper_content:

A simple paper-based optical biosensor for glucose monitoring was developed. As a glucose biosensing principle, a colorimetric glucose assay, using glucose oxidase (GOx) and horseradish peroxidase (HRP), was chosen. The enzymatic glucose assay was implanted on the analytical paper-based device, which is fabricated by the wax printing method. The fabricated device consists of two paper layers. The top layer has a sample loading zone and a detection zone, which are modified with enzymes and chromogens. The bottom layer contains a fluidic channel to convey the solution from the loading zone to the detection zone. Double-sided adhesive tape is used to attach these two layers. In this system, when a glucose solution is dropped onto the loading zone, the solution is transferred to the detection zone, which is modified with GOx, HRP, and chromogenic compounds through the connected fluidic channel. In the presence of GOx-generated H2O2, HRP converts chromogenic compounds into the final product exhibiting a blue color, inducing color change in the detection zone. To confirm the changes in signal intensity in the detection zone, the resulting image was registered by a digital camera from a smartphone. To minimize signal interference from external light, the experiment was performed in a specifically designed light-tight box, which was suited to the smartphone. By using the developed biosensing system, various concentrations of glucose samples (0–20 mM) and human serum (5–17 mM) were precisely analyzed within a few minutes. With the developed system, we could expand the applicability of a smartphone to bioanalytical health care.
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paper_title: A low cost, safe, disposable, rapid and self-sustainable paper-based platform for diagnostic testing: lab-on-paper

paper_content:

There is a strong interest in the use of biopolymers in the electronic and biomedical industries, mainly towards low-cost applications. The possibility of developing entirely new kinds of products based on cellulose is of current interest, in order to enhance and to add new functionalities to conventional paper-based products. We present our results towards the development of paper-based microfluidics for molecular diagnostic testing. Paper properties were evaluated and compared to nitrocellulose, the most commonly used material in lateral flow and other rapid tests. Focusing on the use of paper as a substrate for microfluidic applications, through an eco-friendly wax-printing technology, we present three main and distinct colorimetric approaches: (i) enzymatic reactions (glucose detection); (ii) immunoassays (antibodies anti-Leishmania detection); (iii) nucleic acid sequence identification (Mycobacterium tuberculosis complex detection). Colorimetric glucose quantification was achieved through enzymatic reactions performed within specific zones of the paper-based device. The colouration achieved increased with growing glucose concentration and was highly homogeneous, covering all the surface of the paper reaction zones in a 3D sensor format. These devices showed a major advantage when compared to the 2D lateral flow glucose sensors, where some carryover of the coloured products usually occurs. The detection of anti-Leishmania antibodies in canine sera was conceptually achieved using a paper-based 96-well enzyme-linked immunosorbent assay format. However, optimization is still needed for this test, regarding the efficiency of the immobilization of antigens on the cellulose fibres. The detection of Mycobacterium tuberculosis nucleic acids integrated with a non-cross-linking gold nanoprobe detection scheme was also achieved in a wax-printed 384-well paper-based microplate, by the hybridization with a species-specific probe. The obtained results with the above-mentioned proof-of-concept sensors are thus promising towards the future development of simple and cost-effective paper-based diagnostic devices. (Some figures may appear in colour only in the online journal)
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paper_title: One-touch-activated blood multidiagnostic system using a minimally invasive hollow microneedle integrated with a paper-based sensor

paper_content:

The development of real-time innocuous blood diagnosis has been a long-standing goal in healthcare; an improved, miniature, all-in-one point-of-care testing (POCT) system with low cost and simplified operation is highly desired. Here, we present a one-touch-activated blood multidiagnostic system (OBMS) involving the synergistic integration of a hollow microneedle and paper-based sensor, providing a number of unique characteristics for simplifying the design of microsystems and enhancing user performance. In this OBMS, all functions of blood collection, serum separation, and detection were sequentially automated in one single device that only required one-touch activation by finger-power without additional operations. For the first time, we successfully demonstrated the operation of this system in vivo in glucose and cholesterol diagnosis, showing a great possibility for human clinical application and commercialization. Additionally, this novel system offers a new approach for the use of microneedles and paper sensors as promising intelligent elements in future real-time healthcare monitoring devices.
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paper_title: High throughput method for prototyping three-dimensional, paper-based microfluidic devices

paper_content:

This paper describes an efficient and high throughput method for fabricating three-dimensional (3D) paper-based microfluidic devices. The method avoids tedious alignment and assembly steps and eliminates a major bottleneck that has hindered the development of these types of devices. A single researcher now can prepare hundreds of devices within 1 h.
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paper_title: An animal cell culture monitoring system using a smartphone-mountable paper-based analytical device

paper_content:

Abstract We developed a simple and low-cost cell culture monitoring system utilizing a paper-based analytical device (PAD) and a smartphone. The PAD simultaneously analyses glucose and lactate concentrations in the cell culture medium. Focusing on the fact that animal cells consume glucose and produce lactate under anaerobic conditions, oxidase- and horseradish peroxidase (HRP) enzyme-mediated colorimetric assays were integrated into the PAD. The PAD was designed to have three laminated layers. By using a double-sided adhesive tape as the middle layer and wax coating, a bifurcated fluidic channel was prepared to manipulate sample flow. At the inlet and the outlets of the channel, a sample drop zone and two detection zones for glucose and lactate, respectively, were positioned. When sample solution is loaded onto the drop zone, it flows to the detection zone through the hydrophilic fluidic channel via capillary force. Upon reaching the detection zone, the sample reacts with glucose and lactate oxidases (GOx and LOx) and HRP, immobilized on the detection zone along with colorless chromophores. By the Trinder’s reaction, the colorless chromophore is converted to a blue-colored product, generating concentration-dependent signal. With a gadget designed to aid the image acquisition, the PAD was positioned to the smartphone-embedded camera. Images of the detection zones were acquired using a mobile application and the color intensities were quantified as sensor signals. For the glucose assay using GOx/HRP format, we obtained the limit of detection (LOD ∼0.3 mM) and the limit of quantification (LOQ ∼0.9 mM) values in the dynamic detection range from 0.3 to 8.0 mM of glucose. For lactate assay using LOx/HRP, the LOD (0.02 mM) and the LOQ (0.06 mM) values were registered in the dynamic detection range from 0.02 to 0.50 mM of lactate. With the device, simultaneous analyses of glucose and lactate in cell culture media were conducted, exhibiting highly accurate and reproducible results. Based on the results, we propose that the optical sensing system developed is feasible for practical monitoring of animal cell culture.
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paper_title: Paper-based glucose biosensing system utilizing a smartphone as a signal reader

paper_content:

A simple paper-based optical biosensor for glucose monitoring was developed. As a glucose biosensing principle, a colorimetric glucose assay, using glucose oxidase (GOx) and horseradish peroxidase (HRP), was chosen. The enzymatic glucose assay was implanted on the analytical paper-based device, which is fabricated by the wax printing method. The fabricated device consists of two paper layers. The top layer has a sample loading zone and a detection zone, which are modified with enzymes and chromogens. The bottom layer contains a fluidic channel to convey the solution from the loading zone to the detection zone. Double-sided adhesive tape is used to attach these two layers. In this system, when a glucose solution is dropped onto the loading zone, the solution is transferred to the detection zone, which is modified with GOx, HRP, and chromogenic compounds through the connected fluidic channel. In the presence of GOx-generated H2O2, HRP converts chromogenic compounds into the final product exhibiting a blue color, inducing color change in the detection zone. To confirm the changes in signal intensity in the detection zone, the resulting image was registered by a digital camera from a smartphone. To minimize signal interference from external light, the experiment was performed in a specifically designed light-tight box, which was suited to the smartphone. By using the developed biosensing system, various concentrations of glucose samples (0–20 mM) and human serum (5–17 mM) were precisely analyzed within a few minutes. With the developed system, we could expand the applicability of a smartphone to bioanalytical health care.
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paper_title: Paper and toner three-dimensional fluidic devices: programming fluid flow to improve point-of-care diagnostics

paper_content:

We present a new method for fabricating three-dimensional paper-based fluidic devices that uses toner as a thermal adhesive to bond multiple layers of patterned paper together. The fabrication process is rapid, involves minimal equipment (a laser printer and a laminator) and produces complex channel networks with dimensions down to 1 mm. The devices can run multiple diagnostic assays on one or more samples simultaneously, can incorporate positive and negative controls and can be programmed to display the results of the assays in a variety of patterns. The patterns of the results can encode information, which could be used to identify counterfeit devices, identify samples, encrypt the results for patient privacy or monitor patient compliance.
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paper_title: A low cost, safe, disposable, rapid and self-sustainable paper-based platform for diagnostic testing: lab-on-paper

paper_content:

There is a strong interest in the use of biopolymers in the electronic and biomedical industries, mainly towards low-cost applications. The possibility of developing entirely new kinds of products based on cellulose is of current interest, in order to enhance and to add new functionalities to conventional paper-based products. We present our results towards the development of paper-based microfluidics for molecular diagnostic testing. Paper properties were evaluated and compared to nitrocellulose, the most commonly used material in lateral flow and other rapid tests. Focusing on the use of paper as a substrate for microfluidic applications, through an eco-friendly wax-printing technology, we present three main and distinct colorimetric approaches: (i) enzymatic reactions (glucose detection); (ii) immunoassays (antibodies anti-Leishmania detection); (iii) nucleic acid sequence identification (Mycobacterium tuberculosis complex detection). Colorimetric glucose quantification was achieved through enzymatic reactions performed within specific zones of the paper-based device. The colouration achieved increased with growing glucose concentration and was highly homogeneous, covering all the surface of the paper reaction zones in a 3D sensor format. These devices showed a major advantage when compared to the 2D lateral flow glucose sensors, where some carryover of the coloured products usually occurs. The detection of anti-Leishmania antibodies in canine sera was conceptually achieved using a paper-based 96-well enzyme-linked immunosorbent assay format. However, optimization is still needed for this test, regarding the efficiency of the immobilization of antigens on the cellulose fibres. The detection of Mycobacterium tuberculosis nucleic acids integrated with a non-cross-linking gold nanoprobe detection scheme was also achieved in a wax-printed 384-well paper-based microplate, by the hybridization with a species-specific probe. The obtained results with the above-mentioned proof-of-concept sensors are thus promising towards the future development of simple and cost-effective paper-based diagnostic devices. (Some figures may appear in colour only in the online journal)
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paper_title: One-touch-activated blood multidiagnostic system using a minimally invasive hollow microneedle integrated with a paper-based sensor

paper_content:

The development of real-time innocuous blood diagnosis has been a long-standing goal in healthcare; an improved, miniature, all-in-one point-of-care testing (POCT) system with low cost and simplified operation is highly desired. Here, we present a one-touch-activated blood multidiagnostic system (OBMS) involving the synergistic integration of a hollow microneedle and paper-based sensor, providing a number of unique characteristics for simplifying the design of microsystems and enhancing user performance. In this OBMS, all functions of blood collection, serum separation, and detection were sequentially automated in one single device that only required one-touch activation by finger-power without additional operations. For the first time, we successfully demonstrated the operation of this system in vivo in glucose and cholesterol diagnosis, showing a great possibility for human clinical application and commercialization. Additionally, this novel system offers a new approach for the use of microneedles and paper sensors as promising intelligent elements in future real-time healthcare monitoring devices.
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paper_title: High throughput method for prototyping three-dimensional, paper-based microfluidic devices

paper_content:

This paper describes an efficient and high throughput method for fabricating three-dimensional (3D) paper-based microfluidic devices. The method avoids tedious alignment and assembly steps and eliminates a major bottleneck that has hindered the development of these types of devices. A single researcher now can prepare hundreds of devices within 1 h.
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paper_title: Toward instrument-free digital measurements: a three-dimensional microfluidic device fabricated in a single sheet of paper by double-sided printing and lamination

paper_content:

This study demonstrates a simple approach for fabricating a 3D-μPAD from a single sheet of paper by double-sided printing and lamination. First, a wax printer prints vertically symmetrical and asymmetrical wax patterns onto a double-sided paper surface. Then, a laminator melts the printed wax patterns to form microfluidic channels in the paper sheet. The vertically symmetrical wax patterns form vertical channels when the melted wax patterns make contact with each other. The asymmetrical wax patterns form lateral and vertical channels at the cross section of the paper when the printed wax patterns are melted to a lower height than the thickness of the single sheet of paper. Finally, the two types of wax patterns form a 3D microfluidic network to move fluid laterally and vertically in the single sheet of paper. This method eliminates major technical hurdles related to the complicated and tedious alignment, assembly, bonding, and punching process. This 3D-μPAD can be used in a multiplex digital assay to measure the concentration of a target analyte in a sample solution simply by counting the number of colored bars at a fixed time. It does not require any external instruments to perform digital measurements. Therefore, we expect that this approach could be an instrument-free assay format for use in developing countries.
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paper_title: Paper-based 3D microfluidic device for multiple bioassays

paper_content:

Abstract This study investigates a new paper-based 3D microfluidic analytical device for analyzing multiple biological fluids. A wax-printed and -impregnated device was operated using tip-pinch manipulation of the thumb and index fingers and applied the chemical reaction of a preloaded colorimetric indicator and biological solutions. Chemical sensing of protein and glucose concentrations was quantitatively analyzed by changes in the color intensity of the image taken from three image readout devices including scanner (Epson Perfection V700), microscope (USB-embedded handheld digital microscope), and smartphone (LG Optimus Vu). Paper-based 3D microfluidic analytic device with three image analyzers successfully quantified 1.5–75 μM protein concentrations and 0–900 mg/dL glucose concentrations. Paper-based 3D microfluidic device combined with the smartphone showed the performance in protein bioassay (1.5–75 μM) and glucose bioassay (0–50 mM) including clinically relevant ranges comparable to other devices. An origami-driven paper-based 3D microfluidic analytic is a useful platform with great potential for application in point-of-care diagnostics.
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paper_title: Three-Dimensional Paper-Based Microfluidic Device for Assays of Protein and Glucose in Urine

paper_content:

The first step in curing a disease is being able to detect the disease effectively. Paper-based microfluidic devices are biodegradable and can make diagnosing diseases cost-effective and easy in almost all environments. We created a three-dimesnional (3D) paper device using wax printing fabrication technique and basic principles of origami. This design allows for a versatile fabrication technique over previously reported patterning of SU-8 photoresist on chromatography paper by employing a readily available wax printer. The design also utilizes multiple colorimetric assays that can accommodate one or more analytes including urine, blood, and saliva. In this case to demonstrate the functionality of the 3D paper-based microfluidic system, a urinalysis of protein and glucose assays is conducted. The amounts of glucose and protein introduced to the device are found to be proportional to the color change of each assay. This color change was quantified by use of Adobe Photoshop. Urine samples from participants with no pre-existing health conditions and one person with diabetes were collected and compared against synthetic urine samples with predetermined glucose and protein levels. Utilizing this method, we were able to confirm that both protein and glucose levels were in fact within healthy ranges for healthy participants. For the participant with diabetes, glucose was found to be above the healthy range while the protein level was in the healthy range.
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paper_title: An animal cell culture monitoring system using a smartphone-mountable paper-based analytical device

paper_content:

Abstract We developed a simple and low-cost cell culture monitoring system utilizing a paper-based analytical device (PAD) and a smartphone. The PAD simultaneously analyses glucose and lactate concentrations in the cell culture medium. Focusing on the fact that animal cells consume glucose and produce lactate under anaerobic conditions, oxidase- and horseradish peroxidase (HRP) enzyme-mediated colorimetric assays were integrated into the PAD. The PAD was designed to have three laminated layers. By using a double-sided adhesive tape as the middle layer and wax coating, a bifurcated fluidic channel was prepared to manipulate sample flow. At the inlet and the outlets of the channel, a sample drop zone and two detection zones for glucose and lactate, respectively, were positioned. When sample solution is loaded onto the drop zone, it flows to the detection zone through the hydrophilic fluidic channel via capillary force. Upon reaching the detection zone, the sample reacts with glucose and lactate oxidases (GOx and LOx) and HRP, immobilized on the detection zone along with colorless chromophores. By the Trinder’s reaction, the colorless chromophore is converted to a blue-colored product, generating concentration-dependent signal. With a gadget designed to aid the image acquisition, the PAD was positioned to the smartphone-embedded camera. Images of the detection zones were acquired using a mobile application and the color intensities were quantified as sensor signals. For the glucose assay using GOx/HRP format, we obtained the limit of detection (LOD ∼0.3 mM) and the limit of quantification (LOQ ∼0.9 mM) values in the dynamic detection range from 0.3 to 8.0 mM of glucose. For lactate assay using LOx/HRP, the LOD (0.02 mM) and the LOQ (0.06 mM) values were registered in the dynamic detection range from 0.02 to 0.50 mM of lactate. With the device, simultaneous analyses of glucose and lactate in cell culture media were conducted, exhibiting highly accurate and reproducible results. Based on the results, we propose that the optical sensing system developed is feasible for practical monitoring of animal cell culture.
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paper_title: Highly sensitive colorimetric detection of glucose and uric acid in biological fluids using chitosan-modified paper microfluidic devices

paper_content:

This paper describes the modification of microfluidic paper-based analytical devices (μPADs) with chitosan to improve the analytical performance of colorimetric measurements associated with enzymatic bioassays. Chitosan is a natural biopolymer extensively used to modify biosensing surfaces due to its capability of providing a suitable microenvironment for the direct electron transfer between an enzyme and a reactive surface. This hypothesis was investigated using glucose and uric acid (UA) colorimetric assays as model systems. The best colorimetric sensitivity for glucose and UA was achieved using a chromogenic solution composed of 4-aminoantipyrine and sodium 3,5-dichloro-2-hydroxy-benzenesulfonate (4-AAP/DHBS), which provided a linear response for a concentration range between 0.1 and 1.0 mM. Glucose and UA were successfully determined in artificial serum samples with accuracies between 87 and 114%. The limits of detection (LODs) found for glucose and UA assays were 23 and 37 μM, respectively. The enhanced analytical performance of chitosan-modified μPADs allowed the colorimetric detection of glucose in tear samples from four nondiabetic patients. The achieved concentration levels ranged from 130 to 380 μM. The modified μPADs offered analytical reliability and accuracy as well as no statistical difference from the values achieved through a reference method. Based on the presented results, the proposed μPAD can be a powerful alternative tool for non-invasive glucose analysis.
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paper_title: Paper-fluidic electrochemical biosensing platform with enzyme paper and enzymeless electrodes

paper_content:

Abstract A miniaturized paper-based microfluidic electrochemical enzymatic biosensing platform was developed and the effects of fluidic behaviors in paper substrate on electrochemical sensing were systemically investigated. The biosensor is composed of an enzyme-immobilized pure cellulose paper pad, an enzymeless screen-printed electrode (SPE) modified with platinum nanoparticles (PtNPs), and a pair of clamped acrylonitrile butadiene styrene (ABS) plastic holders to provide good alignment for stable signal sensing. The wicking rate of liquid sample in paper was predicted, using a two-dimensional Fickian-diffusion model, to be 1.0 × 10 −2 cm 2 /s, and was verified experimentally. Dip-coating was used to prepare the enzyme-modified paper pad (EPP), which is amenable for mass manufacturing. The EPP retained excellent hydrophilicity and mechanical properties, with even slightly improved tensile strength and break strain. No significant difference in voltammetric behaviors was observed between measurements made in bulk buffer solution and with different sample volumes applied to EPP beyond its saturation wicking volume. Glucose oxidase (GO x ), an enzyme specific for glucose (Glc) substrate, was used as a model enzyme and its enzymatic reaction product H 2 O 2 was detected by the enzymeless PtNPs-SPE in the presence of ambient electron mediator O 2 . Consequently, Glc was detected with its concentration linearly depending on H 2 O 2 oxidation current with sensitivity of 10.5 μA mM -1 cm -2 and detection limit of 9.3 μM (at S / N = 3). The biosensor can be quickly regenerated with memory effects removed by buffer additions for continuous real-time detection of multiple samples in one run for point-of-care purposes. This integrated platform is also inexpensive since the EPP is easily stored, and enzymeless PtNPs-SPEs can be used multiple times with different EPPs. The green and facile preparation in bulk, excellent mechanical strength, well-maintained enzyme activity, disposability, and good reproducibility and stability make our paper-fluidic biosensor platform suitable for various real-time electrochemical bioassays without any external power for mixing, especially in resource-limited conditions.
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paper_title: Electrochemical detection of glucose from whole blood using paper-based microfluidic devices.

paper_content:

Abstract Electrochemical paper-based analytical devices (ePADs) with integrated plasma isolation for determination of glucose from whole blood samples have been developed. A dumbbell shaped ePAD containing two blood separation zones (VF2 membranes) with a middle detection zone was fabricated using the wax dipping method. The dumbbell shaped device was designed to separate plasma while generating homogeneous flow to the middle detection zone of the ePAD. The proposed ePADs work with whole blood samples with 24–60% hematocrit without dilution, and the plasma was completely separated within 4 min. Glucose in isolated plasma separated was detected using glucose oxidase immobilized on the middle of the paper device. The hydrogen peroxide generated from the reaction between glucose and the enzyme pass through to a Prussian blue modified screen printed electrode (PB-SPEs). The currents measured using chronoamperometry at the optimal detection potential for H 2 O 2 (−0.1 V versus Ag/AgCl reference electrode) were proportional to glucose concentrations in the whole blood. The linear range for glucose assay was in the range 0–33.1 mM ( r 2 = 0.987). The coefficients of variation (CVs) of currents were 6.5%, 9.0% and 8.0% when assay whole blood sample containing glucose concentration at 3.4, 6.3, and 15.6 mM, respectively. Because each sample displayed intra-individual variation of electrochemical signal, glucose assay in whole blood samples were measured using the standard addition method. Results demonstrate that the ePAD glucose assay was not significantly different from the spectrophotometric method ( p = 0.376, paired sample t -test, n = 10).
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paper_title: A hybrid system of carbon ink electrodes and chromatography paper on a CMOS chip and its application to enzymatic glucose sensor

paper_content:

Enzymatic sensors on complementary metal–oxide–semiconductor (CMOS) chips are realized using carbon ink and chromatography paper (ChrPr). Electrodes are fabricated from carbon ink on CMOS chips. The carbon ink electrodes work as well-behaving electrochemical electrodes. Enzyme electrodes are realized by covering the carbon ink electrodes on the CMOS chip with ChrPr supporting enzymes and electron mediators. Such enzyme electrodes successfully give anodic current proportional to the glucose concentration. Good linearity is observed up to 10 mM glucose concentration, which is sufficient for blood glucose testing applications.
---
paper_title: Toward instrument-free digital measurements: a three-dimensional microfluidic device fabricated in a single sheet of paper by double-sided printing and lamination

paper_content:

This study demonstrates a simple approach for fabricating a 3D-μPAD from a single sheet of paper by double-sided printing and lamination. First, a wax printer prints vertically symmetrical and asymmetrical wax patterns onto a double-sided paper surface. Then, a laminator melts the printed wax patterns to form microfluidic channels in the paper sheet. The vertically symmetrical wax patterns form vertical channels when the melted wax patterns make contact with each other. The asymmetrical wax patterns form lateral and vertical channels at the cross section of the paper when the printed wax patterns are melted to a lower height than the thickness of the single sheet of paper. Finally, the two types of wax patterns form a 3D microfluidic network to move fluid laterally and vertically in the single sheet of paper. This method eliminates major technical hurdles related to the complicated and tedious alignment, assembly, bonding, and punching process. This 3D-μPAD can be used in a multiplex digital assay to measure the concentration of a target analyte in a sample solution simply by counting the number of colored bars at a fixed time. It does not require any external instruments to perform digital measurements. Therefore, we expect that this approach could be an instrument-free assay format for use in developing countries.
---
paper_title: Paper-based enzymatic reactors for batch injection analysis of glucose on 3D printed cell coupled with amperometric detection

paper_content:

Abstract This report describes for the first time the development of paper-based enzymatic reactors (PERs) for the detection of glucose (Glu) in artificial serum sample using a 3D printed batch injection analysis (BIA) cell coupled with electrochemical detection. The fabrication of the PERs involved firstly the oxidation of the paper surface with a sodium periodate solution. The oxidized paper was then perforated with a paper punch to create microdisks and activated with a solution containing N -hydroxysuccinimide (NHS) and N -(3-dimethylaminopropyl)- N ′-ethylcarbodiimide hydrochloride (EDC). Glucose oxidase (GOx) enzyme was then covalently immobilized on paper surface to promote the enzymatic assay for the detection of Glu in serum sample. After the addition of Glu on the PER surface placed inside a plastic syringe, the analyte penetrated through the paper surface under vertical flow promoting the enzymatic assay. The reaction product (H 2 O 2 ) was collected with an electronic micropipette in a microtube and analyzed in the 3D BIA cell coupled with screen-printed electrodes (SPEs). The overall preparation time and the cost estimated per PER were 2.5 h and $0.02, respectively. Likewise the PERs, the use of a 3D printer allowed the fabrication of a BIA cell within 4 h at cost of $5. The coupling of SPE with the 3D printed cell exhibited great analytical performance including repeatability and reproducibility lower than 2% as well as high sampling rate (30 injections h −1 ) under low injection volume (10 μL). The limit of detection (LD) and linear range achieved with the proposed approach was 0.11 mmol L −1 and 1–10 mmol L −1 , respectively. Lastly, the glucose concentration level was successfully determined using the proposed method and the values found were not statistically different from the data achieved by a reference method at confidence level of 95%.
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paper_title: A paper-based amperometric glucose biosensor developed with Prussian Blue-modified screen-printed electrodes

paper_content:

a b s t r a c t This paper describes a simple inexpensive paper-based amperometric glucose biosensor developed based on Prussian Blue (PB)-modified screen-printed carbon electrodes (SPCEs). The use of cellulose paper proved to be a simple, "ideal" and green biocompatible immobilization matrix for glucose oxidase (GOx) as it was successfully embedded within the fibre matrix of paper via physical adsorption. The glucose biosensor allowed a small amount (0.5 L) of sample solution for glucose analysis. The biosensor had a linear calibration range between 0.25 mM and 2.00 (R2 = 0.987) and a detection limit of 0.01 mM glucose (S/N = 3). Interference study of selected potential interfering compounds on the biosensor response was investigated. Its analytical performance was demonstrated in the analysis of selected commercial glucose beverages. Despite the simplicity of the immobilization method, the biosensor retained ca. 72% of its activity after a storage period of 45 days. © 2014 Elsevier B.V. All rights reserved.
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paper_title: A “green” cellulose paper based glucose amperometric biosensor

paper_content:

Abstract A simple low cost “green” biosensor configuration comprising of a hydrophilic cellulose paper disk with immobilised glucose oxidase (GOx) via adsorption step, placed on top of a screen printed carbon electrode (SPCE) was developed. This biosensor configuration allowed for low volume of glucose sample (5 μL) to be analysed. Cellulose paper was also used as the pre-storage reagent matrix for 0.1 M phosphate buffer solution (PBS, pH 7.0) and 10 mM soluble ferrocene monocarboxylic acid mediator. This biosensor exhibited a linear dynamic calibration range of 1 to 5 mM glucose ( r 2 = 0.971), with a limit of detection of 0.18 mM and retained 98% of its signal after a period of four months. In addition, its performance was demonstrated in the analysis of selected commercial soda beverages. The glucose concentrations obtained by the biosensor corroborated well with an independent high performance liquid chromatographic (HPLC) method.
---
paper_title: Paper-fluidic electrochemical biosensing platform with enzyme paper and enzymeless electrodes

paper_content:

Abstract A miniaturized paper-based microfluidic electrochemical enzymatic biosensing platform was developed and the effects of fluidic behaviors in paper substrate on electrochemical sensing were systemically investigated. The biosensor is composed of an enzyme-immobilized pure cellulose paper pad, an enzymeless screen-printed electrode (SPE) modified with platinum nanoparticles (PtNPs), and a pair of clamped acrylonitrile butadiene styrene (ABS) plastic holders to provide good alignment for stable signal sensing. The wicking rate of liquid sample in paper was predicted, using a two-dimensional Fickian-diffusion model, to be 1.0 × 10 −2 cm 2 /s, and was verified experimentally. Dip-coating was used to prepare the enzyme-modified paper pad (EPP), which is amenable for mass manufacturing. The EPP retained excellent hydrophilicity and mechanical properties, with even slightly improved tensile strength and break strain. No significant difference in voltammetric behaviors was observed between measurements made in bulk buffer solution and with different sample volumes applied to EPP beyond its saturation wicking volume. Glucose oxidase (GO x ), an enzyme specific for glucose (Glc) substrate, was used as a model enzyme and its enzymatic reaction product H 2 O 2 was detected by the enzymeless PtNPs-SPE in the presence of ambient electron mediator O 2 . Consequently, Glc was detected with its concentration linearly depending on H 2 O 2 oxidation current with sensitivity of 10.5 μA mM -1 cm -2 and detection limit of 9.3 μM (at S / N = 3). The biosensor can be quickly regenerated with memory effects removed by buffer additions for continuous real-time detection of multiple samples in one run for point-of-care purposes. This integrated platform is also inexpensive since the EPP is easily stored, and enzymeless PtNPs-SPEs can be used multiple times with different EPPs. The green and facile preparation in bulk, excellent mechanical strength, well-maintained enzyme activity, disposability, and good reproducibility and stability make our paper-fluidic biosensor platform suitable for various real-time electrochemical bioassays without any external power for mixing, especially in resource-limited conditions.
---
paper_title: Electrochemical detection of glucose from whole blood using paper-based microfluidic devices.

paper_content:

Abstract Electrochemical paper-based analytical devices (ePADs) with integrated plasma isolation for determination of glucose from whole blood samples have been developed. A dumbbell shaped ePAD containing two blood separation zones (VF2 membranes) with a middle detection zone was fabricated using the wax dipping method. The dumbbell shaped device was designed to separate plasma while generating homogeneous flow to the middle detection zone of the ePAD. The proposed ePADs work with whole blood samples with 24–60% hematocrit without dilution, and the plasma was completely separated within 4 min. Glucose in isolated plasma separated was detected using glucose oxidase immobilized on the middle of the paper device. The hydrogen peroxide generated from the reaction between glucose and the enzyme pass through to a Prussian blue modified screen printed electrode (PB-SPEs). The currents measured using chronoamperometry at the optimal detection potential for H 2 O 2 (−0.1 V versus Ag/AgCl reference electrode) were proportional to glucose concentrations in the whole blood. The linear range for glucose assay was in the range 0–33.1 mM ( r 2 = 0.987). The coefficients of variation (CVs) of currents were 6.5%, 9.0% and 8.0% when assay whole blood sample containing glucose concentration at 3.4, 6.3, and 15.6 mM, respectively. Because each sample displayed intra-individual variation of electrochemical signal, glucose assay in whole blood samples were measured using the standard addition method. Results demonstrate that the ePAD glucose assay was not significantly different from the spectrophotometric method ( p = 0.376, paired sample t -test, n = 10).
---
paper_title: A hybrid system of carbon ink electrodes and chromatography paper on a CMOS chip and its application to enzymatic glucose sensor

paper_content:

Enzymatic sensors on complementary metal–oxide–semiconductor (CMOS) chips are realized using carbon ink and chromatography paper (ChrPr). Electrodes are fabricated from carbon ink on CMOS chips. The carbon ink electrodes work as well-behaving electrochemical electrodes. Enzyme electrodes are realized by covering the carbon ink electrodes on the CMOS chip with ChrPr supporting enzymes and electron mediators. Such enzyme electrodes successfully give anodic current proportional to the glucose concentration. Good linearity is observed up to 10 mM glucose concentration, which is sufficient for blood glucose testing applications.
---
paper_title: Paper-based enzymatic reactors for batch injection analysis of glucose on 3D printed cell coupled with amperometric detection

paper_content:

Abstract This report describes for the first time the development of paper-based enzymatic reactors (PERs) for the detection of glucose (Glu) in artificial serum sample using a 3D printed batch injection analysis (BIA) cell coupled with electrochemical detection. The fabrication of the PERs involved firstly the oxidation of the paper surface with a sodium periodate solution. The oxidized paper was then perforated with a paper punch to create microdisks and activated with a solution containing N -hydroxysuccinimide (NHS) and N -(3-dimethylaminopropyl)- N ′-ethylcarbodiimide hydrochloride (EDC). Glucose oxidase (GOx) enzyme was then covalently immobilized on paper surface to promote the enzymatic assay for the detection of Glu in serum sample. After the addition of Glu on the PER surface placed inside a plastic syringe, the analyte penetrated through the paper surface under vertical flow promoting the enzymatic assay. The reaction product (H 2 O 2 ) was collected with an electronic micropipette in a microtube and analyzed in the 3D BIA cell coupled with screen-printed electrodes (SPEs). The overall preparation time and the cost estimated per PER were 2.5 h and $0.02, respectively. Likewise the PERs, the use of a 3D printer allowed the fabrication of a BIA cell within 4 h at cost of $5. The coupling of SPE with the 3D printed cell exhibited great analytical performance including repeatability and reproducibility lower than 2% as well as high sampling rate (30 injections h −1 ) under low injection volume (10 μL). The limit of detection (LD) and linear range achieved with the proposed approach was 0.11 mmol L −1 and 1–10 mmol L −1 , respectively. Lastly, the glucose concentration level was successfully determined using the proposed method and the values found were not statistically different from the data achieved by a reference method at confidence level of 95%.
---
paper_title: A paper-based amperometric glucose biosensor developed with Prussian Blue-modified screen-printed electrodes

paper_content:

a b s t r a c t This paper describes a simple inexpensive paper-based amperometric glucose biosensor developed based on Prussian Blue (PB)-modified screen-printed carbon electrodes (SPCEs). The use of cellulose paper proved to be a simple, "ideal" and green biocompatible immobilization matrix for glucose oxidase (GOx) as it was successfully embedded within the fibre matrix of paper via physical adsorption. The glucose biosensor allowed a small amount (0.5 L) of sample solution for glucose analysis. The biosensor had a linear calibration range between 0.25 mM and 2.00 (R2 = 0.987) and a detection limit of 0.01 mM glucose (S/N = 3). Interference study of selected potential interfering compounds on the biosensor response was investigated. Its analytical performance was demonstrated in the analysis of selected commercial glucose beverages. Despite the simplicity of the immobilization method, the biosensor retained ca. 72% of its activity after a storage period of 45 days. © 2014 Elsevier B.V. All rights reserved.
---
paper_title: A “green” cellulose paper based glucose amperometric biosensor

paper_content:

Abstract A simple low cost “green” biosensor configuration comprising of a hydrophilic cellulose paper disk with immobilised glucose oxidase (GOx) via adsorption step, placed on top of a screen printed carbon electrode (SPCE) was developed. This biosensor configuration allowed for low volume of glucose sample (5 μL) to be analysed. Cellulose paper was also used as the pre-storage reagent matrix for 0.1 M phosphate buffer solution (PBS, pH 7.0) and 10 mM soluble ferrocene monocarboxylic acid mediator. This biosensor exhibited a linear dynamic calibration range of 1 to 5 mM glucose ( r 2 = 0.971), with a limit of detection of 0.18 mM and retained 98% of its signal after a period of four months. In addition, its performance was demonstrated in the analysis of selected commercial soda beverages. The glucose concentrations obtained by the biosensor corroborated well with an independent high performance liquid chromatographic (HPLC) method.
---
paper_title: Development of Electrochemical Paper-based Glucose Sensor Using Cellulose-4-aminophenylboronic Acid-modified Screen-printed Carbon Electrode

paper_content:

The present work describes the fabrication of paper-based analytical devices (μPADs) by immobilization of glucose oxidase onto the screen printed carbon electrodes (SPCEs) for the electrochemical glucose detection. The sensitivity towards glucose was improved by using a SPCE prepared from homemade carbon ink mixed with cellulose acetate. In addition, 4-aminophenylboronic acid (4-APBA) was used as a redox mediator giving a lower detection potential for improvement selectivity. Under optimized condition, the detection limit was 0.86 mM. The proposed device was applied in real samples. This μPAD has many advantages including low sample consumption, rapid analysis method, and low device cost.
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paper_title: Graphene nano-ink biosensor arrays on a microfluidic paper for multiplexed detection of metabolites.

paper_content:

Abstract The development of a miniaturized and low-cost platform for the highly sensitive, selective and rapid detection of multiplexed metabolites is of great interest for healthcare, pharmaceuticals, food science, and environmental monitoring. Graphene is a delicate single-layer, two-dimensional network of carbon atoms with extraordinary electrical sensing capability. Microfluidic paper with printing technique is a low cost matrix. Here, we demonstrated the development of graphene-ink based biosensor arrays on a microfluidic paper for the multiplexed detection of different metabolites, such as glucose, lactate, xanthine and cholesterol. Our results show that the graphene biosensor arrays can detect multiple metabolites on a microfluidic paper sensitively, rapidly and simultaneously. The device exhibits a fast measuring time of less than 2 min, a low detection limit of 0.3 μM, and a dynamic detection range of 0.3–15 μM. The process is simple and inexpensive to operate and requires a low consumption of sample volume. We anticipate that these results could open exciting opportunities for a variety of applications.
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paper_title: A low-cost paper-based inkjet-printed platform for electrochemical analyses

paper_content:

An electrode platform printed on a recyclable low-cost paper substrate was characterized using cyclic voltammetry. The working and counter electrodes were directly printed gold-stripes, while the reference electrode was a printed silver stripe onto which an AgCl layer was deposited electrochemically. The novel paper-based chips showed comparable performance to conventional electrochemical cells. Different types of electrode modifications were carried out to demonstrate that the printed electrodes behave similarly with conventional electrodes. Firstly, a self-assembled monolayer (SAM) of alkanethiols was successfully formed on the Au electrode surface. As a consequence, the peak currents were suppressed and no longer showed clear increase as a function of the scan rate. Such modified electrodes have potential in various sensor applications when terminally substituted thiols are used. Secondly, a polyaniline film was electropolymerized on the working electrode by cyclic voltammetry and used for potentiometric pH sensing. The calibration curve showed close to Nerstian response. Thirdly, a poly(3,4-ethylenedioxythiophene) (PEDOT) layer was electropolymerized both by galvanostatic and cyclic potential sweep method on the working electrode using two different dopants; Cl− to study ion-to-electron transduction on paper-Au/PEDOT system and glucose oxidase in order to fabricate a glucose biosensor. The planar paper-based electrochemical cell is a user-friendly platform that functions with low sample volume and allows the sample to be applied and changed by e.g. pipetting. Low unit cost is achieved with mask- and mesh-free inkjet-printing technology.
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paper_title: Direct writing electrodes using a ball pen for paper-based point-of-care testing

paper_content:

The integration of paper with an electrochemical device has attracted growing attention for point-of-care testing, where it is of great importance to fabricate electrodes on paper in a low-cost, easy and versatile way. In this work, we report a simple strategy for directly writing electrodes on paper using a pressure-assisted ball pen to form a paper-based electrochemical device (PED). This method is demonstrated to be capable of fabricating electrodes on paper with good electrical conductivity and electrochemical performance, holding great potential to be employed in point-of-care applications, such as in human health diagnostics and food safety detection. As examples, the PEDs fabricated using the developed method are applied for detection of glucose in artificial urine and melamine in sample solutions. Furthermore, our developed strategy is also extended to fabricate PEDs with multi-electrode arrays and write electrodes on non-planar surfaces (e.g., paper cup, human skin), indicating the potential application of our method in other fields, such as fabricating biosensors, paper electronics etc.
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paper_title: Fully-drawn origami paper analytical device for electrochemical detection of glucose

paper_content:

Abstract In this work, an origami paper-based analytical device for glucose biosensor by employing fully-drawn pencil electrodes has been reported. The three-electrode system was prepared on paper directly by drawing with nothing more than pencils. By simple printing, two separated zones on paper were designed for the immobilization of the mediator and glucose oxidase (GOx), respectively. The used paper provides a favorable and biocompatible support for maintaining the bioactivities of GOx. With a sandwich-type scheme, the origami biosensor exhibited great analytical performance for glucose sensing including acceptable reproducibility and favorable selectivity against common interferents in physiological fluids. The limit of detection and linear range achieved with the approach was 0.05 mM and 1–12 mM, respectively. Its analytical performance was also demonstrated in the analysis of human blood samples. Such fully-drawn paper-based device is cheap, flexible, portable, disposable, and environmentally friendly, affording great convenience for practical use under resource-limited conditions. We therefore envision that this approach can be extended to generate other functional paper-based devices.
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paper_title: A new approach for paper-based analytical devices with electrochemical detection based on graphite pencil electrodes

paper_content:

Abstract The present work describes for the first time the coupling of graphite pencil electrodes with paper-based analytical devices (μPADs) for glucose biosensing. Electrochemical measurement for μPADs using a two-electrode system was also developed. This dual-electrode configuration on paper provides electrochemical responses similar to those recorded by conventional electrochemical systems (three electrode systems). A wax printing process was used to define hydrophilic circular microzones by inserting hydrophobic patterns on paper. The microzones were employed one for filtration, one for an enzymatic reaction and one for electrochemical detection. By adding 4-aminophenylboronic acid as redox mediator and glucose oxidase to the reaction microzone, it was possible to reach low limits of detection for glucose with graphite pencil electrodes without modifying the electrode. The limit of detection of the proposed μPAD was found to be 0.38 μmol L −1 for glucose. Low sample consumption (40 μL) and fast analysis time (less than 5 min) combined with low cost electrodes and paper-based analytical platforms are attractive properties of the proposed μPAD with electrochemical detection. Artificial blood serum samples containing glucose were analyzed with the proposed device as proof of concept.
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paper_title: Development of Electrochemical Paper-based Glucose Sensor Using Cellulose-4-aminophenylboronic Acid-modified Screen-printed Carbon Electrode

paper_content:

The present work describes the fabrication of paper-based analytical devices (μPADs) by immobilization of glucose oxidase onto the screen printed carbon electrodes (SPCEs) for the electrochemical glucose detection. The sensitivity towards glucose was improved by using a SPCE prepared from homemade carbon ink mixed with cellulose acetate. In addition, 4-aminophenylboronic acid (4-APBA) was used as a redox mediator giving a lower detection potential for improvement selectivity. Under optimized condition, the detection limit was 0.86 mM. The proposed device was applied in real samples. This μPAD has many advantages including low sample consumption, rapid analysis method, and low device cost.
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paper_title: Graphene nano-ink biosensor arrays on a microfluidic paper for multiplexed detection of metabolites.

paper_content:

Abstract The development of a miniaturized and low-cost platform for the highly sensitive, selective and rapid detection of multiplexed metabolites is of great interest for healthcare, pharmaceuticals, food science, and environmental monitoring. Graphene is a delicate single-layer, two-dimensional network of carbon atoms with extraordinary electrical sensing capability. Microfluidic paper with printing technique is a low cost matrix. Here, we demonstrated the development of graphene-ink based biosensor arrays on a microfluidic paper for the multiplexed detection of different metabolites, such as glucose, lactate, xanthine and cholesterol. Our results show that the graphene biosensor arrays can detect multiple metabolites on a microfluidic paper sensitively, rapidly and simultaneously. The device exhibits a fast measuring time of less than 2 min, a low detection limit of 0.3 μM, and a dynamic detection range of 0.3–15 μM. The process is simple and inexpensive to operate and requires a low consumption of sample volume. We anticipate that these results could open exciting opportunities for a variety of applications.
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paper_title: A low-cost paper-based inkjet-printed platform for electrochemical analyses

paper_content:

An electrode platform printed on a recyclable low-cost paper substrate was characterized using cyclic voltammetry. The working and counter electrodes were directly printed gold-stripes, while the reference electrode was a printed silver stripe onto which an AgCl layer was deposited electrochemically. The novel paper-based chips showed comparable performance to conventional electrochemical cells. Different types of electrode modifications were carried out to demonstrate that the printed electrodes behave similarly with conventional electrodes. Firstly, a self-assembled monolayer (SAM) of alkanethiols was successfully formed on the Au electrode surface. As a consequence, the peak currents were suppressed and no longer showed clear increase as a function of the scan rate. Such modified electrodes have potential in various sensor applications when terminally substituted thiols are used. Secondly, a polyaniline film was electropolymerized on the working electrode by cyclic voltammetry and used for potentiometric pH sensing. The calibration curve showed close to Nerstian response. Thirdly, a poly(3,4-ethylenedioxythiophene) (PEDOT) layer was electropolymerized both by galvanostatic and cyclic potential sweep method on the working electrode using two different dopants; Cl− to study ion-to-electron transduction on paper-Au/PEDOT system and glucose oxidase in order to fabricate a glucose biosensor. The planar paper-based electrochemical cell is a user-friendly platform that functions with low sample volume and allows the sample to be applied and changed by e.g. pipetting. Low unit cost is achieved with mask- and mesh-free inkjet-printing technology.
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paper_title: Direct writing electrodes using a ball pen for paper-based point-of-care testing

paper_content:

The integration of paper with an electrochemical device has attracted growing attention for point-of-care testing, where it is of great importance to fabricate electrodes on paper in a low-cost, easy and versatile way. In this work, we report a simple strategy for directly writing electrodes on paper using a pressure-assisted ball pen to form a paper-based electrochemical device (PED). This method is demonstrated to be capable of fabricating electrodes on paper with good electrical conductivity and electrochemical performance, holding great potential to be employed in point-of-care applications, such as in human health diagnostics and food safety detection. As examples, the PEDs fabricated using the developed method are applied for detection of glucose in artificial urine and melamine in sample solutions. Furthermore, our developed strategy is also extended to fabricate PEDs with multi-electrode arrays and write electrodes on non-planar surfaces (e.g., paper cup, human skin), indicating the potential application of our method in other fields, such as fabricating biosensors, paper electronics etc.
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paper_title: Fully-drawn origami paper analytical device for electrochemical detection of glucose

paper_content:

Abstract In this work, an origami paper-based analytical device for glucose biosensor by employing fully-drawn pencil electrodes has been reported. The three-electrode system was prepared on paper directly by drawing with nothing more than pencils. By simple printing, two separated zones on paper were designed for the immobilization of the mediator and glucose oxidase (GOx), respectively. The used paper provides a favorable and biocompatible support for maintaining the bioactivities of GOx. With a sandwich-type scheme, the origami biosensor exhibited great analytical performance for glucose sensing including acceptable reproducibility and favorable selectivity against common interferents in physiological fluids. The limit of detection and linear range achieved with the approach was 0.05 mM and 1–12 mM, respectively. Its analytical performance was also demonstrated in the analysis of human blood samples. Such fully-drawn paper-based device is cheap, flexible, portable, disposable, and environmentally friendly, affording great convenience for practical use under resource-limited conditions. We therefore envision that this approach can be extended to generate other functional paper-based devices.
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paper_title: A new approach for paper-based analytical devices with electrochemical detection based on graphite pencil electrodes

paper_content:

Abstract The present work describes for the first time the coupling of graphite pencil electrodes with paper-based analytical devices (μPADs) for glucose biosensing. Electrochemical measurement for μPADs using a two-electrode system was also developed. This dual-electrode configuration on paper provides electrochemical responses similar to those recorded by conventional electrochemical systems (three electrode systems). A wax printing process was used to define hydrophilic circular microzones by inserting hydrophobic patterns on paper. The microzones were employed one for filtration, one for an enzymatic reaction and one for electrochemical detection. By adding 4-aminophenylboronic acid as redox mediator and glucose oxidase to the reaction microzone, it was possible to reach low limits of detection for glucose with graphite pencil electrodes without modifying the electrode. The limit of detection of the proposed μPAD was found to be 0.38 μmol L −1 for glucose. Low sample consumption (40 μL) and fast analysis time (less than 5 min) combined with low cost electrodes and paper-based analytical platforms are attractive properties of the proposed μPAD with electrochemical detection. Artificial blood serum samples containing glucose were analyzed with the proposed device as proof of concept.
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paper_title: A new insert sample approach to paper spray mass spectrometry: a paper substrate with paraffin barriers

paper_content:

The analytical performance for paper spray (PS) using a new insert sample approach based on paper with paraffin barriers (PS-PB) is presented. The paraffin barrier is made using a simple, fast and cheap method based on the stamping of paraffin onto a paper surface. Typical operation conditions of paper spray such as the solvent volume applied on the paper surface, and the paper substrate type are evaluated. A paper substrate with paraffin barriers shows better performance on analysis of a range of typical analytes when compared to the conventional PS-MS using normal paper (PS-NP) and PS-MS using paper with two rounded corners (PS-RC). PS-PB was applied to detect sugars and their inhibitors in sugarcane bagasse liquors from a second generation ethanol process. Moreover, the PS-PB proved to be excellent, showing results for the quantification of glucose in hydrolysis liquors with excellent linearity (R2 = 0.99), limits of detection (2.77 mmol L−1) and quantification (9.27 mmol L−1). The results are better than for PS-NP and PS-RC. The PS-PB was also excellent in performance when compared with the HPLC-UV method for glucose quantification on hydrolysis of liquor samples.
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paper_title: A paper-based calorimetric microfluidics platform for bio-chemical sensing

paper_content:

In this report, a paper-based micro-calorimetric biochemical detection method is presented. Calorimetric detection of biochemical reactions is demonstrated as an extension of current colorimetric and electrochemical detection mechanisms of paper-based biochemical analytical systems. Reaction and/or binding temperature of glucose/glucose oxidase, DNA/hydrogen peroxide, and biotin/streptavidin, are measured by the paper-based micro-calorimeter. Commercially available glucose calibration samples of 0.05, 0.15 and 0.3% wt/vol concentration are used for comparing the device performance with a commercially available glucose meter (electrochemical detection). The calorimetric glucose detection demonstrates a measurement error less than 2%. The calorimetric detection results of DNA concentrations from 0.9 to 7.3 mg/mL and temperature changes in biotin and streptavidin reaction are presented to demonstrate the feasibility of integrating the calorimetric detection method with paper based microfluidic devices.
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paper_title: Quantum dot-modified paper-based assay for glucose screening

paper_content:

A simple and inexpensive method to fabricate a colloidal CdSe/ZnS quantum dots-modified paper-based assay for glucose is herein reported. The circular paper sheets were uniformly loaded and displayed strong fluorescence under a conventional hand-held UV lamp (365 nm). The assay is based on the use of glucose oxidase enzyme (GOx), which impregnated the paper sheets, producing H2O2 upon the reaction with the glucose contained in the samples. After 20 min of exposure, the fluorescence intensity changed due to the quenching caused by H2O2. To obtain a reading, the paper sheets were photographed under 365 nm excitation using a digital camera. Several parameters, including the amount of QD, sample pH, and amount of GOx were optimized to maximize the response to glucose. The paper-based assay showed a sigmoidal-shaped response with respect to the glucose concentration in the 5-200 mg·dL-1 range (limit of detection of 5 μg·dL-1), demonstrating their potential use for biomedical applications.
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paper_title: Paper membrane-based SERS platform for the determination of glucose in blood samples

paper_content:

In this report, we present a paper membrane-based surface-enhanced Raman scattering (SERS) platform for the determination of blood glucose level using a nitrocellulose membrane as substrate paper, and the microfluidic channel was simply constructed by wax-printing method. The rod-shaped gold nanorod particles were modified with 4-mercaptophenylboronic acid (4-MBA) and 1-decanethiol (1-DT) molecules and used as embedded SERS probe for paper-based microfluidics. The SERS measurement area was simply constructed by dropping gold nanoparticles on nitrocellulose membrane, and the blood sample was dropped on the membrane hydrophilic channel. While the blood cells and proteins were held on nitrocellulose membrane, glucose molecules were moved through the channel toward the SERS measurement area. Scanning electron microscopy (SEM) was used to confirm the effective separation of blood matrix, and total analysis is completed in 5 min. In SERS measurements, the intensity of the band at 1070 cm(-1) which is attributed to B-OH vibration decreased depending on the rise in glucose concentration in the blood sample. The glucose concentration was found to be 5.43 ± 0.51 mM in the reference blood sample by using a calibration equation, and the certified value for glucose was 6.17 ± 0.11 mM. The recovery of the glucose in the reference blood sample was about 88 %. According to these results, the developed paper-based microfluidic SERS platform has been found to be suitable for use for the detection of glucose in blood samples without any pretreatment procedure. We believe that paper-based microfluidic systems may provide a wide field of usage for paper-based applications.
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paper_title: Fast and Effective Turn‐on Paper‐based Phosphorescence Biosensor for Detection of Glucose in Serum

paper_content:

In this study, a turn-on paper-based optical analytical system with a rapid, sensitive and quantitative response for glucose was developed. The luminescence sensing material, crystalline iridium(III)-Zn(II) coordination polymers, or Ir-Zne, was grown electrochemically on stainless steel mesh and then deposited on filter paper. This sensing substrate was subsequently built up under glucose oxidase encapsulated in hydrogel and then immobilized on egg membrane with the layer-by-layer method. Once the glucose solution was dropped onto the paper, the oxygen content was depleted simultaneously with a concomitant increase in the phosphorescence of Ir-Zne. The detection limit for glucose was 0.05 mM. The linear dynamic range for the determination of glucose was 0.05–8.0 mM with a correlation coefficient (R2) of 0.9956 (y=68.11 [glucose]−14.72). The response time was about 0.12 s, and the sample volume was less than 5 μL. The effects of mesh size, buffer concentration, pH, enzyme concentration, temperature, and interference, and the stability of the biosensor, have also been studied in detail. Finally, the biosensor was successfully applied to the determination of glucose in human serum.
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paper_title: Capillary-Driven Toner-Based Microfluidic Devices for Clinical Diagnostics with Colorimetric Detection

paper_content:

The fabrication of toner-based microfluidic devices to perform clinical diagnostics with capillary action and colorimetric detection is described in this report. Test zones and microfluidic channels were drawn in a graphic software package and laser printed on a polyester film. The printed layout and its mirror image were aligned with an intermediary cut-through polyester film and then thermally laminated together at 150 °C at 60 cm/min to obtain a channel with ca. 100-μm depth. Colorimetric assays for glucose, protein, and cholesterol were successfully performed using a desktop scanner. The limit of detection (LD) values found for protein, cholesterol, and glucose were 8, 0.2, and 0.3 mg/mL, respectively. The relative standard deviation (RSD) values for an interdevices comparison were 6%, 1%, and 3% for protein, cholesterol, and glucose, respectively. Bioassays were successfully performed on toner-based devices stored at different temperatures during five consecutive days without loss of activity.
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paper_title: Development of the smartphone-based colorimetry for multi-analyte sensing arrays

paper_content:

Here we report development of a smartphone app (application) that digitizes the colours of a colorimetric sensor array. A conventional colorimetric sensor array consists of multiple paper-based sensors, and reports the detection results in terms of colour change. Evaluation of the colour changes is normally done by the naked eye, which may cause uncertainties due to personal subjectivity and the surrounding conditions. Solutions have been particularly sought in smartphones as they are capable of spectrometric functions. Our report specifically focuses on development of a practical app for immediate point-of-care (POC) multi-analyte sensing without additional devices. First, the individual positions of the sensors are automatically identified by the smartphone; second, the colours measured at each sensor are digitized based on a correction algorithm; and third, the corrected colours are converted to concentration values by pre-loaded calibration curves. All through these sequential processes, the sensor array taken in a smartphone snapshot undergoes laboratory-level spectrometry. The advantages of inexpensive and convenient paper-based colorimetry and the ubiquitous smartphone are tied to achieve a ready-to-go POC diagnosis.
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Title: A Review on Microfluidic Paper-Based Analytical Devices for Glucose Detection

Section 1: Introduction
Description 1: Summarize the significance of glucose detection, point-of-care tests, and the advantages of microfluidic paper-based analytical devices (µPADs).

Section 2: Fabrication Process of Colorimetric Glucose µPADs
Description 2: Discuss the various methods for fabricating colorimetric glucose µPADs, including the use of glucose oxidase and horseradish peroxidase, along with commonly used color indicators.

Section 3: Alternative Color Indicators for Glucose µPADs
Description 3: Review the use of different organics and nanoparticles as alternative color indicators to improve color signal strength and detection limits.

Section 4: 3D-µPADs
Description 4: Describe the development and advantages of three-dimensional microfluidic paper-based analytical devices, and their fabrication techniques.

Section 5: Electrochemical Detection of Glucose
Description 5: Present an overview of electrochemical detection methods integrated with µPADs for glucose detection, emphasizing their low cost, high sensitivity, and minimal sample preparation.

Section 6: Advanced Fabrications of Electrochemical Glucose µPADs
Description 6: Cover advanced fabrication techniques for electrochemical glucose µPADs, including the use of screen-printed electrodes and complementary metal-oxide-semiconductor chips.

Section 7: Electrochemical Glucose µPADs with Printed Electrodes
Description 7: Explore methods of fabricating electrodes directly on paper substrates for electrochemical glucose detection, highlighting various innovative approaches.

Section 8: Other Glucose Detection Platforms
Description 8: Review alternative glucose detection techniques applied to µPADs, such as luminescence, fluorescence, calorimetric, mass spectrum, and surface-enhanced Raman spectroscopy.

Section 9: Conclusions
Description 9: Summarize the progress and potential future developments in µPADs for glucose detection, highlighting their importance in point-of-care diagnostics and potential commercialization.