Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review
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paper_title: Simultaneous high speed optical and impedance analysis of single particles with a microfluidic cytometer.

paper_content:

We describe a microfluidic cytometer that performs simultaneous optical and electrical characterisation of particles. The microfluidic chip measures side scattered light, signal extinction and fluorescence using integrated optical fibres coupled to photomultiplier tubes. The channel is 80 μm high and 200 μm wide, and made from SU-8 patterned and sandwiched between glass substrates. Particles were focused into the analysis region using 1-D hydrodynamic focusing and typical particle velocities were 0.1 ms(-1). Excitation light is coupled into the detection channel with an optical fibre and focused into the channel using an integrated compound air lens. The electrical impedance of particles is measured at 1 MHz using micro-electrodes fabricated on the channel top and bottom. This data is used to accurately size the particles. The system is characterised using a range of different sized polystyrene beads (fluorescent and non-fluorescent). Single and mixed populations of beads were measured and the data compared with a conventional flow cytometer.
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paper_title: Developing optofluidic technology through the fusion of microfluidics and optics

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We describe devices in which optics and fluidics are used synergistically to synthesize novel functionalities. Fluidic replacement or modification leads to reconfigurable optical systems, whereas the implementation of optics through the microfluidic toolkit gives highly compact and integrated devices. We categorize optofluidics according to three broad categories of interactions: fluid–solid interfaces, purely fluidic interfaces and colloidal suspensions. We describe examples of optofluidic devices in each category.
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paper_title: Commercialization of microfluidic devices

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Microfluidic devices offer automation and high-throughput screening, and operate at low volumes of consumables. Although microfluidics has the potential to reduce turnaround times and costs for analytical devices, particularly in medical, veterinary, and environmental sciences, this enabling technology has had limited diffusion into consumer products. This article analyzes the microfluidics market, identifies issues, and highlights successful commercialization strategies. Addressing niche markets and establishing compatibility with existing workflows will accelerate market penetration.
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paper_title: The intersection of flow cytometry with microfluidics and microfabrication

paper_content:

A modern flow cytometer can analyze and sort particles on a one by one basis at rates of 50,000 particles per second. Flow cytometers can also measure as many as 17 channels of fluorescence, several angles of scattered light, and other non-optical parameters such as particle impedance. More specialized flow cytometers can provide even greater analysis power, such as single molecule detection, imaging, and full spectral collection, at reduced rates. These capabilities have made flow cytometers an invaluable tool for numerous applications including cellular immunophenotyping, CD4+ T-cell counting, multiplex microsphere analysis, high-throughput screening, and rare cell analysis and sorting. Many bio-analytical techniques have been influenced by the advent of microfluidics as a component in analytical tools and flow cytometry is no exception. Here we detail the functions and uses of a modern flow cytometer, review the recent and historical contributions of microfluidics and microfabricated devices to field of flow cytometry, examine current application areas, and suggest opportunities for the synergistic application of microfabrication approaches to modern flow cytometry.
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paper_title: Recent advancements in optofluidics-based single-cell analysis: optical on-chip cellular manipulation, treatment, and property detection

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Cellular analysis plays important roles in various biological applications, such as cell biology, drug development, and disease diagnosis. Conventional cellular analysis usually measures the average response from a whole cell group. However, bulk measurements may cause misleading interpretations due to cell heterogeneity. Another problem is that current cellular analysis may not be able to differentiate various subsets of cell populations, each exhibiting a different behavior than the others. Single-cell analysis techniques are developed to analyze cellular properties, conditions, or functional responses in a large cell population at the individual cell level. Integrating optics with microfluidic platforms provides a well-controlled microenvironment to precisely control single cell conditions and perform non-invasive high-throughput analysis. This paper reviews recent developments in optofluidic technologies for various optics-based single-cell analyses, which involve single cell manipulation, treatment, and property detection. Finally, we provide our views on the future development of integrated optics with microfluidics for single-cell analysis and discuss potential challenges and opportunities of this emerging research field in biological applications.
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paper_title: A microfluidic impedance flow cytometer for identification of differentiation state of stem cells.

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This paper presents a microfluidic electrical impedance flow cytometer (FC) for identifying the differentiation state of single stem cells. This device is comprised of a novel dual micropore design, which not only enhances the processing throughput, but also allows the associated electrodes to be used as a reference for one another. A signal processing algorithm, based on the support vector machine (SVM) theory, and a data classification method were developed to automate the identification of sample types and cell differentiation state based on measured impedance values. The device itself was fabricated using a combination of standard and soft lithography techniques to generate a PDMS-gold electrode construct. Experimental testing with non-biological particles and mouse embryonic carcinoma cells (P19, undifferentiated and differentiated) was carried out using a range of excitation frequencies. The effects of the frequency and the interrogation parameters on sample identification performance were investigated. It was found that the real and imaginary part of the detected impedance signal were adequate for distinguishing the undifferentiated P19 cells from non-biological polystyrene beads at all tested frequencies. A higher frequency and an opacity index were required to resolve the undifferentiated and differentiated P19 cells by capturing capacitive changes in electrophysiological properties arising from differentiation. The experimental results demonstrated salient accuracy of the device and algorithm, and established its feasibility for non-invasive, label-free identification of the differentiation state of the stem cells.
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paper_title: The intersection of flow cytometry with microfluidics and microfabrication

paper_content:

A modern flow cytometer can analyze and sort particles on a one by one basis at rates of 50,000 particles per second. Flow cytometers can also measure as many as 17 channels of fluorescence, several angles of scattered light, and other non-optical parameters such as particle impedance. More specialized flow cytometers can provide even greater analysis power, such as single molecule detection, imaging, and full spectral collection, at reduced rates. These capabilities have made flow cytometers an invaluable tool for numerous applications including cellular immunophenotyping, CD4+ T-cell counting, multiplex microsphere analysis, high-throughput screening, and rare cell analysis and sorting. Many bio-analytical techniques have been influenced by the advent of microfluidics as a component in analytical tools and flow cytometry is no exception. Here we detail the functions and uses of a modern flow cytometer, review the recent and historical contributions of microfluidics and microfabricated devices to field of flow cytometry, examine current application areas, and suggest opportunities for the synergistic application of microfabrication approaches to modern flow cytometry.
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paper_title: Hydrodynamic focusing for impedance-based detection of specifically bound microparticles and cells: Implications of fluid dynamics on tunable sensitivity

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Abstract A 4-electrode impedance-based microfluidic sensor was designed to achieve tunable sensitivity, while simultaneously decreasing the channel clogging and nonspecific binding that often adversely impact device function. Hydrodynamic focusing – a characteristic of laminar flow at low Reynold's number – provides highly controllable sensitivity in impedance-based microfluidic biosensors, by creating a “virtual” microchannel with soft walls and adjustable dimensions. Enhanced sensitivity, limited nonspecific binding, and a reduction in microchannel clogging have been achieved using hydrodynamic focusing within a 250 μm deep by 1 mm wide microchannel. The microfluidic sensor was able to detect microparticles as small as 5 μm in diameter specifically bound between co-planar platinum micro-electrodes. However, detection of submicron particles and E . coli cells in similar fashion proved to be challenging. A theoretical analysis of forces exerted by the fluids within the microchannel allowed specification of flow rates amenable to decreased nonspecific binding of interfering cells or microparticles, while ensuring minimal disruption to specifically bound targets. Simulations revealed that complex flow behavior of the hydrodynamically focused streams around specifically bound micron-sized particles reduces sensitivity. An improved understanding is thus presented of the parameters that impact the sensitivity of impedance-based biosensors implementing hydrodynamic focusing and specific target binding.
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paper_title: 3D microfabrication with inclined/rotated UV lithography

paper_content:

Abstract This paper presents a novel microfabrication technology of three-dimensional (3D) microstructures with inclined/rotated UV lithography using negative thick photoresist, SU-8. In exposure process, a photomask and a SU-8 coated substrate are fixed together, and tilted or tilted and rotated to a UV source. The reflected UV at the interface between the resist and the substrate is also exploited as well as the incident UV. With the 3D microfabrication technology, various 3D microstructures are easily fabricated such as oblique cylinders, embedded channels, bridges, V-grooves, truncated cones, and so on. The angles between fabricated structures and normal lines are 19.5°, when the incident angle of UV in air is 32°. Thus, the refractive index of SU-8 and the maximum refractive angle at the interface between SU-8 and air are expected about 1.6 and 39°, respectively.
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paper_title: Emerging optofluidic technologies for point-of-care genetic analysis systems: a review.

paper_content:

This review describes recently emerging optical and microfluidic technologies suitable for point-of-care genetic analysis systems. Such systems must rapidly detect hundreds of mutations from biological samples with low DNA concentration. We review optical technologies delivering multiplex sensitivity and compatible with lab-on-chip integration for both tagged and non-tagged optical detection, identifying significant source and detector technology emerging from telecommunications technology. We highlight the potential for improved hybridization efficiency through careful microfluidic design and outline some novel enhancement approaches using target molecule confinement. Optimization of fluidic parameters such as flow rate, channel height and time facilitates enhanced hybridization efficiency and consequently detection performance as compared with conventional assay formats (e.g. microwell plates). We highlight lab-on-chip implementations with integrated microfluidic control for "sample-to-answer" systems where molecular biology protocols to realize detection of target DNA sequences from whole blood are required. We also review relevant technology approaches to optofluidic integration, and highlight the issue of biomolecule compatibility. Key areas in the development of an integrated optofluidic system for DNA hybridization are optical/fluidic integration and the impact on biomolecules immobilized within the system. A wide range of technology platforms have been advanced for detection, quantification and other forms of characterization of a range of biomolecules (e.g. RNA, DNA, protein and whole cell). Owing to the very different requirements for sample preparation, manipulation and detection of the different types of biomolecules, this review is focused primarily on DNA-DNA interactions in the context of point-of-care analysis systems.
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paper_title: Microflow Cytometer for optical analysis of phytoplankton.

paper_content:

Analysis of the intrinsic fluorescence profiles of individual marine algae can be used in general classification of organisms based on cell size and fluorescence properties. We describe the design and fabrication of a Microflow Cytometer on a chip for characterization of phytoplankton. The Microflow Cytometer measured distinct side scatter and fluorescence properties of Synechococcus sp., Nitzschia d., and Thalassiosira p.; measurements were confirmed using the benchtop Accuri C6 flow cytometer. The Microflow Cytometer proved sensitive enough to detect and characterize picoplankton with diameter approximately 1 μm and larger phytoplankton of up to 80 μm in length. The wide range in size discrimination coupled with detection of intrinsic fluorescent pigments suggests that this Microflow Cytometer will be able to distinguish different populations of phytoplankton on unmanned underwater vehicles.
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paper_title: Microfluidic Mixing: A Review

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The aim of microfluidic mixing is to achieve a thorough and rapid mixing of multiple samples in microscale devices. In such devices, sample mixing is essentially achieved by enhancing the diffusion effect between the different species flows. Broadly speaking, microfluidic mixing schemes can be categorized as either "active", where an external energy force is applied to perturb the sample species, or "passive", where the contact area and contact time of the species samples are increased through specially-designed microchannel configurations. Many mixers have been proposed to facilitate this task over the past 10 years. Accordingly, this paper commences by providing a high level overview of the field of microfluidic mixing devices before describing some of the more significant proposals for active and passive mixers.
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paper_title: Optofluidic restricted imaging, spectroscopy and counting of nanoparticles by evanescent wave using immiscible liquids

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Conventional flow cytometry (FC) suffers from the diffraction limit for the detection of nanoparticles smaller than 100 nm, whereas traditional total internal reflection (TIR) microscopy can only detect few samples near the solid-liquid interface mostly in static states. Here we demonstrate a novel on-chip optofluidic technique using evanescent wave sensing for single nanoparticle real time detection by combining hydrodynamic focusing and TIR using immiscible flows. The immiscibility of the high-index sheath flow and the low-index core flow naturally generate a smooth, flat and step-index interface that is ideal for the TIR effect, whose evanescent field can penetrate the full width of the core flow. Hydrodynamic focusing can focus on all the nanoparticles in the extreme centre of the core flow with a width smaller than 1 μm. This technique enables us to illuminate every single sample in the running core flow by the evanescent field, leaving none unaffected. Moreover, it works well for samples much smaller than the diffraction limit. We have successfully demonstrated the scattering imaging and counting of 50 nm and 100 nm Au nanoparticles and also the fluorescence imaging and counting of 200 nm beads. The effective counting speeds are estimated as 1500, 2300 and 2000 particles per second for the three types of nanoparticles, respectively. The optical scattering spectra were also measured to determine the size of individual Au nanoparticles. This provides a new technique to detect nanoparticles and we foresee its application in the detection of molecules for biomedical analyses.
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paper_title: The origins and the future of microfluidics

paper_content:

The manipulation of fluids in channels with dimensions of tens of micrometres--microfluidics--has emerged as a distinct new field. Microfluidics has the potential to influence subject areas from chemical synthesis and biological analysis to optics and information technology. But the field is still at an early stage of development. Even as the basic science and technological demonstrations develop, other problems must be addressed: choosing and focusing on initial applications, and developing strategies to complete the cycle of development, including commercialization. The solutions to these problems will require imagination and ingenuity.
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paper_title: Concise Review: Microfluidic Technology Platforms: Poised to Accelerate Development and Translation of Stem Cell-Derived Therapies

paper_content:

Stem cells are a powerful resource for producing a variety of cell types with utility in clinically associated applications, including preclinical drug screening and development, disease and developmental modeling, and regenerative medicine. Regardless of the type of stem cell, substantial barriers to clinical translation still exist and must be overcome to realize full clinical potential. These barriers span processes including cell isolation, expansion, and differentiation; purification, quality control, and therapeutic efficacy and safety; and the economic viability of bioprocesses for production of functional cell products. Microfluidic systems have been developed for a myriad of biological applications and have the intrinsic capability of controlling and interrogating the cellular microenvironment with unrivalled precision; therefore, they have particular relevance to overcoming such barriers to translation. Development of microfluidic technologies increasingly utilizes stem cells, addresses stem cell-relevant biological phenomena, and aligns capabilities with translational challenges and goals. In this concise review, we describe how microfluidic technologies can contribute to the translation of stem cell research outcomes, and we provide an update on innovative research efforts in this area. This timely convergence of stem cell translational challenges and microfluidic capabilities means that there is now an opportunity for both disciplines to benefit from increased interaction.
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paper_title: The intersection of flow cytometry with microfluidics and microfabrication

paper_content:

A modern flow cytometer can analyze and sort particles on a one by one basis at rates of 50,000 particles per second. Flow cytometers can also measure as many as 17 channels of fluorescence, several angles of scattered light, and other non-optical parameters such as particle impedance. More specialized flow cytometers can provide even greater analysis power, such as single molecule detection, imaging, and full spectral collection, at reduced rates. These capabilities have made flow cytometers an invaluable tool for numerous applications including cellular immunophenotyping, CD4+ T-cell counting, multiplex microsphere analysis, high-throughput screening, and rare cell analysis and sorting. Many bio-analytical techniques have been influenced by the advent of microfluidics as a component in analytical tools and flow cytometry is no exception. Here we detail the functions and uses of a modern flow cytometer, review the recent and historical contributions of microfluidics and microfabricated devices to field of flow cytometry, examine current application areas, and suggest opportunities for the synergistic application of microfabrication approaches to modern flow cytometry.
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paper_title: Lab-on-chip technologies: making a microfluidic network and coupling it into a complete microsystem—a review

paper_content:

Microfluidics is an emerging field that has given rise to a large number of scientific and technological developments over the last few years. This review reports on the use of various materials, such as silicon, glass and polymers, and their related technologies for the manufacturing of simple microchannels and complex systems. It also presents the main application fields concerned with the different technologies and the most significant results reported by academic and industrial teams. Finally, it demonstrates the advantage of developing approaches for associating polymer technologies for manufacturing of fluidic elements with integration of active or sensitive elements, particularly silicon devices.
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paper_title: Microfluidics for flow cytometric analysis of cells and particles.

paper_content:

This review describes recent developments in microfabricated flow cytometers and related microfluidic devices that can detect, analyze, and sort cells or particles. The high-speed analytical capabilities of flow cytometry depend on the cooperative use of microfluidics, optics and electronics. Along with the improvement of other components, replacement of conventional glass capillary-based fluidics with microfluidic sample handling systems operating in microfabricated structures enables volume- and power-efficient, inexpensive and flexible analysis of particulate samples. In this review, we present various efforts that take advantage of novel microscale flow phenomena and microfabrication techniques to build microfluidic cell analysis systems.
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paper_title: Hydrodynamic Flow Confinement Technology in Microfluidic Perfusion Devices

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Hydrodynamically confined flow device technology is a young research area with high practical application potential in surface processing, assay development, and in various areas of single cell research. Several variants have been developed, and most recently, theoretical and conceptual studies, as well as fully developed automated systems, were presented. In this article we review concepts, fabrication strategies, and application areas of hydrodynamically confined flow (HCF) devices.
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paper_title: Highly efficient dual-channel cytometric-detection of micron-sized particles in microfluidic device

paper_content:

Abstract To demonstrate the ability to efficiently count and identify suspended micron-sized particles by simultaneously detecting their fluorescence emission and light scattering in microfabricated channel, a compact configuration that used a polydimethylsiloxane (PDMS) microfabricated channel as interrogation component, hydrodynamic focusing for particle control, and a simple free-space optical setup for signal detection, was accordingly developed. Subsequently, a quantitative count of 1.013 μm diameter fluorescently labeled beads in suspension was implemented in a microfluidic device employing both fluorescence emission and light scattering at average particle throughput ranging from 83 to 416 particles/s. As a result, the detection efficiencies above 88% for both signals and correlation percentages above 97% between them were routinely achieved. In addition, it was shown that effective differentiation of 1.013 μm fluorescently labeled beads from various unlabeled beads in mixed populations of high mixing ratios had been successfully realized in this microfluidic-device-based instrumentation. Finally, the demonstrated system was used to detect fluorescein isothiocyanate (FITC) labeled nonpathogenic bacteria of Escherichia coli ( E. coli ) DH5α. The results showed the detection efficiencies above 89.7% for fluorescence emission and 94.5% for light scattering signals, and a correlation of 94.9% between the two signals at an average throughput of 350 cells/s have been obtained. As a comparison, the detection accuracies of the dual-channel cytometric detection of the FITC-labeled E. coli DH5α cells in the microfluidic device are approximately 84.3% and 88.8% for fluorescence emission and light scattering respectively when compared against a manual cell count using a haemocytometer as a standard.
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paper_title: A high-discernment microflow cytometer with microweir structure.

paper_content:

Using a simple and reliable isotropic wet etching process, we fabricated a microflow cytometer in which cells/particles are concentrated in the center of the sample stream using a 2-D hydrodynamic focusing technique and an microweir structure. Having focused the cells/particles, they are detected and counted using a LIF method. The experimental and numerical results confirm the effectiveness of the hydrodynamic sheath flows in squeezing the cells/particles into a narrow stream in the horizontal X-Y plane. Furthermore, it is shown numerically that the microweir structure results in the separation of the cells/particles in the vertical X-Z plane such that they pass through the detection region in a sequential fashion and can therefore be counted with a high degree of precision. The experimental results obtained using fluorescent polystyrene beads with diameters of 5 and 10 microm, respectively, confirm the suitability of the proposed device for microfluidic applications requiring the high-precision counting of particles or cells within a sample flow.
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paper_title: Flow cytometry of Escherichia coli on microfluidic devices.

paper_content:

Flow cytometry of the bacterium Escherichia coli was demonstrated on a microfabricated fluidic device (microchip). The channels were coated with poly(dimethylacrylamide) to prevent cell adhesion, and the cells were transported electrophoretically by applying potentials to the fluid reservoirs. The cells were electrophoretically focused at the channel cross and detected by coincident light scattering and fluorescence. The E. coli were labeled with a membrane-permeable nucleic acid stain (Syto15), a membrane-impermeable nucleic acid stain (propidium iodide), or a fluorescein-labeled antibody and counted at rates from 30 to 85 Hz. The observed labeling efficiencies for the dyes and antibody were greater than 94%.
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paper_title: Microflow Cytometer for optical analysis of phytoplankton.

paper_content:

Analysis of the intrinsic fluorescence profiles of individual marine algae can be used in general classification of organisms based on cell size and fluorescence properties. We describe the design and fabrication of a Microflow Cytometer on a chip for characterization of phytoplankton. The Microflow Cytometer measured distinct side scatter and fluorescence properties of Synechococcus sp., Nitzschia d., and Thalassiosira p.; measurements were confirmed using the benchtop Accuri C6 flow cytometer. The Microflow Cytometer proved sensitive enough to detect and characterize picoplankton with diameter approximately 1 μm and larger phytoplankton of up to 80 μm in length. The wide range in size discrimination coupled with detection of intrinsic fluorescent pigments suggests that this Microflow Cytometer will be able to distinguish different populations of phytoplankton on unmanned underwater vehicles.
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paper_title: PDMS-based opto-fluidic micro flow cytometer with two-color, multi-angle fluorescence detection capability using PIN photodiodes

paper_content:

This research describes the design and performance of a microfluidic observation channel capable of performing flow cytometry measurements using solid-state lasers and PIN-based photodetectors. The observation cell, fabricated using poly(dimethylsiloxane) (PDMS)-based soft lithography, is designed with integrated microgrooves that permit the physical registration of optical fibers that serve as optical waveguides for laser excitation and fluorescence detection. The optical fiber arrangement is designed to permit simultaneous multi-color excitation of sample particles, at a single interrogation point in the observation channel, using multiple angles of excitation and detection. The multiple angles can be used to increase signal-to-noise ratio ( S/N) by performing two independent fluorescence measurements simultaneously, and to permit multiple simultaneous cytometric measurements to occur at a single interrogation point. Moreover, the multi-angle micro flow cytometer is proven to provide consistent color differentiation that allows the design to achieve spectrometer capabilities not possible on a conventional flow cytometer. The micro flow cytometer performance was evaluated by considering the detection of fluorescent microspheres (15m) and nucleic acid labeled fungus (Saccharomyces cerevisiae). Multiple angles (45 ◦ , 135 ◦ , and 180 ◦ ) between fluorescence excitation and detection were investigated with respect to S/N, S/N coefficient of variation, and particle recovery. The experiments demonstrate the feasibility of developing a high S/N single-cell fluorescence detection system based exclusively on silicon-based PIN photodiodes coupled with the lock-in amplification technique. The technique is applicable to the detection of scatter and fluorescent signals emanating from inorganic particles and biological cells, and is highly amenable to economical manufacturing, miniaturization, and low-power operation. Consequently, the design approach and performance results described in this investigation have applicability to the development of practical and low-cost cytometric micro total analysis systems (TAS). © 2003 Elsevier B.V. All rights reserved.
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paper_title: Hydrodynamic Focusing for a Micromachined Flow Cytometer

paper_content:

This paper describes hydrodynamic focusing inside a micromachined flow cytometer. Flow cytometry is a process whereby cells are analyzed and sorted based on hydrodynamic focusing phenomenon and specific cellular characteristics. In this study, the hydrodynamic focusing phenomenon is first modeled by employing potential flow theory. Then the flow field inside the flow cytometer is numerically simulated. The effect of the device geometry and relative sheath and sample flow rate on the focusing of the center flow is explored systematically. At last, a micromachine-based flow chamber is designed and fabricated on plastic substrates as a micro flow cytometer. Hydrodynamic focusing is verified with the use of microscopic visualization of water sheath flows and dyecontaining sample flow. Experimental data indicate that the size of focused sample stream can be reduced to about 3mm, which is applicable to cell sorting and counting. @DOI: 10.1115/1.1385514#
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paper_title: Microflow Cytometers with Integrated Hydrodynamic Focusing

paper_content:

This study demonstrates the suitability of microfluidic structures for high throughput blood cell analysis. The microfluidic chips exploit fully integrated hydrodynamic focusing based on two different concepts: Two-stage cascade focusing and spin focusing (vortex) principle. The sample--A suspension of micro particles or blood cells--is injected into a sheath fluid streaming at a substantially higher flow rate, which assures positioning of the particles in the center of the flow channel. Particle velocities of a few m/s are achieved as required for high throughput blood cell analysis. The stability of hydrodynamic particle positioning was evaluated by measuring the pulse heights distributions of fluorescence signals from calibration beads. Quantitative assessment based on coefficient of variation for the fluorescence intensity distributions resulted in a value of about 3% determined for the micro-device exploiting cascade hydrodynamic focusing. For the spin focusing approach similar values were achieved for sample flow rates being 1.5 times lower. Our results indicate that the performances of both variants of hydrodynamic focusing suit for blood cell differentiation and counting. The potential of the micro flow cytometer is demonstrated by detecting immunologically labeled CD3 positive and CD4 positive T-lymphocytes in blood.
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paper_title: Micro flow cytometer with self-aligned 3D hydrodynamic focusing

paper_content:

A micro flow cytometer with a single step 3D hydrodynamic flow focusing has been developed. The proposed design is capable to create a single-file particle stream that is self-aligned with an integrated optical fiber-based detection system, regardless of the flow rate ratio between the focusing and core liquids. The design approach provides the ability to adjust the stream size while keeping the position of the focused stream centered with respect to the focusing channel. The device has been fabricated by direct micro milling of PMMA sheets. Experimental validation of the hydrodynamic sheath focusing effect has been presented and sample stream with tuneable size from about 18 to 50 μm was measured. Flow cytometry measurements have been performed by using 10-23 μm fluorescent particles. From the analysis of the signals collected at each transit event we can confirm that the device was capable to align and measure microparticles with a good coefficient of variance.
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paper_title: Simultaneous optical and impedance analysis of single cells: A comparison of two microfluidic sensors with sheath flow focusing

paper_content:

Systems with disposable microfluidic cartridges are demanded for in vitro point-of-care diagnostics. Here, we developed two microfluidic sensors for flow cytometry cell differentiation based on the combined analysis of electrical impedance and optical properties of microparticles and blood cells. Both microdevices incorporate 1D sheath flow focusing to confine particles in a defined plane. For one sensor, a novel approach based on electroplating of gold was used in combination with lithography in SU-8. Electrodes for differential measurements are integrated into the flow channel perpendicularly to the plane of the substrate. For the manufacturing of the second sensor, we applied double-etched glass structures with integrated sputtered Pt electrode pairs. Stability of hydrodynamic focusing was benchmarked by measuring the coefficients of variations of calibration beads with specified size and fluorescence intensities. Sensitivity of electrical impedance measured in the MHz frequency range allowed detection of beads with a diameter of 1 μm when using side scatter as trigger signal. Measured particles were moving at a velocity of up to 6 m/s. Differentiation of leukocytes was achieved by simultaneous detection of impedance signals, correlated with intrinsic electrical properties of cells, and fluorescence measurements using standard protocols for immunophenotyping of white blood cells. The resulting separation of cell clusters is similar to that of microdevices with 2D hydrodynamic focusing and routine flow cytometers.
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paper_title: 3D microfabrication with inclined/rotated UV lithography

paper_content:

Abstract This paper presents a novel microfabrication technology of three-dimensional (3D) microstructures with inclined/rotated UV lithography using negative thick photoresist, SU-8. In exposure process, a photomask and a SU-8 coated substrate are fixed together, and tilted or tilted and rotated to a UV source. The reflected UV at the interface between the resist and the substrate is also exploited as well as the incident UV. With the 3D microfabrication technology, various 3D microstructures are easily fabricated such as oblique cylinders, embedded channels, bridges, V-grooves, truncated cones, and so on. The angles between fabricated structures and normal lines are 19.5°, when the incident angle of UV in air is 32°. Thus, the refractive index of SU-8 and the maximum refractive angle at the interface between SU-8 and air are expected about 1.6 and 39°, respectively.
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paper_title: Multiplexed detection of bacteria and toxins using a microflow cytometer.

paper_content:

A microfabricated flow cytometer was used to demonstrate multiplexed detection of bacteria and toxins using fluorescent coded microspheres. Antibody-coated microspheres bound biothreat targets in a sandwich immunoassay format. The microfluidic cytometer focused the microspheres in three dimensions within the laser interrogation region using passive groove structures to surround the sample stream with sheath fluid. Optical analysis at four different wavelengths identified the coded microspheres and quantified target bound by the presence of phycoerythrin tracer. The multiplexed assays in the microflow cytometer had performance approaching that of a commercial benchtop flow cytometer. The respective limits of detection for bacteria (Escherichia coli, Listeria, and Salmonella) were found to be 10(3), 10(5), and 10(4) cfu/mL for the microflow cytometer and 10(3), 10(6), and 10(5) cfu/mL for the commercial system. Limits of detection for the toxins (cholera toxin, staphylococcal enterotoxin B, and ricin) were 1.6, 0.064, and 1.6 ng/mL for the microflow cytometer and 1.6, 0.064, and 8.0 ng/mL for the commercial system.
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paper_title: Three-dimensional hydrodynamic focusing in polydimethylsiloxane (PDMS) microchannels

paper_content:

This paper presents a generalization of the hydrodynamic focusing technique to three-dimensions. Three-dimensional (3-D) hydrodynamic focusing offers the advantages of precision positioning of molecules in both vertical and lateral dimensions and minimizing the interaction of the sample fluid with the surfaces of the channel walls. In an ideal approach, 3-D hydrodynamic focusing could be achieved by completely surrounding the sample flow by a cylindrical sheath flow that constrains the sample flow to the center of the channel in both the lateral and the vertical dimensions. We present here design and simulation, 3-D fabrication, and experimental results from a piecewise approximation to such a cylindrical flow. Two-dimensional (2-D) and 3-D hydrodynamic focusing chips were fabricated using micromolding methods with polydimethylsiloxane (PDMS). Three-dimensional hydrodynamic focusing chips were fabricated using the "membrane sandwich" method. Laser scanning confocal microscopy was used to study the hydrodynamic focusing experiments performed in the 2-D and 3-D chips with Rhodamine 6G solution as the sample fluid and water as the sheath fluid.
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paper_title: Multi-wavelength microflow cytometer using groove-generated sheath flow

paper_content:

A microflow cytometer was developed that ensheathed the sample (core) fluid on all sides and interrogated each particle in the sample stream at four different wavelengths. Sheathing was achieved by first sandwiching the core fluid with the sheath fluid laterally via fluid focusing. Chevron-shaped groove features fabricated in the top and bottom of the channel directed sheath fluid from the sides to the top and bottom of the channel, completely surrounding the sample stream. Optical fibers inserted into guide channels provided excitation light from diode lasers at 532 and 635 nm and collected the emission wavelengths. Two emission collection fibers were connected to PMTs through a multimode fiber splitter and optical filters for detection at 635 nm (scatter), 665 nm and 700 nm (microsphere identification) and 565 nm (phycoerythrin tracer). The cytometer was capable of discriminating microspheres with different amounts of the fluorophores used for coding and detecting the presence of a phycoerythrin antibody complex on the surface of the microspheres. Assays for Escherichia coli were compared with a commercial Luminex flow cytometer.
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paper_title: Improved inclined multi-lithography using water as exposure medium and its 3D mixing microchannel application

paper_content:

This paper presents the inclined UV lithography in water medium and 3D mixing microchannel application. The theoretical limit of the inclination angle of the structure is determined by the difference of the refractive index between air and SU-8. In order to reduce the mismatch of the refractive index between air and photoresist, DI water was used as exposure medium instead of air. As a result, the maximum angle of the inclined structure was improved from 38.7° (in air) to 56.2° (in water). Fine and high aspect ratio SU-8 structures were obtained using UV exposure in water medium. As a microfluidic application, a microchannel having 3D slanted grooves was designed and fabricated. 45° slanted grooves were formed successfully on the sidewalls of the microchannel by using inclined UV exposure in water medium. Mixing efficiency of this microchannel was evaluated by CFD simulation and cross-sectional fluorescence image detection using confocal microscope.
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paper_title: An efficient 3-dimensional hydrodynamic focusing microfluidic device by means of locally increased aspect ratio

paper_content:

In this paper, we have designed, fabricated and characterized an efficient 3-dimensional hydrodynamic focusing microfluidic device (3D-HFMD) in numerical and experimental ways. The 3D-HFMD achieved vertical focusing of core sample flow effectively in the pressure driven flow with the help of locally increased aspect ratio of thickness to width without any horizontal separation wall, thereby enabling us to realize 3D focusing after simple horizontal focusing. Extensive numerical analysis shows that the 3D-HFMD enhances the performance of vertical focusing compared with the previous designs over a wide range of the Reynolds numbers, even in the case of a the global aspect ratio less than 0.5. Experimental results from the fabricated 3D-HFMD confirmed that 3D hydrodynamic focusing could be successfully achieved, even when the dimension of the channel was kept consistent with that of general microfluidic channels. The 3D-HFMD suggested in this study could be applied to various integrated biological or chemical microfluidic devices.
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paper_title: Sub-micrometer-precision, three-dimensional (3D) hydrodynamic focusing via “microfluidic drifting”

paper_content:

In this article, we demonstrate single-layered, “microfluidic drifting” based three-dimensional (3D) hydrodynamic focusing devices with particle/cell focal positioning approaching submicron precision along both lateral and vertical directions. By systematically optimizing channel geometries and sample/sheath flow rates, a series of “microfluidic drifting” based 3D hydrodynamic focusing devices with different curvature angles are designed and fabricated. Their performances are then evaluated using confocal microscopy, fast camera imaging, and side-view imaging techniques. Using a device with a curvature angle of 180°, we have achieved a standard deviation of ±0.45 μm in particle focal position and a coefficient of variation (CV) of 2.37% in flow cytometric measurements. To the best of our knowledge, this is the best CV that has been achieved using a microfluidic flow cytometry device. Moreover, the device showed the capability to distinguish 8 peaks when subjected to a stringent 8-peak rainbow calibration test, signifying the ability to perform sensitive, accurate tests similar to commercial flow cytometers. We have further tested and validated our device by detection of HEK-293 cells. With its advantages in simple fabrication (i.e., single-layered device), precise 3D hydrodynamic focusing (i.e., submicrometer precision along both lateral and vertical directions), and high detection resolution (i.e., low CV), our method could serve as an important basis for high-performance, mass-producible microfluidic flow cytometry.
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paper_title: Micro flow cytometer with self-aligned 3D hydrodynamic focusing

paper_content:

A micro flow cytometer with a single step 3D hydrodynamic flow focusing has been developed. The proposed design is capable to create a single-file particle stream that is self-aligned with an integrated optical fiber-based detection system, regardless of the flow rate ratio between the focusing and core liquids. The design approach provides the ability to adjust the stream size while keeping the position of the focused stream centered with respect to the focusing channel. The device has been fabricated by direct micro milling of PMMA sheets. Experimental validation of the hydrodynamic sheath focusing effect has been presented and sample stream with tuneable size from about 18 to 50 μm was measured. Flow cytometry measurements have been performed by using 10-23 μm fluorescent particles. From the analysis of the signals collected at each transit event we can confirm that the device was capable to align and measure microparticles with a good coefficient of variance.
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paper_title: Investigations of micrometer sample stream profiles in a three-dimensional hydrodynamic focusing device

paper_content:

Abstract In this work we describe the design and fabrication of a novel three-dimensional flow focusing device with channel dimensions of 50 μm × 50 μm at the focusing region. The device is suitable for one-by-one cell or particle detection near the channel bottom, in for example a Coulter counter or for optical near field detection. The device comprises a silicon-glass sandwich with SU-8 resist in between. The sample flow is hydrodynamically focused between three sheath flows. At the fourth side the sample flow is moving along the channel bottom. We investigated the focusing limitation of the sample flow and found that the minimum width amounts to 2.5 μm at a channel width of 50 μm. Confocal laser scanning measurements were constituted to obtain the sample flow profile. We show the experimental and numerical images of the focused stream shape from a cross-section perspective. Furthermore, we analyzed the measured fluorescent color intensity of the sample stream and from that, calculated the sample flow height. These results were compared with the corresponding confocal images. Additionally, the influence of the hydrodynamic focusing on the sample profile and its uniformity over a certain distance was investigated.
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paper_title: The good, the bad, and the tiny: a review of microflow cytometry

paper_content:

Recent developments in microflow cytometry have concentrated on advancing technology in four main areas: (1) focusing the particles to be analyzed in the microfluidic channel, (2) miniaturization of the fluid-handling components, (3) miniaturization of the optics, and (4) integration and applications development. Strategies for focusing particles in a narrow path as they pass through the detection region include the use of focusing fluids, nozzles, and dielectrophoresis. Strategies for optics range from the use of microscope objectives to polymer waveguides or optical fibers embedded on-chip. While most investigators use off-chip fluidic control, there are a few examples of integrated valves and pumps. To date, demonstrations of applications are primarily used to establish that the microflow systems provide data of the same quality as laboratory systems, but new capabilities—such as automated sample staining—are beginning to emerge. Each of these four areas is discussed in detail in terms of the progress of development, the continuing limitations, and potential future directions for microflow cytometers.
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paper_title: Standing surface acoustic wave ( SSAW )-based microfluidic cytometer †

paper_content:

Trapping and sorting of micro-sized objects is one important application of lab on a chip devices, with the use of acoustic bubbles emerging as an effective, non-contact method. Acoustically actuated bubbles are known to exert a secondary radiation force (FSR) on micro-particles and stabilize them on the bubble surface, when this radiation force exceeds the external hydrodynamic forces that act to keep the particles in motion. While the theoretical expression of FSR has been derived by Nyborg decades ago, no direct experimental validation of this force has been performed, and the relationship between FSR and the bubble's ability to trap particles in a given lab on a chip device remains largely empirical. In order to quantify the connection between the bubble oscillation and the resultant FSR, we experimentally measure the amplitude of bubble oscillations that give rise to FSR and observe the trapping and release of a single microsphere in the presence of the mean flow at the corresponding acoustic parameters using an acoustofluidic device. By combining well-developed theories that connect bubble oscillations to the acoustic actuation, we derive the expression for the critical input voltage that leads to particle release into the flow, in good agreement with the experiments.
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paper_title: Sorting drops and cells with acoustics: acoustic microfluidic fluorescence-activated cell sorter

paper_content:

We describe a versatile microfluidic fluorescence-activated cell sorter that uses acoustic actuation to sort cells or drops at ultra-high rates. Our acoustic sorter combines the advantages of traditional fluorescence-activated cell (FACS) and droplet sorting (FADS) and is applicable for a multitude of objects. We sort aqueous droplets, at rates as high as several kHz, into two or even more outlet channels. We can also sort cells directly from the medium without prior encapsulation into drops; we demonstrate this by sorting fluorescently labeled mouse melanoma cells in a single phase fluid. Our acoustic microfluidic FACS is compatible with standard cell sorting cytometers, yet, at the same time, enables a rich variety of more sophisticated applications.
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paper_title: Three-dimensional magnetic focusing of particles and cells in ferrofluid flow through a straight microchannel

paper_content:

Focusing particles and cells into a tight stream is often required in order for continuous flow detection, counting and sorting. So far a variety of particle focusing methods have been developed in microfluidic devices, among which magnetic focusing is still relatively new. We?develop in this work an approach to embedding symmetrically two repulsive permanent magnets about a straight rectangular microchannel in a PDMS-based microfluidic device. The closest distance between the magnets is limited only by the sizes of the embedded and holder magnets involved in the fabrication process. The developed device is used to implement and investigate the three-dimensional magnetic focusing of polystyrene particles in ferrofluid microflow with both the top- and side-view visualizations. The effects of flow speed and particle size on the particle focusing effectiveness are studied. The developed device is also applied to magnetically focus yeast cells in ferrofluid, which proves to be biocompatible as verified by a cell viability test. In addition, an analytical model is developed and found to be able to predict the experimentally observed particle and cell focusing behaviors with reasonable agreement.
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paper_title: High-efficiency magnetic particle focusing using dielectrophoresis and magnetophoresis in a microfluidic device

paper_content:

We describe a novel technique that utilizes simultaneous implementation of dielectrophoresis (DEP) and magnetophoresis (MAP) to focus magnetic particles into streams for optical analysis of biological samples. This technique does not require sheath flow and utilizes a novel interdigitated electrode array chip that yields multiple streams of flowing magnetic particles in single-file columns. The MAP force placed particles in close proximity to the microelectrodes where they were subjected to a strong DEP force that generated the particle focusing effect. Particle focusing efficiency was improved using this combination DEP?MAP technique compared to DEP alone: particle stream widths were reduced ~47% and stream width variability was reduced 80% for focused streams of 8.5 ?m diameter magnetic particles. 3 ?m diameter magnetic particles were strongly focused with DEP?MAP (~4 ?m wide streams with sub-?m variability in stream width) while DEP alone provided minimal focusing. Additional components of a prototype detection system were also demonstrated including an integrated magnetic pelleting component, a hand-held MHz frequency signal generator and a bench-top near-confocal microscope for optical analysis of flowing particles. Preliminary testing of a sandwich assay performed on the surface of magnetic particles showed 50 ppb detection levels of a surrogate biotoxin (ovalbumin) in a raw milk sample.
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paper_title: Lab-on-Chip Cytometry Based on Magnetoresistive Sensors for Bacteria Detection in Milk

paper_content:

Flow cytometers have been optimized for use in portable platforms, where cell separation, identification and counting can be achieved in a compact and modular format. This feature can be combined with magnetic detection, where magnetoresistive sensors can be integrated within microfluidic channels to detect magnetically labelled cells. This work describes a platform for in-flow detection of magnetically labelled cells with a magneto-resistive based cell cytometer. In particular, we present an example for the validation of the platform as a magnetic counter that identifies and quantifies Streptococcus agalactiae in milk.
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paper_title: Real-time control of inertial focusing in microfluidics using dielectrophoresis (DEP)

paper_content:

In this paper, we propose a novel hybrid microfluidic device, for the first time, which integrates and fully couples both dielectrophoresis (DEP) and inertial focusing. A DEP force is coupled with inertial lift force to adjust particle's equilibrium position in the vertical direction in real time. The focusing pattern and position of the particles along the horizontal plane can be concurrently adjusted by adjusting their vertical position with DEP forces. The magnitude of secondary flow drag dramatically increases when particles are levitated towards the centre of the channel in the vertical direction. The paper analytically and experimentally investigates the mechanism of this tuneable DEP-inertial microfluidic device. The proposed hybrid device possesses the advantages of both DEP and inertial microfluidic devices, working in a high-throughput manner, as well as having precise controllability in real-time. This DEP-inertial microfluidic device is potentially a versatile and robust platform for a feedback-controlled manipulation and separation of particles and cells.
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paper_title: Three-Dimensional Electro-Sonic Flow Focusing Ionization Microfluidic Chip for Mass Spectrometry

paper_content:

Increasing research efforts have been recently devoted to the coupling of microfluidic chip-integrated ionization sources to mass spectrometry (MS). Considering the limitations of microfluidic chips coupled with MS such as liquid spreading, dead volume, and manufacturing troubles, this paper proposed a new three-dimensional (3D) flow focusing (FF)-based microfluidic ionizing source. This source was fabricated by using the two-layer soft lithography method with the nozzle placed inside the chip. The proposed FF microfluidic chip can realize two-phase FF with liquid in air regardless of the viscosity ratio of the continuous and dispersed phases. MS results indicated that the proposed FF microfluidic chip can work as a typical electrical ionization source when supplied with high voltage and can serve as a sonic ionization source without high voltage. The electro-sonic FF ionization microfluidic chip is expected to have various applications, particularly in the integrated and portable applications of ionization sources coupling with portable MS in the future.
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paper_title: Dielectrophoretic focusing of particles in a microchannel constriction using DC-biased AC flectric fields.

paper_content:

This paper presents a fundamental study of particle electrokinetic focusing in a single microchannel constriction. Through both experiments and simulations, we demonstrate that such dielectrophoresis-induced particle focusing can be implemented in a much smaller magnitude of DC-biased AC electric fields (10 kV/m in total) as compared to pure DC electric fields (up to 100 kV/m). This is attributed to the increase in the ratio of cross-stream particle dielectrophoretic velocity to streamwise electrokinetic velocity as only the DC field component contributes to the latter. The effects of the 1 kHz frequency AC to DC electric field ratio on particle trajectories and velocity variations through the microchannel constriction are also examined, which are found to agree with the simulation results.
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paper_title: Focusing microparticles in a microfluidic channel with standing surface acoustic waves (SSAW)

paper_content:

We introduce a novel on-chip microparticle focusing technique using standing surface acoustic waves (SSAW). Our method is simple, fast, dilution-free, and applicable to virtually any type of microparticle.
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paper_title: Vertical focusing device utilizing dielectrophoretic force and its application on microflow cytometer

paper_content:

Focusing of particles/cells in the vertical direction inside a micromachined flow cytometer is a critical issue while using an embedded optical detection system aligned with microchannels. Even if the particles/cells have been focused centrally in the horizontal direction using coplanar sheath flows, appreciable errors may still arise if they are randomly distributed in the vertical direction. This work presents a vertical focusing device utilizing dielectrophoretic (DEP) forces and its application on micromachined flow cytometer. A pair of parallel microelectrodes is deposited on the upper and bottom surface of the microfluidic channel to drive particles/cells into the vertical center of the sample flow. This new microfluidic device is capable of three-dimensional (3-D) focusing of microparticles/cells and thus improves the uniformity of the optical detection signals. This 3-D focusing feature of the sample flow is realized utilizing the combination of dielectrophoretic and hydrodynamic forces. Initially, two sheath flows are used to focus the sample flow horizontally by means of hydrodynamic forces, and then two embedded planar electrodes apply negative DEP forces to focus the particles/cells vertically. A new micromachined flow cytometer integrated with an embedded optical detection mechanism is then demonstrated. Numerical simulation is used to analyze the operation conditions and the dimension of the microelectrodes for DEP manipulation. The dynamic trace of the moving particles/cells within a flow stream under the DEP manipulation is calculated numerically. Micro polystyrene beads and diluted human red blood cells (RBC) are used to test the performance of the proposed device. The experimental results confirm the suitability of the proposed device for applications requiring precise counting of particles or cells. Experimental data indicates the proposed method can provide more stable signals over the other types of micromachined flow cytometers that were previously reported.
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paper_title: Three-dimensional continuous particle focusing in a microfluidic channel via standing surface acoustic waves (SSAW)

paper_content:

Three-dimensional (3D) continuous microparticle focusing has been achieved in a single-layer polydimethylsiloxane (PDMS) microfluidic channel using a standing surface acoustic wave (SSAW). The SSAW was generated by the interference of two identical surface acoustic waves (SAWs) created by two parallel interdigital transducers (IDTs) on a piezoelectric substrate with a microchannel precisely bonded between them. To understand the working principle of the SSAW-based 3D focusing and investigate the position of the focal point, we computed longitudinal waves, generated by the SAWs and radiated into the fluid media from opposite sides of the microchannel, and the resultant pressure and velocity fields due to the interference and reflection of the longitudinal waves. Simulation results predict the existence of a focusing point which is in good agreement with our experimental observations. Compared with other 3D focusing techniques, this method is non-invasive, robust, energy-efficient, easy to implement, and applicable to nearly all types of microparticles.
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paper_title: Simultaneous high speed optical and impedance analysis of single particles with a microfluidic cytometer.

paper_content:

We describe a microfluidic cytometer that performs simultaneous optical and electrical characterisation of particles. The microfluidic chip measures side scattered light, signal extinction and fluorescence using integrated optical fibres coupled to photomultiplier tubes. The channel is 80 μm high and 200 μm wide, and made from SU-8 patterned and sandwiched between glass substrates. Particles were focused into the analysis region using 1-D hydrodynamic focusing and typical particle velocities were 0.1 ms(-1). Excitation light is coupled into the detection channel with an optical fibre and focused into the channel using an integrated compound air lens. The electrical impedance of particles is measured at 1 MHz using micro-electrodes fabricated on the channel top and bottom. This data is used to accurately size the particles. The system is characterised using a range of different sized polystyrene beads (fluorescent and non-fluorescent). Single and mixed populations of beads were measured and the data compared with a conventional flow cytometer.
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paper_title: Scattering detection using a photonic-microfluidic integrated device with on-chip collection capabilities.

paper_content:

SU-8-based photonic-microfluidic integrated devices with on-chip beam shaping and collection capabilities were demonstrated in a scattering detection and counting application. Through the proper deployment of the tailored beam geometries via the on-chip excitation optics, excellent CV values were measured for 1, 2, and 5 μm blank beads, 16.4, 11.0, and 12.5%, respectively, coupled with a simple free-space optical detection scheme. The performance of these devices was found dependent on the combination of on-chip, lens-shaped beam geometry and bead size. While very low CVs were obtained when the combination was ideal, a nonideal combination could still result in acceptable CVs for flow cytometry; the reliability was confirmed via devices being able to resolve separate populations of 2.0 and 5.0 μm beads from their mixture with low CV values of 15.9 and 18.5%, respectively. On-chip collection using integrated on-chip optical waveguides was shown to be very reliable in comparison with a free-space collection scheme, yielding a coincident rate of 94.2%. A CV as low as 19.2% was obtained from the on-chip excitation and collection of 5 μm beads when the on-chip lens-shaped beam had a 6.0-μm beam waist.
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paper_title: PDMS-based opto-fluidic micro flow cytometer with two-color, multi-angle fluorescence detection capability using PIN photodiodes

paper_content:

This research describes the design and performance of a microfluidic observation channel capable of performing flow cytometry measurements using solid-state lasers and PIN-based photodetectors. The observation cell, fabricated using poly(dimethylsiloxane) (PDMS)-based soft lithography, is designed with integrated microgrooves that permit the physical registration of optical fibers that serve as optical waveguides for laser excitation and fluorescence detection. The optical fiber arrangement is designed to permit simultaneous multi-color excitation of sample particles, at a single interrogation point in the observation channel, using multiple angles of excitation and detection. The multiple angles can be used to increase signal-to-noise ratio ( S/N) by performing two independent fluorescence measurements simultaneously, and to permit multiple simultaneous cytometric measurements to occur at a single interrogation point. Moreover, the multi-angle micro flow cytometer is proven to provide consistent color differentiation that allows the design to achieve spectrometer capabilities not possible on a conventional flow cytometer. The micro flow cytometer performance was evaluated by considering the detection of fluorescent microspheres (15m) and nucleic acid labeled fungus (Saccharomyces cerevisiae). Multiple angles (45 ◦ , 135 ◦ , and 180 ◦ ) between fluorescence excitation and detection were investigated with respect to S/N, S/N coefficient of variation, and particle recovery. The experiments demonstrate the feasibility of developing a high S/N single-cell fluorescence detection system based exclusively on silicon-based PIN photodiodes coupled with the lock-in amplification technique. The technique is applicable to the detection of scatter and fluorescent signals emanating from inorganic particles and biological cells, and is highly amenable to economical manufacturing, miniaturization, and low-power operation. Consequently, the design approach and performance results described in this investigation have applicability to the development of practical and low-cost cytometric micro total analysis systems (TAS). © 2003 Elsevier B.V. All rights reserved.
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paper_title: A Review of Single-Mode Fiber Optofluidics

paper_content:

We review the field we describe as “single-mode fiber optofluidics” which combines the technologies of microfluidics with single-mode fiber optics for delivering new implementations of well-known single-mode optical fiber devices. The ability of a fluid to be easily shaped to different geometries plus the ability to have its optical properties easily changed via concentration changes or an applied electrical or magnetic field offers potential benefits such as no mechanical moving parts, miniaturization, increased sensitivity, and lower costs. However, device fabrication and operation can be more complex than in established single-mode fiber optic devices.
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paper_title: Fiber-Based, Injection-Molded Optofluidic Systems: Improvements in Assembly and Applications

paper_content:

We present a method to fabricate polymer optofluidic systems by means of injection molding that allow the insertion of standard optical fibers. The chip fabrication and assembly methods produce large numbers of robust optofluidic systems that can be easily assembled and disposed of, yet allow precise optical alignment and improve delivery of optical power. Using a multi-level chip fabrication process, complex channel designs with extremely vertical sidewalls, and dimensions that range from few tens of nanometers to hundreds of microns can be obtained. The technology has been used to align optical fibers in a quick and precise manner, with a lateral alignment accuracy of 2.7 ± 1.8 μm. We report the production, assembly methods, and the characterization of the resulting injection-molded chips for Lab-on-Chip (LoC) applications. We demonstrate the versatility of this technology by carrying out two types of experiments that benefit from the improved optical system: optical stretching of red blood cells (RBCs) and Raman spectroscopy of a solution loaded into a hollow core fiber. The advantages offered by the presented technology are intended to encourage the use of LoC technology for commercialization and educational purposes.
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paper_title: Fabrication and Performance of a Photonic-Microfluidic Integrated Device

paper_content:

Fabrication and performance of a functional photonic-microfluidic flow cytometer is demonstrated. The devices are fabricated on a Pyrex substrate by photolithographically patterning the microchannels and optics in a SU-8 layer that is sealed via a poly(dimethylsiloxane) (PDMS) layer through a unique chemical bonding method. The resulting devices eliminate the free-space excitation optics through integration of microlenses onto the chip to mimic conventional cytometry excitation. Devices with beam waists of 6 μm and 12 μm in fluorescent detection and counting tests using 2.5 and 6 μm beads-show CVs of 9%–13% and 23% for the two devices, respectively. These results are within the expectations for a conventional cytometer (5%–15%) and demonstrate the ability to integrate the photonic components for excitation onto the chip and the ability to maintain the level of reliable detection.
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paper_title: Optofluidic waveguide as a transformation optics device for lightwave bending and manipulation

paper_content:

Transformation optics represents a new paradigm for designing light-manipulating devices, such as cloaks and field concentrators, through the engineering of electromagnetic space using materials with spatially variable parameters. Here we analyse liquid flowing in an optofluidic waveguide as a new type of controllable transformation optics medium. We show that a laminar liquid flow in an optofluidic channel exhibits spatially variable dielectric properties that support novel wave-focussing and interference phenomena, which are distinctively different from the discrete diffraction observed in solid waveguide arrays. our work provides new insight into the unique optical properties of optofluidic waveguides and their potential applications.
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paper_title: Versatile liquid-core optofluidic waveguides fabricated in hydrophobic silica aerogels by femtosecond-laser ablation

paper_content:

Abstract We report on the fabrication and characterization of versatile light waveguides exploiting filaments of a polar liquid confined within hydrophobic silica aerogels. Aerogels are highly porous materials with extremely low refractive index which makes them suitable as rigid cladding of liquid-core optofluidic waveguides based on total internal reflection of light. In this article, we introduce a new microfabrication technique that allows direct and precise processing of monolithic silica aerogels by ablation with femtosecond laser pulses. Using fast scanning of the focused laser ablation beam synchronized with the motion of the processed aerogel sample, we created high-quality straight microchannels of ∼5 mm length with controlled cross-sections inside monolithic aerogels. After the ablation, we filled the channels with high-refractive index ethylene glycol, forming multimode liquid core – solid cladding optofluidic waveguides. Subsequently, we carried out light-guiding experiments to measure overall optical attenuation of these waveguides. The characterization of waveguide transmission yielded values of propagation losses lower than 10 dB cm - 1 , demonstrating that the liquid-core waveguides with laser-ablated aerogel cladding represent an attractive alternative in optofluidic applications targeting controlled routing of light along arbitrary three-dimensional paths.
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paper_title: Fluorescent liquid-core/air-cladding waveguides towards integrated optofluidic light sources.

paper_content:

We have demonstrated fluorescent liquid-core/air-cladding (LA) waveguides suitable for use as integrated optofluidic light sources. These waveguides were fabricated by conventional soft lithography using poly(dimethylsiloxane) (PDMS). Two-phase stratified flows of air and ethylene glycol with fluorescent dye were generated along the PDMS channel. Compared to the liquid-core/liquid-cladding (L(2)) waveguide, the larger refractive index contrast of the LA waveguide resulted in stronger optical confinement. Specifically, the larger refractive index contrast led to experimentally achievable captured fractions (the amount of light to be coupled into the liquid core) as high as 22.8% and the measured propagation losses as low as 0.14 dB cm(-1). Furthermore, in our LA waveguides, diffusional mixing of the core and cladding fluids did not occur and the size of the core stream could be reversibly tuned simply by adjusting the flow rates of the two contiguous phases.
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paper_title: Soap Films as 1D waveguides

paper_content:

Laser light is injected in a free standing hori-zontal draining soap lm through the glass frame sustain-ing the lm. Two propagation regimes are clearly identi-ed depending on the lm thickness. At the beginning of the drainage, the soap lm behaves as a multimode-one dimensional optoouidic waveguide. In particular, we ob-serve that the injected light creates a bottleneck in the lm and part of the injected light is refracted leading to whiskers. At the end of the drainage where the lm thick-ness is below µm, there is a strong selection among the various possible optical modes in the lm, and part of the light is deeected. This leads to a self selection of the mode propagation inside the lm.
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paper_title: Tunable self-imaging effect using hybrid optofluidic waveguides

paper_content:

Multimode interference (MMI) is a typical self-imaging phenomenon and has been widely exploited for optical devices like couplers and splitters. Usually, it utilizes solid waveguides only and thus has very limited tunability in self-imaging properties. This paper reports our original work on tunable MMI using a hybrid optofluidic waveguide. It is generated by the diffusion between miscible flows in a microchannel and consists of two parts: gradient-index liquid–liquid waveguide for light modulation and step-index liquid–solid waveguide for image formation. Simulation and experiments have shown successful realization of the real-time tuning of self-imaging properties. For detail, the image point (focal spot) width could be modulated from 7 μm to 16 μm, and the self-imaging period changes in the range of 500 μm by varying the fluid properties. It is foreseeable that such a hybrid optofluidic waveguide may find wider applications in lab-on-a-chip systems and optical devices.
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paper_title: Integrated real-time optofluidic SERS via a liquid-core/liquid-cladding waveguide

paper_content:

This paper describes the realization of highly sensitive surface-enhanced Raman spectroscopy (SERS) via an integrated three-dimensional liquid-core/liquid-cladding waveguide. The cladding flow enclosed the core flow in both the horizontal and vertical directions within a single-layered microfluidic channel, and the laser beam was guided through the core stream. Deionized water was used to suspend SERS-active silver nanocolloids to form the core fluid, and 2,2,2-trifluoroethanol was used as the cladding fluid. Dipicolinic acid, commonly used as a biomarker for the detection of Bacillus anthracis, was employed as the target analyte. The sensitivity of the SERS signal was enhanced through this design because the signals backscattered from the analytes accumulated along the core stream and increased the SERS detection volume. The SERS data was evaluated in terms of the analyte concentration in the core fluid, the irradiated laser power, and the cross-sectional area of the core stream. The present data were compared with a conventional SERS detection approach in order to quantify the enhancement factor. The detection limit of concentration, 50 nM at an acquisition time of 6 s, was a factor of 35 smaller than that of conventional methods.
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paper_title: Reconfigurable liquid-core/liquid-cladding optical waveguides with dielectrophoresis-driven virtual microchannels on an electromicrofluidic platform

paper_content:

An electrically reconfigurable liquid-core/liquid-cladding (L2) optical waveguide with core liquid γ-butyrolactone (GBL, ncore = 1.4341, ecore = 39) and silicone oil (ncladding = 1.401, ecladding = 2.5) as cladding liquid is accomplished using dielectrophoresis (DEP) that attracts and deforms the core liquid with the greater permittivity to occupy the region of strong electric field provided by Teflon-coated ITO electrodes between parallel glass plates. Instead of continuously flowing core and cladding liquids along a physical microchannel, the DEP-formed L2 optical waveguide guides light in a stationary virtual microchannel that requires liquids of limited volume without constant supply and creates stable liquid/liquid interfaces for efficient light guidance in a simply fabricated microfluidic device. We designed and examined (1) stationary and (2) moving L2 optical waveguides on the parallel-plate electromicrofluidic platform. In the stationary L-shaped waveguide, light was guided in a GBL virtual microchannel core for a total of 27.85 mm via a 90° bend (radius 5 mm) before exiting from the light outlet of cross-sectional area 100 μm × 100 μm. For the stationary spiral waveguide, light was guided in a GBL core containing Rhodamine 6G (R6G, 1 mM) and through a series of 90° bends with decreasing radii from 5 mm to 2.5 mm. With the stationary straight waveguide, the propagation loss was measured to be 2.09 dB cm−1 in GBL with R6G (0.01 mM). The moving L-shaped waveguide was implemented on a versatile electromicrofluidic platform on which electrowetting and DEP were employed to generate a precise GBL droplet and form a waveguide core. On sequentially applying appropriate voltage to one of three parallel L-shaped driving electrodes, the GBL waveguide core was shifted; the guided light was switched at a speed of up to 0.929 mm s−1 (switching period 70 ms, switching rate 14.3 Hz) when an adequate electric signal (173.1 VRMS, 100 kHz) was applied.
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paper_title: A Solid-Cladding/Liquid-Core/Liquid-Cladding Sandwich Optical Waveguide for the Study of Dynamic Extraction of Dye by Ionic Liquid BmimPF6

paper_content:

We present a sandwich liquid-core waveguide for the study of the dynamic extraction of dye molecules by ionic liquid (IL). The incident light was introduced into the core of the IL phase directly via a fused silica prism to form total reflection at the IL/cladding interface. The changes in light intensity and absorbance spectra over time were monitored during the extraction process by maintaining an optical focus on the IL/water interface. The dynamic extraction of the dye Eriochrome Black-T (EBT) by the IL 1-n-butyl-3-methylimidazolium hexafluorophosphate (BmimPF6) at different concentrations and various pH conditions was investigated. The results indicated that the accumulation of the dye at the IL/water interface and the dynamic extraction of the dye into the IL phase both contributed to the increase in absorbance. The increased signal along with the increase in pH suggests that electrostatic interaction might be the main driving force for the extraction of EBT by BmimPF6, and the micro-circumstance provided by IL also has a decisive effect on the characteristics of the spectra recorded by the waveguide system.
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paper_title: Fabrication and Performance of a Photonic-Microfluidic Integrated Device

paper_content:

Fabrication and performance of a functional photonic-microfluidic flow cytometer is demonstrated. The devices are fabricated on a Pyrex substrate by photolithographically patterning the microchannels and optics in a SU-8 layer that is sealed via a poly(dimethylsiloxane) (PDMS) layer through a unique chemical bonding method. The resulting devices eliminate the free-space excitation optics through integration of microlenses onto the chip to mimic conventional cytometry excitation. Devices with beam waists of 6 μm and 12 μm in fluorescent detection and counting tests using 2.5 and 6 μm beads-show CVs of 9%–13% and 23% for the two devices, respectively. These results are within the expectations for a conventional cytometer (5%–15%) and demonstrate the ability to integrate the photonic components for excitation onto the chip and the ability to maintain the level of reliable detection.
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paper_title: A tunable 3D optofluidic waveguide dye laser via two centrifugal Dean flow streams

paper_content:

This paper presents a tunable optofluidic waveguide dye laser utilizing two centrifugal Dean flows. The centrifugal Dean flow increases the light confinement of the dye laser by shaping a three-dimensional (3D) liquid waveguide from curved microchannels. The active medium with the laser dye is dissolved in the liquid core and pumped with an external pump laser to produce stimulated emission. The laser's Fabry–Perot microcavity is formed with a pair of aligned gold-coated fiber facets to amplify the fluorescent emission. The advantage of the 3D optofluidic waveguide dye laser is its higher efficiency, thus to obtain lasing at a reduced threshold (60%) with higher output energy. The demonstrated slope efficiency is at least 3-fold higher than its traditional two-dimensional equivalent. In addition, the laser output energy can be varied on demand by tuning the flow rates of the two flows. This technique provides a versatile platform for high potential applications microfluidic biosensor and bioanalysis.
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paper_title: Integration of optical components on-chip for scattering and fluorescence detection in an optofluidic device

paper_content:

An optofluidic device is demonstrated with photonic components integrated onto the chip for use in fluorescence and scatter detection and counting applications. The device is fabricated by integrating the optical and fluidic components in a single functional layer. Optical excitation on-chip is accomplished via a waveguide integrated with a system of lenses that reforms the geometry of the beam in the microfluidic channel into a specific shape that is more suitable for reliable detection. Separate counting tests by detecting fluorescence and scattered signals from 2.5 and 6.0 μm beads were performed and found to show detection reliability comparable to that of conventional means of excitation and an improvement over other microchip-based designs.
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paper_title: Scattering detection using a photonic-microfluidic integrated device with on-chip collection capabilities.

paper_content:

SU-8-based photonic-microfluidic integrated devices with on-chip beam shaping and collection capabilities were demonstrated in a scattering detection and counting application. Through the proper deployment of the tailored beam geometries via the on-chip excitation optics, excellent CV values were measured for 1, 2, and 5 μm blank beads, 16.4, 11.0, and 12.5%, respectively, coupled with a simple free-space optical detection scheme. The performance of these devices was found dependent on the combination of on-chip, lens-shaped beam geometry and bead size. While very low CVs were obtained when the combination was ideal, a nonideal combination could still result in acceptable CVs for flow cytometry; the reliability was confirmed via devices being able to resolve separate populations of 2.0 and 5.0 μm beads from their mixture with low CV values of 15.9 and 18.5%, respectively. On-chip collection using integrated on-chip optical waveguides was shown to be very reliable in comparison with a free-space collection scheme, yielding a coincident rate of 94.2%. A CV as low as 19.2% was obtained from the on-chip excitation and collection of 5 μm beads when the on-chip lens-shaped beam had a 6.0-μm beam waist.
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paper_title: Formation and characterization of an ideal excitation beam geometry in an optofluidic device

paper_content:

An optimal excitation beam shape is necessary to perform reliable flow cytometric analysis but has so far not been implemented in a photonic-microfluidic integrated (i.e. optofluidic) device. We have achieved this feature by integrating a 1D lens system with planar waveguides and microfluidic channel on a substrate using one patterning material via a one-shot process. In this paper, we report the method of design and the performance of specifically formed excitation regions shaped to be ideal for reducing double detections, improving SNR, and for reliable detection in a flow cytometry application. Demonstration of different sizes via changes to lens design shows the ability to control the width of the shaped beam according to a targeted detection.
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paper_title: A photonic-microfluidic integrated device for reliable fluorescence detection and counting.

paper_content:

A photonic-microfluidic integrated device is demonstrated with excellent and reliable fluorescence detection performance. CV values of 8% for 2.5-μm beads and 14% for 6-μm beads were achieved through the correct deployment of carefully formed excitation beam shapes via integrated on-chip optics even without the use of 3D hydrodynamic focusing or a high-quality laser source and single mode beam propagation. The devices are fabricated in a monolithic planar fashion using a system of microlenses and waveguides integrated with microfluidic channels on-chip and packaged using a high-quality and low-cost channel sealing and high-performance interconnecting technology developed from our earlier works. Beam geometry in the excitation region is shown to affect the variation of fluorescence intensity from specimens, hence configurations of beam geometry targeted for a specific bead sizes are examined to ensure proper deployment of the lens designs. The formed high-quality optical excitation regions ensure reliable detection even with relaxed hydrodynamic focusing to ensure applicability with multiple specimen sizes. Device performance with each bead size was found to be acceptable for a range of beam geometries with a different ideal configuration for each bead size. These device designs help to form a device that will supplement conventional flow cytometry in point-of-care and remote detection applications by performing specific detections with an inexpensive and replaceable device.
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paper_title: Fabrication and Performance of a Photonic-Microfluidic Integrated Device

paper_content:

Fabrication and performance of a functional photonic-microfluidic flow cytometer is demonstrated. The devices are fabricated on a Pyrex substrate by photolithographically patterning the microchannels and optics in a SU-8 layer that is sealed via a poly(dimethylsiloxane) (PDMS) layer through a unique chemical bonding method. The resulting devices eliminate the free-space excitation optics through integration of microlenses onto the chip to mimic conventional cytometry excitation. Devices with beam waists of 6 μm and 12 μm in fluorescent detection and counting tests using 2.5 and 6 μm beads-show CVs of 9%–13% and 23% for the two devices, respectively. These results are within the expectations for a conventional cytometer (5%–15%) and demonstrate the ability to integrate the photonic components for excitation onto the chip and the ability to maintain the level of reliable detection.
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paper_title: A method for detecting forward scattering signals on-chip with a photonic-microfluidic integrated device

paper_content:

A photonic integrated microfluidic device is demonstrated to perform optical excitation and forward scatter collection all on-chip in a planar format. Integrated on-chip optics formed a tailored beam geometry for optimal excitation of particles while a special design modification allowed for on-chip forward collection with the beam shaping capabilities. A notch was placed in the lens system that caused a dark spot on the facet of a collection waveguide while not affecting the beam geometry at the point of interrogation. The modified device with the ability to form a 10 μm beam geometry was demonstrated to detect the forward scatter from blank 5 μm diameter polystyrene beads. Free-space collection of side scatter signals was performed simultaneously with the on-chip collection and the designs demonstrated and enhanced SNR while the reliability of detection was determined to be appropriate for many applications. Excellent performance was confirmed via a false positive rate of 0.4%, a missed events rate of 6.8%, and a coincident rate of 96.3% as determined between simultaneously performed free-space and on-chip detection schemes.
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paper_title: The good, the bad, and the tiny: a review of microflow cytometry

paper_content:

Recent developments in microflow cytometry have concentrated on advancing technology in four main areas: (1) focusing the particles to be analyzed in the microfluidic channel, (2) miniaturization of the fluid-handling components, (3) miniaturization of the optics, and (4) integration and applications development. Strategies for focusing particles in a narrow path as they pass through the detection region include the use of focusing fluids, nozzles, and dielectrophoresis. Strategies for optics range from the use of microscope objectives to polymer waveguides or optical fibers embedded on-chip. While most investigators use off-chip fluidic control, there are a few examples of integrated valves and pumps. To date, demonstrations of applications are primarily used to establish that the microflow systems provide data of the same quality as laboratory systems, but new capabilities—such as automated sample staining—are beginning to emerge. Each of these four areas is discussed in detail in terms of the progress of development, the continuing limitations, and potential future directions for microflow cytometers.
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paper_title: Flexible planar microfluidic chip employing a light emitting diode and a PIN-photodiode for portable flow cytometers

paper_content:

Detection of fluorescence particles is a key method of flow cytometry. We evaluate the performance of a design for a microfluidic fluorescence particle detection device. Due to the planar design with low layer thicknesses, we avoid optical components such as lenses or dichroic mirrors and substitute them with a shadow mask and colored film filters. A commercially available LED is used as the light source and a PIN-photodiode as detector. This design approach reduces component cost and power consumption and enables supplying the device with power from a standard USB port. From evaluation of this design, we obtain a maximum particle detection frequency of up to 600 particles per second at a sensitivity of better than 4.7 × 105 MESF (molecules of equivalent soluble fluorochrome) measured with particles for FITC sensitivity calibration. Lowering the flow rate increases the instrument sensitivity by an order of magnitude enabling the detection of particles with 4.5 × 104 MESF.
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paper_title: PDMS-based opto-fluidic micro flow cytometer with two-color, multi-angle fluorescence detection capability using PIN photodiodes

paper_content:

This research describes the design and performance of a microfluidic observation channel capable of performing flow cytometry measurements using solid-state lasers and PIN-based photodetectors. The observation cell, fabricated using poly(dimethylsiloxane) (PDMS)-based soft lithography, is designed with integrated microgrooves that permit the physical registration of optical fibers that serve as optical waveguides for laser excitation and fluorescence detection. The optical fiber arrangement is designed to permit simultaneous multi-color excitation of sample particles, at a single interrogation point in the observation channel, using multiple angles of excitation and detection. The multiple angles can be used to increase signal-to-noise ratio ( S/N) by performing two independent fluorescence measurements simultaneously, and to permit multiple simultaneous cytometric measurements to occur at a single interrogation point. Moreover, the multi-angle micro flow cytometer is proven to provide consistent color differentiation that allows the design to achieve spectrometer capabilities not possible on a conventional flow cytometer. The micro flow cytometer performance was evaluated by considering the detection of fluorescent microspheres (15m) and nucleic acid labeled fungus (Saccharomyces cerevisiae). Multiple angles (45 ◦ , 135 ◦ , and 180 ◦ ) between fluorescence excitation and detection were investigated with respect to S/N, S/N coefficient of variation, and particle recovery. The experiments demonstrate the feasibility of developing a high S/N single-cell fluorescence detection system based exclusively on silicon-based PIN photodiodes coupled with the lock-in amplification technique. The technique is applicable to the detection of scatter and fluorescent signals emanating from inorganic particles and biological cells, and is highly amenable to economical manufacturing, miniaturization, and low-power operation. Consequently, the design approach and performance results described in this investigation have applicability to the development of practical and low-cost cytometric micro total analysis systems (TAS). © 2003 Elsevier B.V. All rights reserved.
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paper_title: A microfluidic device for continuous, real time blood plasma separation

paper_content:

A microfluidic device for continuous, real time blood plasma separation is introduced. The principle of the blood plasma separation from blood cells is supported by the Zweifach–Fung effect and was experimentally demonstrated using simple microchannels. The blood plasma separation device is composed of a blood inlet, a bifurcating region which leads to a purified plasma outlet, and a concentrated blood cell outlet. It was designed to separate blood plasma from an initial blood sample of up to 45% inlet hematocrit (volume percentage of cells). The microfluidic network was designed using an analogous electrical circuit, as well as analytical and numerical studies. The functionality of this device was demonstrated using defibrinated sheep blood. During 30 minutes of continuous blood infusion through the device, all the erythrocytes (red blood cells) traveled through the device toward the concentrated blood outlet while only the plasma was separated at the bifurcating regions and flowed towards the plasma outlet. The device has been operated continuously without any clogging or hemolysis of cells. The experimentally determined plasma selectivity with respect to blood hematocrit level was almost 100% regardless of the inlet hematocrit. The total plasma separation volume percent varied from 15% to 25% with increasing inlet hematocrit. Due to the device’s simple structure and control mechanism, this microdevice is expected to be used for highly efficient continuous, real time cell-free blood plasma separation from blood samples for use in lab on a chip applications.
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paper_title: An integrated microfluidic chip enabling control and spatially resolved monitoring of temperature in micro flow reactors

paper_content:

A strength of microfluidic chip laboratories is the rapid heat transfer that, in principle, enables a very homogeneous temperature distribution in chemical processes. In order to exploit this potential, we present an integrated chip system where the temperature is precisely controlled and monitored at the microfluidic channel level. This is realized by integration of a luminescent temperature sensor layer into the fluidic structure together with inkjet-printed micro heating elements. This allows steering of the temperature at the microchannel level and monitoring of the reaction progress simultaneously. A fabrication procedure is presented that allows for straightforward integration of thin polymer layers with optical sensing functionality in microchannels of glass–polydimethylsiloxane (PDMS) chips of only 150 μm width and 29 μm height. Sensor layers consisting of polyacrylonitrile and a temperature-sensitive ruthenium tris-phenanthroline probe with film thicknesses of about 0.5 to 6 μm were generated by combining blade coating and abrasion techniques. Optimal coating procedures were developed and evaluated. The chip-integrated sensor layers were calibrated and investigated with respect to stability, reproducibility, and response times. These microchips allowed observation of temperature in a wide range with a signal change of around 1.6 % per K and a maximum resolution of around 0.07 K. The device is employed to study temperature-controlled continuous micro flow reactions. This is demonstrated exemplarily for the tryptic cleavage of coumarin-modified peptides via fluorescence detection.
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paper_title: Microfluidics for flow cytometric analysis of cells and particles.

paper_content:

This review describes recent developments in microfabricated flow cytometers and related microfluidic devices that can detect, analyze, and sort cells or particles. The high-speed analytical capabilities of flow cytometry depend on the cooperative use of microfluidics, optics and electronics. Along with the improvement of other components, replacement of conventional glass capillary-based fluidics with microfluidic sample handling systems operating in microfabricated structures enables volume- and power-efficient, inexpensive and flexible analysis of particulate samples. In this review, we present various efforts that take advantage of novel microscale flow phenomena and microfabrication techniques to build microfluidic cell analysis systems.
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paper_title: A simple low-cost method to enhance luminescence and fluorescence signals in PDMS-based microfluidic devices

paper_content:

We present a simple and low-cost method to increase the signal intensity originating from poly(dimethyl)siloxane (PDMS) based microfluidic devices by modifying the optical properties of the polymer. The addition of titanium dioxide results in an opaque, white composite with enhanced light reflection properties. The composite can be used to increase the collected light from fluorescent and luminescent moieties inside microfluidic channels by up to 11-fold, as well as reducing the background signal in luminescence studies. Both of these effects increase sensitivity and lower detection limits in analytical setups based on PDMS microfluidic devices. Furthermore, the production of the composite material is simple, low-cost and can be implemented in standard PDMS device fabrication.
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paper_title: Microfluidic oxygen imaging using integrated optical sensor layers and a color camera

paper_content:

In this work we present a high resolution oxygen imaging approach, which can be used to study 2D oxygen distribution inside microfluidic environments. The presented setup comprises a fabrication process of microfluidic chips with integrated luminescent sensing films combined with referenced oxygen imaging applying a color CCD-camera. Enhancement of the sensor performance was achieved by applying the principle of light harvesting. This principle enabled ratiometric imaging employing the red and the green channel of a color CCD-camera. The oxygen sensitive emission of platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorphenyl)-porphyrin (PtTFPP) was detected by the red channel, while the emission of a reference dye was detected by the green channel. This measurement setup allowed for accurate real-time 2D oxygen imaging with superior quality compared to intensity imaging. The sensor films were subsequently used to measure the respiratory activity of human cell cultures (HeLa carcinoma cells and normal human dermal fibroblasts) in a microfluidic system. The sensor setup is well suited for different applications from spatially and temporally resolving oxygen concentration inside microfluidic channels to parallelization of oxygen measurements and paves the way to novel cell based assays, e.g. in tissue engineering, tumor biology and hypoxia reperfusion phenomena.
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paper_title: Time-Domain Microfluidic Fluorescence Lifetime Flow Cytometry for High-Throughput Förster Resonance Energy Transfer Screening

paper_content:

Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Forster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5–5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving fluorescence lifetime measurements, thereby providing statistically significant quantitative data for analysis of large cell populations. © 2014 International Society for Advancement of Cytometry
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paper_title: Highly efficient dual-channel cytometric-detection of micron-sized particles in microfluidic device

paper_content:

Abstract To demonstrate the ability to efficiently count and identify suspended micron-sized particles by simultaneously detecting their fluorescence emission and light scattering in microfabricated channel, a compact configuration that used a polydimethylsiloxane (PDMS) microfabricated channel as interrogation component, hydrodynamic focusing for particle control, and a simple free-space optical setup for signal detection, was accordingly developed. Subsequently, a quantitative count of 1.013 μm diameter fluorescently labeled beads in suspension was implemented in a microfluidic device employing both fluorescence emission and light scattering at average particle throughput ranging from 83 to 416 particles/s. As a result, the detection efficiencies above 88% for both signals and correlation percentages above 97% between them were routinely achieved. In addition, it was shown that effective differentiation of 1.013 μm fluorescently labeled beads from various unlabeled beads in mixed populations of high mixing ratios had been successfully realized in this microfluidic-device-based instrumentation. Finally, the demonstrated system was used to detect fluorescein isothiocyanate (FITC) labeled nonpathogenic bacteria of Escherichia coli ( E. coli ) DH5α. The results showed the detection efficiencies above 89.7% for fluorescence emission and 94.5% for light scattering signals, and a correlation of 94.9% between the two signals at an average throughput of 350 cells/s have been obtained. As a comparison, the detection accuracies of the dual-channel cytometric detection of the FITC-labeled E. coli DH5α cells in the microfluidic device are approximately 84.3% and 88.8% for fluorescence emission and light scattering respectively when compared against a manual cell count using a haemocytometer as a standard.
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paper_title: Sealing SU-8 microfluidic channels using PDMS

paper_content:

A simple method of irreversibly sealing SU-8 microfluidic channels using PDMS is reported in this paper. The method is based on inducing a chemical reaction between PDMS and SU-8 by first generating amino groups on PDMS surface using N2 plasma treatment, then allowing the amino groups to react with the residual epoxy groups on SU-8 surface at an elevated temperature. The N2 plasma treatment of PDMS can be conducted using an ordinary plasma chamber and high purity N2, while the residual epoxy groups on SU-8 surface can be preserved by post-exposure baking SU-8 at a temperature no higher than 95 °C. The resultant chemical bonding between PDMS and SU-8 using the method create an interface that can withstand a stress that is greater than the bulk strength of PDMS. The bond is permanent and is long-term resistant to water. The method was applied in fabricating SU-8 microfluidi-photonic integrated devices, and the obtained devices were tested to show desirable performance.
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paper_title: A Simple and Reliable PDMS and SU-8 Irreversible Bonding Method and Its Application on a Microfluidic-MEA Device for Neuroscience Research

paper_content:

Polydimethylsiloxane (PDMS) and SU-8 are currently two very commonly used polymeric materials in the microfluidics field for biological applications. However; there is a pressing need to find a simple, reliable, irreversible bonding method between these two materials for their combined use in innovative integrated microsystems. In this paper, we attempt to investigate the aminosilane-mediated irreversible bonding method for PDMS and SU-8 with X-Ray Photoelectron Spectroscopy (XPS) surface analysis and bonding strength tests. Additionally, the selected bonding method was applied in fabricating a microelectrode array (MEA) device, including microfluidic features, which allows electrophysiological observations on compartmentalized neuronal cultures. As there is a growing trend towards microfluidic devices for neuroscience research, this type of integrated microdevice, which can observe functional alterations on compartmentalized neuronal culture, can potentially be used for neurodegenerative disease research and pharmaceutical development.
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paper_title: Advances in Microfluidic Materials, Functions, Integration, and Applications

paper_content:

Microfluidics consist of microfabricated structures for liquid handling, with cross-sections in the 1–500 μm range, and small volume capacity (fL-nL). Capillary tubes connected with fittings,1 although utilizing small volumes, are not considered microfluidics for the purposes of this paper since they are not microfabricated. Likewise, millifluidic systems, made by conventional machining tools, are excluded due to their larger feature sizes (>500 μm). ::: ::: Though micromachined systems for gas chromatography were introduced in the 1970’s,2 the field of microfluidics did not gain much traction until the 1990’s.3 Silicon and glass were the original materials used, but then the focus shifted to include polymer substrates, and in particular, polydimethylsiloxane (PDMS). Since then the field has grown to encompass a wide variety of materials and applications. The successful demonstration of electrophoresis and electroosmotic pumping in a microfluidic device provided a nonmechanical method for both fluid control and separation.4 Laser induced fluorescence (LIF) enabled sensitive detection of fluorophores or fluorescently labeled molecules. The expanded availability of low-cost printing allowed for cheaper and quicker mask fabrication for use in soft lithography.5 Commercial microfluidic systems are now available from Abbott, Agilent, Caliper, Dolomite, Micralyne, Microfluidic Chip Shop, Micrux Technologies and Waters, as a few prominent examples. For a more thorough description of the history of microfluidics, we refer the reader to a number of comprehensive, specialized reviews,3, 6–11 as well as a more general 2006 review.12 ::: ::: The field of microfluidics offers many advantages compared to carrying out processes through bulk solution chemistry, the first of which relates to a lesson taught to every first-year chemistry student. Simply stated, diffusion is slow! Thus, the smaller the distance required for interaction, the faster it will be. Smaller channel dimensions also lead to smaller sample volumes (fL-nL), which can reduce the amount of sample or reagents required for testing and analysis. Reduced dimensions can also lead to portable devices to enable on-site testing (provided the associated hardware is similarly portable). Finally, integration of multiple processes (like labeling, purification, separation and detection) in a microfluidic device can be highly enabling for many applications. ::: ::: Microelectromechanical systems (MEMS) contain integrated electrical and mechanical parts that create a sensor or system. Applications of MEMS are ubiquitous, including automobiles, phones, video games and medical and biological sensors.13 Micro-total analysis systems, also known as labs-on-a-chip, are the chemical analogue of MEMS, as integrated microfluidic devices that are capable of automating multiple processes relevant to laboratory sciences. For example, a typical lab-on-a-chip system might selectively purify a complex mixture (through filtering, antibody capture, etc.), then separate target components and detect them. ::: ::: Microfluidic devices consist of a core of common components. Areas defined by empty space, such as reservoirs (wells), chambers and microchannels, are central to microfluidic systems. Positive features, created by areas of solid material, add increased functionality to a chip and can consist of membranes, monoliths, pneumatic controls, beams and pillars. Given the ubiquitous nature of negative components, and microchannels in particular, we focus here on a few of their properties. Microfluidic channels have small overall volumes, laminar flow and a large surface-to-volume ratio. Dimensions of a typical separation channel in microchip electrophoresis (μCE) are: 50 μm width, 15 μm height and 5 cm length for a volume of 37.5 nL. Flow in these devices is normally nonturbulent due to low Reynolds numbers. For example, water flowing at 20°C in the above channel at 1 μL/min (2.22 cm/s) results in a Reynolds number of ~0.5, where <2000 is laminar flow. Since flow is nonturbulent, mixing is normally diffusion-limited. Small channel sizes also have a high surface-to-volume ratio, leading to different characteristics from what are commonly found in bulk volumes. The material surface can be used to manipulate fluid movement (such as by electroosmotic flow, EOF) and surface interactions. For a solution in contact with a charged surface, a double layer of charge is created as oppositely charged ions are attracted to the surface charges. This electrical double layer consists of an inner rigid or Stern Layer and an outer diffuse layer. An electrostatic potential known as the zeta potential is formed, with the magnitude of the potential decreasing as distance from the surface increases. The electrical double layer is the basis for EOF, wherein an applied voltage causes the loosely bound diffuse layer to move towards an electrode, dragging the bulk solution along. Charges on the exposed surface also exert a greater influence on the fluid in a channel as its size decreases. Larger surface-to-volume ratios are more prone to nonspecific adsorption and surface fouling. In particular, non-charged and hydrophobic microdevice surfaces can cause proteins in solution to denature and stick. ::: ::: We focus our review on advances in microfluidic systems since 2008. In doing this, we occasionally must cover foundational work in microfluidics that is considerably less recent. We do not focus on chemical synthesis applications of microfluidics although it is an expanding area, nor do we delve into lithography, device fabrication or production costs. Our specific emphasis herein is on four areas within microfluidics: properties and applications of commonly used materials, basic functions, integration, and selected applications. For each of these four topics we provide a concluding section on opportunities for future development, and at the end of this review, we offer general conclusions and prospective for future work in the field. Due to the considerable scope of the field of microfluidics, we limit our discussion to selected examples from each area, but cite in-depth reviews for the reader to turn to for further information about specific topics. We also refer the reader to recent comprehensive reviews on advances in lab-on-a-chip systems by Arora et al.10 and Kovarik et al.14
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paper_title: Formation and characterization of an ideal excitation beam geometry in an optofluidic device

paper_content:

An optimal excitation beam shape is necessary to perform reliable flow cytometric analysis but has so far not been implemented in a photonic-microfluidic integrated (i.e. optofluidic) device. We have achieved this feature by integrating a 1D lens system with planar waveguides and microfluidic channel on a substrate using one patterning material via a one-shot process. In this paper, we report the method of design and the performance of specifically formed excitation regions shaped to be ideal for reducing double detections, improving SNR, and for reliable detection in a flow cytometry application. Demonstration of different sizes via changes to lens design shows the ability to control the width of the shaped beam according to a targeted detection.
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paper_title: Three-Dimensional Paper Microfluidic Devices Assembled Using the Principles of Origami

paper_content:

We report a method, based on the principles of origami (paper folding), for fabricating three-dimensional (3-D) paper microfluidic devices. The entire 3-D device is fabricated on a single sheet of flat paper in a single photolithographic step. It is assembled by simply folding the paper by hand. Following analysis, the device can be unfolded to reveal each layer. The applicability of the device to chemical analysis is demonstrated by colorimetric and fluorescence assays using multilayer microfluidic networks.
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paper_title: Lab-on-chip technologies: making a microfluidic network and coupling it into a complete microsystem—a review

paper_content:

Microfluidics is an emerging field that has given rise to a large number of scientific and technological developments over the last few years. This review reports on the use of various materials, such as silicon, glass and polymers, and their related technologies for the manufacturing of simple microchannels and complex systems. It also presents the main application fields concerned with the different technologies and the most significant results reported by academic and industrial teams. Finally, it demonstrates the advantage of developing approaches for associating polymer technologies for manufacturing of fluidic elements with integration of active or sensitive elements, particularly silicon devices.
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paper_title: A Simple and Reliable PDMS and SU-8 Irreversible Bonding Method and Its Application on a Microfluidic-MEA Device for Neuroscience Research

paper_content:

Polydimethylsiloxane (PDMS) and SU-8 are currently two very commonly used polymeric materials in the microfluidics field for biological applications. However; there is a pressing need to find a simple, reliable, irreversible bonding method between these two materials for their combined use in innovative integrated microsystems. In this paper, we attempt to investigate the aminosilane-mediated irreversible bonding method for PDMS and SU-8 with X-Ray Photoelectron Spectroscopy (XPS) surface analysis and bonding strength tests. Additionally, the selected bonding method was applied in fabricating a microelectrode array (MEA) device, including microfluidic features, which allows electrophysiological observations on compartmentalized neuronal cultures. As there is a growing trend towards microfluidic devices for neuroscience research, this type of integrated microdevice, which can observe functional alterations on compartmentalized neuronal culture, can potentially be used for neurodegenerative disease research and pharmaceutical development.
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paper_title: Formation and characterization of an ideal excitation beam geometry in an optofluidic device

paper_content:

An optimal excitation beam shape is necessary to perform reliable flow cytometric analysis but has so far not been implemented in a photonic-microfluidic integrated (i.e. optofluidic) device. We have achieved this feature by integrating a 1D lens system with planar waveguides and microfluidic channel on a substrate using one patterning material via a one-shot process. In this paper, we report the method of design and the performance of specifically formed excitation regions shaped to be ideal for reducing double detections, improving SNR, and for reliable detection in a flow cytometry application. Demonstration of different sizes via changes to lens design shows the ability to control the width of the shaped beam according to a targeted detection.
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paper_title: Standing surface acoustic wave ( SSAW )-based microfluidic cytometer †

paper_content:

Trapping and sorting of micro-sized objects is one important application of lab on a chip devices, with the use of acoustic bubbles emerging as an effective, non-contact method. Acoustically actuated bubbles are known to exert a secondary radiation force (FSR) on micro-particles and stabilize them on the bubble surface, when this radiation force exceeds the external hydrodynamic forces that act to keep the particles in motion. While the theoretical expression of FSR has been derived by Nyborg decades ago, no direct experimental validation of this force has been performed, and the relationship between FSR and the bubble's ability to trap particles in a given lab on a chip device remains largely empirical. In order to quantify the connection between the bubble oscillation and the resultant FSR, we experimentally measure the amplitude of bubble oscillations that give rise to FSR and observe the trapping and release of a single microsphere in the presence of the mean flow at the corresponding acoustic parameters using an acoustofluidic device. By combining well-developed theories that connect bubble oscillations to the acoustic actuation, we derive the expression for the critical input voltage that leads to particle release into the flow, in good agreement with the experiments.
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paper_title: Highly efficient dual-channel cytometric-detection of micron-sized particles in microfluidic device

paper_content:

Abstract To demonstrate the ability to efficiently count and identify suspended micron-sized particles by simultaneously detecting their fluorescence emission and light scattering in microfabricated channel, a compact configuration that used a polydimethylsiloxane (PDMS) microfabricated channel as interrogation component, hydrodynamic focusing for particle control, and a simple free-space optical setup for signal detection, was accordingly developed. Subsequently, a quantitative count of 1.013 μm diameter fluorescently labeled beads in suspension was implemented in a microfluidic device employing both fluorescence emission and light scattering at average particle throughput ranging from 83 to 416 particles/s. As a result, the detection efficiencies above 88% for both signals and correlation percentages above 97% between them were routinely achieved. In addition, it was shown that effective differentiation of 1.013 μm fluorescently labeled beads from various unlabeled beads in mixed populations of high mixing ratios had been successfully realized in this microfluidic-device-based instrumentation. Finally, the demonstrated system was used to detect fluorescein isothiocyanate (FITC) labeled nonpathogenic bacteria of Escherichia coli ( E. coli ) DH5α. The results showed the detection efficiencies above 89.7% for fluorescence emission and 94.5% for light scattering signals, and a correlation of 94.9% between the two signals at an average throughput of 350 cells/s have been obtained. As a comparison, the detection accuracies of the dual-channel cytometric detection of the FITC-labeled E. coli DH5α cells in the microfluidic device are approximately 84.3% and 88.8% for fluorescence emission and light scattering respectively when compared against a manual cell count using a haemocytometer as a standard.
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paper_title: Simultaneous high speed optical and impedance analysis of single particles with a microfluidic cytometer.

paper_content:

We describe a microfluidic cytometer that performs simultaneous optical and electrical characterisation of particles. The microfluidic chip measures side scattered light, signal extinction and fluorescence using integrated optical fibres coupled to photomultiplier tubes. The channel is 80 μm high and 200 μm wide, and made from SU-8 patterned and sandwiched between glass substrates. Particles were focused into the analysis region using 1-D hydrodynamic focusing and typical particle velocities were 0.1 ms(-1). Excitation light is coupled into the detection channel with an optical fibre and focused into the channel using an integrated compound air lens. The electrical impedance of particles is measured at 1 MHz using micro-electrodes fabricated on the channel top and bottom. This data is used to accurately size the particles. The system is characterised using a range of different sized polystyrene beads (fluorescent and non-fluorescent). Single and mixed populations of beads were measured and the data compared with a conventional flow cytometer.
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paper_title: Scattering detection using a photonic-microfluidic integrated device with on-chip collection capabilities.

paper_content:

SU-8-based photonic-microfluidic integrated devices with on-chip beam shaping and collection capabilities were demonstrated in a scattering detection and counting application. Through the proper deployment of the tailored beam geometries via the on-chip excitation optics, excellent CV values were measured for 1, 2, and 5 μm blank beads, 16.4, 11.0, and 12.5%, respectively, coupled with a simple free-space optical detection scheme. The performance of these devices was found dependent on the combination of on-chip, lens-shaped beam geometry and bead size. While very low CVs were obtained when the combination was ideal, a nonideal combination could still result in acceptable CVs for flow cytometry; the reliability was confirmed via devices being able to resolve separate populations of 2.0 and 5.0 μm beads from their mixture with low CV values of 15.9 and 18.5%, respectively. On-chip collection using integrated on-chip optical waveguides was shown to be very reliable in comparison with a free-space collection scheme, yielding a coincident rate of 94.2%. A CV as low as 19.2% was obtained from the on-chip excitation and collection of 5 μm beads when the on-chip lens-shaped beam had a 6.0-μm beam waist.
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paper_title: Microflow Cytometers with Integrated Hydrodynamic Focusing

paper_content:

This study demonstrates the suitability of microfluidic structures for high throughput blood cell analysis. The microfluidic chips exploit fully integrated hydrodynamic focusing based on two different concepts: Two-stage cascade focusing and spin focusing (vortex) principle. The sample--A suspension of micro particles or blood cells--is injected into a sheath fluid streaming at a substantially higher flow rate, which assures positioning of the particles in the center of the flow channel. Particle velocities of a few m/s are achieved as required for high throughput blood cell analysis. The stability of hydrodynamic particle positioning was evaluated by measuring the pulse heights distributions of fluorescence signals from calibration beads. Quantitative assessment based on coefficient of variation for the fluorescence intensity distributions resulted in a value of about 3% determined for the micro-device exploiting cascade hydrodynamic focusing. For the spin focusing approach similar values were achieved for sample flow rates being 1.5 times lower. Our results indicate that the performances of both variants of hydrodynamic focusing suit for blood cell differentiation and counting. The potential of the micro flow cytometer is demonstrated by detecting immunologically labeled CD3 positive and CD4 positive T-lymphocytes in blood.
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paper_title: Sub-micrometer-precision, three-dimensional (3D) hydrodynamic focusing via “microfluidic drifting”

paper_content:

In this article, we demonstrate single-layered, “microfluidic drifting” based three-dimensional (3D) hydrodynamic focusing devices with particle/cell focal positioning approaching submicron precision along both lateral and vertical directions. By systematically optimizing channel geometries and sample/sheath flow rates, a series of “microfluidic drifting” based 3D hydrodynamic focusing devices with different curvature angles are designed and fabricated. Their performances are then evaluated using confocal microscopy, fast camera imaging, and side-view imaging techniques. Using a device with a curvature angle of 180°, we have achieved a standard deviation of ±0.45 μm in particle focal position and a coefficient of variation (CV) of 2.37% in flow cytometric measurements. To the best of our knowledge, this is the best CV that has been achieved using a microfluidic flow cytometry device. Moreover, the device showed the capability to distinguish 8 peaks when subjected to a stringent 8-peak rainbow calibration test, signifying the ability to perform sensitive, accurate tests similar to commercial flow cytometers. We have further tested and validated our device by detection of HEK-293 cells. With its advantages in simple fabrication (i.e., single-layered device), precise 3D hydrodynamic focusing (i.e., submicrometer precision along both lateral and vertical directions), and high detection resolution (i.e., low CV), our method could serve as an important basis for high-performance, mass-producible microfluidic flow cytometry.
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paper_title: Optical microflow cytometer for particle counting, sizing and fluorescence detection

paper_content:

A novel on-chip optical flow cytometer concept is reported for fluorescence detection, enumeration, and sizing of microparticles in a poly-dimethylsiloxane (PDMS) microchip. The detection system integrates a pair of external optical fibers and other optical components for particle counting, sizing and fluorescence analysis in each measurement simultaneously. The scattered light signal indicates the total number and the size of the particles passing through the detection window, whereas the concurrent backward fluorescence signal shows only the number of fluorescence particles. In the experiments, microparticles of four different sizes with diameters ranging from 3.2 to 10.2 µm were discriminated and counted based on the fluorescence and scattered light intensity. The relative percentage of the fluorescence-labeled particles can be analyzed by the ratio of the events of fluorescence signals to forward scattered signals.
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paper_title: Micro flow cytometer with self-aligned 3D hydrodynamic focusing

paper_content:

A micro flow cytometer with a single step 3D hydrodynamic flow focusing has been developed. The proposed design is capable to create a single-file particle stream that is self-aligned with an integrated optical fiber-based detection system, regardless of the flow rate ratio between the focusing and core liquids. The design approach provides the ability to adjust the stream size while keeping the position of the focused stream centered with respect to the focusing channel. The device has been fabricated by direct micro milling of PMMA sheets. Experimental validation of the hydrodynamic sheath focusing effect has been presented and sample stream with tuneable size from about 18 to 50 μm was measured. Flow cytometry measurements have been performed by using 10-23 μm fluorescent particles. From the analysis of the signals collected at each transit event we can confirm that the device was capable to align and measure microparticles with a good coefficient of variance.
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paper_title: A photonic-microfluidic integrated device for reliable fluorescence detection and counting.

paper_content:

A photonic-microfluidic integrated device is demonstrated with excellent and reliable fluorescence detection performance. CV values of 8% for 2.5-μm beads and 14% for 6-μm beads were achieved through the correct deployment of carefully formed excitation beam shapes via integrated on-chip optics even without the use of 3D hydrodynamic focusing or a high-quality laser source and single mode beam propagation. The devices are fabricated in a monolithic planar fashion using a system of microlenses and waveguides integrated with microfluidic channels on-chip and packaged using a high-quality and low-cost channel sealing and high-performance interconnecting technology developed from our earlier works. Beam geometry in the excitation region is shown to affect the variation of fluorescence intensity from specimens, hence configurations of beam geometry targeted for a specific bead sizes are examined to ensure proper deployment of the lens designs. The formed high-quality optical excitation regions ensure reliable detection even with relaxed hydrodynamic focusing to ensure applicability with multiple specimen sizes. Device performance with each bead size was found to be acceptable for a range of beam geometries with a different ideal configuration for each bead size. These device designs help to form a device that will supplement conventional flow cytometry in point-of-care and remote detection applications by performing specific detections with an inexpensive and replaceable device.
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paper_title: Simultaneous optical and impedance analysis of single cells: A comparison of two microfluidic sensors with sheath flow focusing

paper_content:

Systems with disposable microfluidic cartridges are demanded for in vitro point-of-care diagnostics. Here, we developed two microfluidic sensors for flow cytometry cell differentiation based on the combined analysis of electrical impedance and optical properties of microparticles and blood cells. Both microdevices incorporate 1D sheath flow focusing to confine particles in a defined plane. For one sensor, a novel approach based on electroplating of gold was used in combination with lithography in SU-8. Electrodes for differential measurements are integrated into the flow channel perpendicularly to the plane of the substrate. For the manufacturing of the second sensor, we applied double-etched glass structures with integrated sputtered Pt electrode pairs. Stability of hydrodynamic focusing was benchmarked by measuring the coefficients of variations of calibration beads with specified size and fluorescence intensities. Sensitivity of electrical impedance measured in the MHz frequency range allowed detection of beads with a diameter of 1 μm when using side scatter as trigger signal. Measured particles were moving at a velocity of up to 6 m/s. Differentiation of leukocytes was achieved by simultaneous detection of impedance signals, correlated with intrinsic electrical properties of cells, and fluorescence measurements using standard protocols for immunophenotyping of white blood cells. The resulting separation of cell clusters is similar to that of microdevices with 2D hydrodynamic focusing and routine flow cytometers.
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paper_title: Fabrication and Performance of a Photonic-Microfluidic Integrated Device

paper_content:

Fabrication and performance of a functional photonic-microfluidic flow cytometer is demonstrated. The devices are fabricated on a Pyrex substrate by photolithographically patterning the microchannels and optics in a SU-8 layer that is sealed via a poly(dimethylsiloxane) (PDMS) layer through a unique chemical bonding method. The resulting devices eliminate the free-space excitation optics through integration of microlenses onto the chip to mimic conventional cytometry excitation. Devices with beam waists of 6 μm and 12 μm in fluorescent detection and counting tests using 2.5 and 6 μm beads-show CVs of 9%–13% and 23% for the two devices, respectively. These results are within the expectations for a conventional cytometer (5%–15%) and demonstrate the ability to integrate the photonic components for excitation onto the chip and the ability to maintain the level of reliable detection.
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paper_title: Sub-micrometer-precision, three-dimensional (3D) hydrodynamic focusing via “microfluidic drifting”

paper_content:

In this article, we demonstrate single-layered, “microfluidic drifting” based three-dimensional (3D) hydrodynamic focusing devices with particle/cell focal positioning approaching submicron precision along both lateral and vertical directions. By systematically optimizing channel geometries and sample/sheath flow rates, a series of “microfluidic drifting” based 3D hydrodynamic focusing devices with different curvature angles are designed and fabricated. Their performances are then evaluated using confocal microscopy, fast camera imaging, and side-view imaging techniques. Using a device with a curvature angle of 180°, we have achieved a standard deviation of ±0.45 μm in particle focal position and a coefficient of variation (CV) of 2.37% in flow cytometric measurements. To the best of our knowledge, this is the best CV that has been achieved using a microfluidic flow cytometry device. Moreover, the device showed the capability to distinguish 8 peaks when subjected to a stringent 8-peak rainbow calibration test, signifying the ability to perform sensitive, accurate tests similar to commercial flow cytometers. We have further tested and validated our device by detection of HEK-293 cells. With its advantages in simple fabrication (i.e., single-layered device), precise 3D hydrodynamic focusing (i.e., submicrometer precision along both lateral and vertical directions), and high detection resolution (i.e., low CV), our method could serve as an important basis for high-performance, mass-producible microfluidic flow cytometry.
---
paper_title: Simultaneous optical and impedance analysis of single cells: A comparison of two microfluidic sensors with sheath flow focusing

paper_content:

Systems with disposable microfluidic cartridges are demanded for in vitro point-of-care diagnostics. Here, we developed two microfluidic sensors for flow cytometry cell differentiation based on the combined analysis of electrical impedance and optical properties of microparticles and blood cells. Both microdevices incorporate 1D sheath flow focusing to confine particles in a defined plane. For one sensor, a novel approach based on electroplating of gold was used in combination with lithography in SU-8. Electrodes for differential measurements are integrated into the flow channel perpendicularly to the plane of the substrate. For the manufacturing of the second sensor, we applied double-etched glass structures with integrated sputtered Pt electrode pairs. Stability of hydrodynamic focusing was benchmarked by measuring the coefficients of variations of calibration beads with specified size and fluorescence intensities. Sensitivity of electrical impedance measured in the MHz frequency range allowed detection of beads with a diameter of 1 μm when using side scatter as trigger signal. Measured particles were moving at a velocity of up to 6 m/s. Differentiation of leukocytes was achieved by simultaneous detection of impedance signals, correlated with intrinsic electrical properties of cells, and fluorescence measurements using standard protocols for immunophenotyping of white blood cells. The resulting separation of cell clusters is similar to that of microdevices with 2D hydrodynamic focusing and routine flow cytometers.
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Title: Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review
Section 1: Introduction
Description 1: Introduction to the development and current status of flow cytometry, including the transition to microflow cytometers and the emergence of optofluidic devices.

Section 2: The Principles of Flow Cytometry
Description 2: Explanation of the basic principles and techniques of flow cytometry, including particle focusing, beam shaping, signal detection, and data analysis.

Section 3: Microflow Cytometer
Description 3: Overview of the advantages, fabrication techniques, and applications of microflow cytometers, contrasted with conventional flow cytometers.

Section 4: Major Components of an Optofluidic Microflow Cytometer
Description 4: Discussion of the four principal areas in the design of optofluidic microflow cytometers: flow control, optical design, microfabrication, and system integration.

Section 5: Flow Control
Description 5: Details about methods of controlling fluid flow in microflow cytometers, including 2D and 3D hydrodynamic flow focusing, as well as alternative focusing methods like acoustics, dielectrophoresis, and magnetophoresis.

Section 6: Light Guide and Collection
Description 6: Information on the light guiding and collection mechanisms within optofluidic microflow cytometers, including the types of waveguides and on-chip lens systems used.

Section 7: Data Collection
Description 7: Overview of the optical detectors used for data collection in optofluidic microflow cytometers, including PMTs, APDs, PIN photodiodes, CCD cameras, and CMOS arrays.

Section 8: Data Analysis
Description 8: Description of the process and tools used to analyze the light signals collected from particles or cells in a microflow cytometer, including electrical pulse characteristics and histogram analyses.

Section 9: Materials
Description 9: Examination of various materials used for constructing microflow cytometers, such as silicon, glass, polymers like PDMS, SU-8, and other emerging materials like paper.

Section 10: Device Integration
Description 10: Explanation of the processes and techniques for integrating different layers and components of an optofluidic microflow cytometer into a functional device.

Section 11: Performance of Optofluidic Microflow Cytometers
Description 11: Review of recent advancements in optofluidic microflow cytometers' performance, compared to conventional benchtop cytometers, focusing on sensitivity, throughput, and coefficient of variation (CV).

Section 12: Conclusions and Future Perspective
Description 12: Summary of the current state, advantages, and potential future developments in the field of optofluidic microflow cytometers, including pathways for further miniaturization and commercialization.