Microfluidics-Based Lab-on-Chip Systems in DNA-Based Biosensing: An Overview
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paper_title: Design Automation and Test Solutions for Digital Microfluidic Biochips

paper_content:

Microfluidics-based biochips are revolutionizing high-throughput sequencing, parallel immunoassays, blood chemistry for clinical diagnostics, and drug discovery. These devices enable the precise control of nanoliter volumes of biochemical samples and reagents. They combine electronics with biology, and they integrate various bioassay operations, such as sample preparation, analysis, separation, and detection. Compared to conventional laboratory procedures, which are cumbersome and expensive, miniaturized biochips offer the advantages of higher sensitivity, lower cost due to smaller sample and reagent volumes, system integration, and less likelihood of human error. This tutorial paper provides an overview of droplet-based ?digital? microfluidic biochips. It describes emerging computer-aided design (CAD) tools for the automated synthesis and optimization of biochips from bioassay protocols. Recent advances in fluidic-operation scheduling, module placement, droplet routing, pin-constrained chip design, and testing are presented. These CAD techniques allow biochip users to concentrate on the development of nanoscale bioassays, leaving chip optimization and implementation details to design-automation tools.
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paper_title: Microfluidic diagnostic technologies for global public health

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

paper_content:

Polymers have assumed the leading role as substrate materials for microfluidic devices in recent years. They offer a broad range of material parameters as well as material and surface chemical properties which enable microscopic design features that cannot be realised by any other class of materials. A similar range of fabrication technologies exist to generate microfluidic devices from these materials. This review will introduce the currently relevant microfabrication technologies such as replication methods like hot embossing, injection molding, microthermoforming and casting as well as photodefining methods like lithography and laser ablation for microfluidic systems and discuss academic and industrial considerations for their use. A section on back-end processing completes the overview.
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paper_title: Microfluidics for Biological Applications

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Microfluidics for Biological Applications provides researchers and scientists in the biotechnology, pharmaceutical, and life science industries with an introduction to the basics of microfluidics and also discusses how to link these technologies to various biological applications at the industrial and academic level. Readers will gain insight into a wide variety of biological applications for microfluidics. The material presented here is divided into four parts, Part I gives perspective on the history and development of microfluidic technologies, Part II presents overviews on how microfluidic systems have been used to study and manipulate specific classes of components, Part III focuses on specific biological applications of microfluidics: biodefense, diagnostics, high throughput screening, and tissue engineering and finally Part IV concludes with a discussion of emerging trends in the microfluidics field and the current challenges to the growth and continuing success of the field.
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paper_title: A 1.5 µL microbial fuel cell for on-chip bioelectricity generation

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We have developed a dual-chamber microfluidic microbial fuel cell (MFC) system that allows on-chip bacterial culture and conversion of bacterial metabolism into electricity. The micro-MFC contains a vertically stacked 1.5 µL anode chamber and 4 µL cathode chamber, and represents the smallest MFC device to our knowledge. Microfluidic deliveries of growth medium and catholyte were achieved in separate flow channels without cross-channel mass exchange. After inoculation of electrogenic Shewanella oneidensis strain MR-1, current generation was observed on an external load for up to two weeks. Current production was repeatable with replenishment of organic substrates. A maximum current density of 1300 A/m3 and power density of 15 W/m3 were achieved. Electron microscopic studies confirmed large-scale, uniform biofilm growth on the gold anode, and suggested that the enhanced cell/anode interaction in the small volume may accelerate start-up. Our result demonstrates a versatile platform for studying the fundamental issues in MFCs on the micro-scale, and suggests the possibility of powering nanodevices using on-chip bioenergy.
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paper_title: Capillary electrophoresis chips with a sheath-flow supported electrochemical detection system.

paper_content:

Microfabricated capillary electrophoresis chips containing an integrated sheath-flow electrochemical detector are developed with the goal of minimizing the influence of separation voltages on end-column detection while maintaining optimum performance. The microdevice consists of an upper glass wafer carrying the etched separation, injection, and sheath-flow channels and a lower glass wafer on which gold- and silver-plated electrodes have been fabricated. The sheath-flow channels join the end of the separation channel from each side, and gravity-driven flow carries the analytes to the electrochemical detector placed at working distances of 100, 150, 200, and 250 microm from the separation channel exit. The performance of this detector is evaluated using catechol and a detection limit of 4.1 microM obtained at a working distance of 250 microm. Detection of DNA restriction fragments and PCR product sizing is demonstrated using the electroactive intercalating dye, iron phenanthroline. Additionally, an allele-specific, PCR-based single-nucleotide polymorphism typing assay for the C282Y substitution diagnostic for hereditary hemochromatosis is developed and evaluated using ferrocene-labeled primers. This study advances the feasibility of high-speed, high-throughput chemical and genetic analysis using microchip electrochemical detection.
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paper_title: High Purity DNA Extraction with a SPE Microfluidic Chip Using KI as the Binding Salt

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Based on solid phase extraction method, a novel silicon-PDMS-glass microchip for high purity DNA extraction has been developed by using KI as the binding salt. The microfluidic chip fabricated by MEMS technology was composed of a silicon substrate with a coiled channel and a compounded PDMS-glass cover. With this microfluidic chip, the wall of the coiled channel was used as solid phase matrix for binding DNA and DNA was extracted by the fluxion of the binding buffer, washing buffer and elution buffer. KI as a substitute for guanidine, was used successfully as binding salt for purification DNA, obtaining higher purity of genomic DNA and about 13.9 ng DNA from 1 μL rat whole blood in 35 minutes.
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paper_title: Mammalian electrophysiology on a microfluidic platform

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Abstract ::: The recent development of automated patch clamp technology has increased the throughput of electrophysiology but at the expense of visual access to the cells being studied. To improve visualization and the control of cell position, we have developed a simple alternative patch clamp technique based on microfluidic junctions between a main chamber and lateral recording capillaries, all fabricated by micromolding of polydimethylsiloxane (PDMS). PDMS substrates eliminate the need for vibration isolation and allow direct cell visualization and manipulation using standard microscopy. Microfluidic integration allows recording capillaries to be arrayed 20 μm apart, for a total chamber volume of <0.5 nl. The geometry of the recording capillaries permits high-quality, stable, whole-cell seals despite the hydrophobicity of the PDMS surface. Using this device, we are able to demonstrate reliable whole-cell recording of mammalian cells on an inexpensive microfluidic platform. Recordings of activation of the voltage-sensitive potassium channel Kv2.1 in mammalian cells compare well with traditional pipette recordings. The results make possible the integration of whole-cell electrophysiology with easily manufactured microfluidic lab-on-a-chip devices. ::: ::: microfluidics ::: patch clamp ::: drug screening ::: single-cell assay
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paper_title: Microfluidics-based systems biology.

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Systems biology seeks to develop a complete understanding of cellular mechanisms by studying the functions of intra- and inter-cellular molecular interactions that trigger and coordinate cellular events. However, the complexity of biological systems causes accurate and precise systems biology experimentation to be a difficult task. Most biological experimentation focuses on highly detailed investigation of a single signaling mechanism, which lacks the throughput necessary to reconstruct the entirety of the biological system, while high-throughput testing often lacks the fidelity and detail necessary to fully comprehend the mechanisms of signal propagation. Systems biology experimentation, however, can benefit greatly from the progress in the development of microfluidic devices. Microfluidics provides the opportunity to study cells effectively on both a single- and multi-cellular level with high-resolution and localized application of experimental conditions with biomimetic physiological conditions. Additionally, the ability to massively array devices on a chip opens the door for high-throughput, high fidelity experimentation to aid in accurate and precise unraveling of the intertwined signaling systems that compose the inner workings of the cell.
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paper_title: Automated Design and Programming of a Microfluidic DNA Computer

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Previously, we described ways to implement the functions AND and OR in a DNA computer consisting of microreactors with attached heating elements that control annealing of DNA. Based on these findings, we have devised a similar device that can solve a satisfiability problem in any form. The device occupies linear space and operates in quadratic time, while a previously described competing device is built in quadratic space and operates in quadratic time or greater. Reducing the number of reactors in a DNA computer reduces the loss of DNA through binding to the surfaces of the system.
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paper_title: A thermopneumatic dispensing micropump

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Abstract A micropump for dispensing microliter liquid volumes is realized by warming a thermopneumatic fluid that expands a membrane to pressurize a liquid reservoir with an outlet flow restrictor. The temperature of this liquid–vapor perfluorocarbon mixture is controlled by a thin film heater. The outlet flow restrictors, used in this work, are 75–100 μm silica capillaries, which result in microliter per minute flow rates at 7–42 kPa liquid pressure levels. We investigate both open loop designs and closed loop designs with pressure feedback. The closed loop designs offer increased transient and steady state flow control and allow a much larger range of pump geometries compared to open loop designs. The micropump dispenses 1.4 μl/min for 4.5 h with an average power of 200 mW. Due to its design with no moving parts, this thermopneumatic pump is low cost and simple in construction. The basic principles appear suited to MEMS fabrication, and so would have potential applications for micro total chemical analysis systems (μTAS), biosensors, and “lab-on-a-chip” devices.
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paper_title: Microfluidics: Fluid physics at the nanoliter scale

paper_content:

Microfabricated integrated circuits revolutionized computation by vastly reducing the space, labor, and time required for calculations. Microfluidic systems hold similar promise for the large-scale automation of chemistry and biology, suggesting the possibility of numerous experiments performed rapidly and in parallel, while consuming little reagent. While it is too early to tell whether such a vision will be realized, significant progress has been achieved, and various applications of significant scientific and practical interest have been developed. Here a review of the physics of small volumes (nanoliters) of fluids is presented, as parametrized by a series of dimensionless numbers expressing the relative importance of various physical phenomena. Specifically, this review explores the Reynolds number Re, addressing inertial effects; the Peclet number Pe, which concerns convective and diffusive transport; the capillary number Ca expressing the importance of interfacial tension; the Deborah, Weissenberg, and elasticity numbers De, Wi, and El, describing elastic effects due to deformable microstructural elements like polymers; the Grashof and Rayleigh numbers Gr and Ra, describing density-driven flows; and the Knudsen number, describing the importance of noncontinuum molecular effects. Furthermore, the long-range nature of viscous flows and the small device dimensions inherent in microfluidics mean that the influence of boundaries is typically significant. A variety of strategies have been developed to manipulate fluids by exploiting boundary effects; among these are electrokinetic effects, acoustic streaming, and fluid-structure interactions. The goal is to describe the physics behind the rich variety of fluid phenomena occurring on the nanoliter scale using simple scaling arguments, with the hopes of developing an intuitive sense for this occasionally counterintuitive world.
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paper_title: A PMMA valveless micropump using electromagnetic actuation

paper_content:

We have fabricated and characterized a polymethylmethacrylate (PMMA) valveless micropump. The pump consists of two diffuser elements and a polydimethylsiloxane (PDMS) membrane with an integrated composite magnet made of NdFeB magnetic powder. A large-stroke membrane deflection (~200 μm) is obtained using external actuation by an electromagnet. We present a detailed analysis of the magnetic actuation force and the flow rate of the micropump. Water is pumped at flow rates of up to 400 µl/min and backpressures of up to 12 mbar. We study the frequency-dependent flow rate and determine a resonance frequency of 12 and 200 Hz for pumping of water and air, respectively. Our experiments show that the models for valveless micropumps of A. Olsson et al. (J Micromech Microeng 9:34, 1999) and L.S. Pan et al. (J Micromech Microeng 13:390, 2003) correctly predict the resonance frequency, although additional modeling of losses is necessary.
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paper_title: A valveless micro impedance pump driven by electromagnetic actuation

paper_content:

Over the past two decades, a variety of micropumps have been explored for various applications in microfluidics such as control of pico- and nanoliter flows for drug delivery as well as chemical mixing and analysis. We present the fabrication and preliminary experimental studies of flow performance on the micro impedance pump, a previously unexplored method of pumping fluid on the microscale. The micro impedance pump was constructed of a simple thin-walled tube coupled at either end to glass capillary tubing and actuated electromagnetically. Through the cumulative effects of wave propagation and reflection originating from an excitation located asymmetrically along the length of the elastic tube, a pressure head can be established to drive flow. Flow rates were observed to be reversible and highly dependent on the profile of the excitation. Micro impedance pump flow studies were conducted in open and closed circuit flow configurations. Maximum flow rates of 16 ml min-1 have been achieved under closed loop flow conditions with an elastic tube diameter of 2 mm. Two size scales with channel diameters of 2 mm and 250 µm were also examined in open circuit flow, resulting in flow rates of 191 µl min-1 and 17 µl min-1, respectively.
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paper_title: A Soft-Polymer Piezoelectric Bimorph Cantilever-Actuated Peristaltic Micropump

paper_content:

For this work, a peristaltic micropump was fabricated. Actuation of the micropump was accomplished with piezoelectric cantilevers. To date, a minimal number of soft polymer-based micropump designs, have explored the use of piezoelectric materials as actuators. The fluidic channel for the micropump was fabricated using PDMS and soft lithography. A novel and very simple template fabrication process was employed, where the use of a mask and clean room facilities was not required. Replica molding to the template produces both, a channel measuring ∼95μm in height, and a rounded cross-sectional geometry, the latter of which is known to be favorable for complete valve shutoff. Clamps were adhered to the tips of the cantilevers, and used to secure in place aluminum valves. The valves had finely machined tips [3mm×200μm(L×W)] on one surface. These tips served as contact points for the valve making contact with the PDMS membrane surface, and were used for the purpose of opening and closing the channels. The cantilevers were secured in place with in-house manufactured micropositioners, which were used to position the valves directly over the PDMS channel. The micropump was thoroughly tested where the variables characterized were maximum attainable backpressure, flow rate, valve open/close characteristics, and valve leakage. The effect of the phase difference (60°, 90°, and 120°) between the square wave signals delivered to each of the three cantilevers was investigated for flow rate and maximum attainable backpressure. Of the three signal phases, the 120° signal demonstrated the largest flow rate range of 52–575 nL/min (0.1–25 Hz), as well as the highest attainable backpressure value of 36,800 Pa (5.34 psi). The valve shutoff characteristics for this micropump was also examined. Fluorescein was trapped inside the microchannel, where the fluorescent signal was monitored throughout the valves open/close cycle with the aid of an epifluorescent microscope. It was found that the fluorescent signal went to zero with the valve fully closed, supporting the conclusion that the valve completely closes off the channel. Further evidence of this claim was demonstrated by observing the valve leakage characteristics. An electronic pressure sensor was used to collect data for this experiment, where it was found the valve was able to hold off 36,800 Pa (5.34 psi), only loosing 2% of this pressure over 10 minutes. In conclusion, it has been shown this micropump outperforms many existing micropump designs, and is suitable for integration into a variety of both macro, and microdevice platforms. Experiments are currently underway to examine how the flow and valving characteristics change for valves with different tip dimensions. A discussion will also be given for improved fabrication techniques, where injection molding is currently being used as the fabrication method to examine the performance changes associated with different cross-sectional PDMS channel geometries. The end goal for use of this micropump is twofold; 1) integration into a micro-free flow separation device, and 2) integration into a capillary electrophoresis instrument for use in direct-sampling neuroscience experiments.
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paper_title: Microfluidic on-chip fluorescence-activated interface control system

paper_content:

A microfluidic dynamic fluorescence-activated interface control system was developed for lab-on-a-chip applications. The system consists of a straight rectangular microchannel, a fluorescence excitation source, a detection sensor, a signal conversion circuit, and a high-voltage feedback system. Aqueous NaCl as conducting fluid and aqueous glycerol as nonconducting fluid were introduced to flow side by side into the straight rectangular microchannel. Fluorescent dye was added to the aqueous NaCl to work as a signal representing the interface position. Automatic control of the liquid interface was achieved by controlling the electroosmotic effect that exists only in the conducting fluid using a high-voltage feedback system. A LABVIEW program was developed to control the output of high-voltage power supply according the actual interface position, and then the interface position is modified as the output of high-voltage power supply. At last, the interface can be moved to the desired position automatically using this feedback system. The results show that the system presented in this paper can control an arbitrary interface location in real time. The effects of viscosity ratio, flow rates, and polarity of electric field were discussed. This technique can be extended to switch the sample flow and droplets automatically.
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paper_title: Nucleic Acid-based Detection of Bacterial Pathogens Using Integrated Microfluidic Platform Systems

paper_content:

The advent of nucleic acid-based pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques, driving the development of portable, integrated biosensors. The miniaturization and automation of integrated detection systems presents a significant advantage for rapid, portable field-based testing. In this review, we highlight current developments and directions in nucleic acid-based micro total analysis systems for the detection of bacterial pathogens. Recent progress in the miniaturization of microfluidic processing steps for cell capture, DNA extraction and purification, polymerase chain reaction, and product detection are detailed. Discussions include strategies and challenges for implementation of an integrated portable platform.
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paper_title: Mixing with bubbles: a practical technology for use with portable microfluidic devices

paper_content:

This paper demonstrates a methodology for micromixing that is sufficiently simple that it can be used in portable microfluidic devices. It illustrates the use of the micromixer by incorporating it into an elementary, portable microfluidic system that includes sample introduction, sample filtration, and valving. This system has the following characteristics: (i) it is powered with a single hand-operated source of vacuum, (ii) it allows samples to be loaded easily by depositing them into prefabricated wells, (iii) the samples are filtered in situ to prevent clogging of the microchannels, (iv) the structure of the channels ensure mixing of the laminar streams by interaction with bubbles of gas introduced into the channels, (v) the device is prepared in a single-step soft-lithographic process, and (vi) the device can be prepared to be resistant to the adsorption of proteins, and can be used with or without surface-active agents.
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paper_title: Investigation of active interface control of pressure driven two-fluid flow in microchannels

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We report a novel concept to control the interface location of a pressure-driven multi-phase flow in a microchannel by using electroosmotic flow effects. This concept has potential applications in flow switching and cell sorting in bio-analytical systems. In an H-shaped microchannel structure, aqueous sodium chloride (NaCl) solution and glycerol diluted with water were pumped through two inlets at the same flow rate. The electric field was applied on the electrolyte solution side. Adjusting the magnitude and direction of electric field has successfully controlled the interface position between the two phases. This technique provides a new approach to control the interface position between the two fluids.
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paper_title: Droplet microfluidics for characterizing the neurotoxin-induced responses in individual Caenorhabditis elegans

paper_content:

A droplet-based microfluidic device integrated with a novel floatage-based trap array and a tapered immobilization channel array was presented for characterizing the neurotoxin-induced multiple responses in individual Caenorhabditis elegans (C. elegans) continuously. The established device enabled the evaluations of movement and fluorescence imaging analysis of individual C. elegans simultaneously. The utility of this device was demonstrated by the pharmacological evaluation of neurotoxin (6-hydroxydopamine, 6-OHDA) triggered mobility defects, neuron degeneration and oxidative stress in individual worms. Exposure of living worms to 6-OHDA could cause obvious mobility defects, selective degeneration of dopaminergic (DAergic) neurons, and increased oxidative stress in a dose dependent manner. These results are important towards the understanding of mechanisms leading to DAergic toxicity by neurotoxin and will be of benefit for the screening of new therapeutics for neurodegenerative diseases. This device was simple, stable and easy to operate, with the potential to facilitate whole-animal assays and drug screening in a high throughput manner at single animal resolution.
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paper_title: Development and modeling of electrically triggered hydrogels for microfluidic applications

paper_content:

In this paper, we present progress in the development of electrically triggered hydrogels as components in microfluidic systems. Stimuli-responsive hydrogels are fabricated using liquid-phase photopolymerization techniques and are subjected to different voltage signals in order to determine their volume change response characteristics. A chemoelectromechanical model has been developed to predict the swelling and deswelling kinetics of these hydrogels. The Nernst-Planck equation, Poisson equation, and mechanical equations are the basic governing relationships, and these are solved in an iterative manner to compute the deformation of the hydrogel in response to varied electrical input.
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paper_title: A simple PDMS-based microfluidic channel design that removes bubbles for long-term on-chip culture of mammalian cells

paper_content:

This report shows methods to fabricate polydimethylsiloxane (PDMS) microfluidic systems for long-term (up to 10 day) cell culture. Undesired bubble accumulation in microfluidic channels abruptly changes the microenvironment of adherent cells and leads to the damage and death of cells. Existing bubble trapping approaches have drawbacks such as the need to pause fluid flow, requirement for external vacuum or pressure source, and possible cytotoxicity. This study reports two kinds of integrated bubble trap (IBT) which have excellent properties, including simplicity in structure, ease in fabrication, no interference with the flow, and long-term stability. IBT-A provides the simplest solution to prevent bubbles from entering microfluidic channels. In situ time-lapse imaging experiments indicate that IBT-B is an excellent device both for bubble trapping and debubbling in cell-loaded microfluidics. MC 3T3 E1 cells, cultured in a long and curved microfluidic channel equipped with IBT-B, showed high viability and active proliferation after 10 days of continuous fluid flow. The comprehensive measures taken in our experiments have led to successful long-term, bubble-free, on-chip culture of cells.
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paper_title: A membrane micropump electrostatically actuated across the working fluid

paper_content:

A novel electrostatically actuated valveless micropump is presented whereby an actuation voltage is applied across a working fluid, which takes advantage of the higher relative electrical permittivity of water and many other fluids with respect to air. The device is fabricated in silicon and the diaphragm is made of electroplated nickel, while the assembly is carried out using flip–chip bonding. A reduced-order model is used to describe the micropump's performance in terms of electrical properties of the fluid, the residual stress in the diaphragm, geometrical features and the actuation voltage. The tested prototype featured a ~1 µl min−1 flow rate at 50 V actuation voltage. The model predictions show the possibility of achieving flow rates >1 µl min−1 with the actuation voltage <10 V for devices with 3 mm diaphragm size.
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paper_title: Logic control of microfluidics with smart colloid.

paper_content:

We report the successful realization of a microfluidic chip with switching and corresponding inverting functionalities. The chips are identical logic control components incorporating a type of smart colloid, giant electrorheological fluid (GERF), which possesses reversible characteristics via a liquid-solid phase transition under external electric field. Two pairs of electrodes embedded on the sides of two microfluidic channels serve as signal input and output, respectively. One, located in the GERF micro-channel is used to control the flow status of GERF, while another one in the ither micro-fluidic channel is used to detect the signal generated with a passing-by droplet (defined as a signal droplet). Switching of the GERF from the suspended state (off-state) to the flowing state (on-state) or vice versa in the micro-channel is controlled by the appearance of signal droplets whenever they pass through the detection electrode. The output on-off signals can be easily demonstrated, clearly matching with GERF flow status. Our results show that such a logic switch is also a logic IF gate, while its inverter functions as a NOT gate.
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paper_title: Point-of-care testing of proteins

paper_content:

Point-of-care testing (POCT) is a fast developing area in clinical diagnostics that is considered to be one of the main driving forces for the future in vitro diagnostic market. POCT means decentralized testing at the site of patient care. The most important POCT devices are handheld blood glucose sensors. In some of these sensors, after the application of less than 1 microl whole blood, the results are displayed in less than 10 s. For protein determination, the most commonly used devices are based on lateral flow technology. Although these devices are convenient to use, the results are often only qualitative or semiquantitative. The review will illuminate some of the current methods employed in POCT for proteins and will discuss the outlook for techniques (e.g., electrochemical immunosensors) that could have a great impact on future POCT of proteins.
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paper_title: Design Tools for Digital Microfluidic Biochips: Toward Functional Diversification and More Than Moore

paper_content:

Microfluidics-based biochips enable the precise control of nanoliter volumes of biochemical samples and reagents. They combine electronics with biology, and they integrate various bioassay operations, such as sample preparation, analysis, separation, and detection. Compared to conventional laboratory procedures, which are cumbersome and expensive, miniaturized biochips offer the advantages of higher sensitivity, lower cost due to smaller sample and reagent volumes, system integration, and less likelihood of human error. This paper first describes the droplet-based “digital” microfluidic technology platform and emerging applications. The physical principles underlying droplet actuation are next described. Finally, the paper presents computer-aided design tools for simulation, synthesis and chip optimization. These tools target modeling and simulation, scheduling, module placement, droplet routing, pin-constrained chip design, and testing.
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paper_title: Microfluidics: Fluid physics at the nanoliter scale

paper_content:

Microfabricated integrated circuits revolutionized computation by vastly reducing the space, labor, and time required for calculations. Microfluidic systems hold similar promise for the large-scale automation of chemistry and biology, suggesting the possibility of numerous experiments performed rapidly and in parallel, while consuming little reagent. While it is too early to tell whether such a vision will be realized, significant progress has been achieved, and various applications of significant scientific and practical interest have been developed. Here a review of the physics of small volumes (nanoliters) of fluids is presented, as parametrized by a series of dimensionless numbers expressing the relative importance of various physical phenomena. Specifically, this review explores the Reynolds number Re, addressing inertial effects; the Peclet number Pe, which concerns convective and diffusive transport; the capillary number Ca expressing the importance of interfacial tension; the Deborah, Weissenberg, and elasticity numbers De, Wi, and El, describing elastic effects due to deformable microstructural elements like polymers; the Grashof and Rayleigh numbers Gr and Ra, describing density-driven flows; and the Knudsen number, describing the importance of noncontinuum molecular effects. Furthermore, the long-range nature of viscous flows and the small device dimensions inherent in microfluidics mean that the influence of boundaries is typically significant. A variety of strategies have been developed to manipulate fluids by exploiting boundary effects; among these are electrokinetic effects, acoustic streaming, and fluid-structure interactions. The goal is to describe the physics behind the rich variety of fluid phenomena occurring on the nanoliter scale using simple scaling arguments, with the hopes of developing an intuitive sense for this occasionally counterintuitive world.
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paper_title: Biomicrofluidics: Recent trends and future challenges

paper_content:

Biomicrofluidics is an active area of research at present, exploring the synergy of microfluidics with cellular and molecular biology, biotechnology, and biomedical engineering. The present article outlines the recent advancements in these areas, including the development of novel lab-on-a-chip based applications. Particular emphasis is given on the microfluidics-based handling of DNA, cells, and proteins, as well as fundamental microfluidic considerations for design of biomedical microdevices. Future directions of research on these topics are also discussed.
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paper_title: Design Tools for Digital Microfluidic Biochips: Toward Functional Diversification and More Than Moore

paper_content:

Microfluidics-based biochips enable the precise control of nanoliter volumes of biochemical samples and reagents. They combine electronics with biology, and they integrate various bioassay operations, such as sample preparation, analysis, separation, and detection. Compared to conventional laboratory procedures, which are cumbersome and expensive, miniaturized biochips offer the advantages of higher sensitivity, lower cost due to smaller sample and reagent volumes, system integration, and less likelihood of human error. This paper first describes the droplet-based “digital” microfluidic technology platform and emerging applications. The physical principles underlying droplet actuation are next described. Finally, the paper presents computer-aided design tools for simulation, synthesis and chip optimization. These tools target modeling and simulation, scheduling, module placement, droplet routing, pin-constrained chip design, and testing.
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paper_title: Laminar flow and convective transport processes : scaling principles and asymptotic analysis

paper_content:

Basic Principles Unidirectional Flows Creeping Flows Further Results in the Creeping Flow Limit Asymptotic Approximations for Unidirectional, One-Dimensional, and Nearly Unidirectional Flows Thin Films, Lubrication, and Related Problems Weak Convection Effects Strong Convection Effects in Heat and Mass Transfer at Low Reynolds Number Laminar Boundary-Layer theory Thermal Boundary-Layer Theory at Large Reynolds Number Natural and Mixed Convection Flows.
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paper_title: RNA biosensor for the rapid detection of viable Escherichia coli in drinking water.

paper_content:

A highly sensitive and specific RNA biosensor was developed for the rapid detection of viable Escherichia coli as an indicator organism in water. The biosensor is coupled with protocols developed earlier for the extraction and amplification of mRNA molecules from E. coli [Anal. Biochem. 303 (2002) 186]. However, in contrast to earlier detection methods, the biosensor allows the rapid detection and quantification of E. coli mRNA in only 15-20 min. In addition, the biosensor is portable, inexpensive and very easy to use, which makes it an ideal detection system for field applications. Viable E. coli are identified and quantified via a 200 nt-long target sequence from mRNA (clpB) coding for a heat shock protein. For sample preparation, a heat shock is applied to the cells prior to disruption. Then, mRNA is extracted, purified and finally amplified using the isothermal amplification technique Nucleic acid sequence-based amplification (NASBA). The amplified RNA is then quantified with the biosensor. The biosensor is a membrane-based DNA/RNA hybridization system using liposome amplification. The various biosensor components such as DNA probe sequences and concentration, buffers, incubation times have been optimized, and using a synthetic target sequence, a detection limit of 5 fmol per sample was determined. An excellent correlation to a much more elaborate and expensive laboratory based detection system was demonstrated, which can detect as few as 40 E. coli cfu/ml. Finally, the assay was tested regarding its specificity; no false positive signals were obtained from other microorganisms or from nonviable E. coli cells.
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paper_title: Electrical detection of viral DNA using ultramicroelectrode arrays.

paper_content:

A fully electrical array for voltammetric detection of redox molecules produced by enzyme-labeled affinity binding complexes is shown. The electronic detection is based on ultramicroelectrode arrays manufactured in silicon technology. The 200-μm circular array positions have 800-nm-wide interdigitated gold ultramicroelectrodes embedded in silicon dioxide. Immobilization of oligonucleotide capture probes onto the gold electrodes surfaces is accomplished via thiol−gold self-assembling. Spatial separation of probes at different array positions is controlled by polymeric rings around each array position. The affinity bound complexes are labeled with alkaline phosphatase, which converts the electrochemically inactive substrate 4-aminophenyl phosphate into the active 4-hydroxyaniline (HA). The nanoscaled electrodes are used to perform a sensitive detection of enzyme activity by signal enhancing redox recycling of HA resulting in local and position-specific current signals. Multiplexing and serial readout is rea...
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paper_title: Development of a microfluidic biosensor module for pathogen detection

paper_content:

The development of a microfluidic biosensor module with fluorescence detection for the identification of pathogenic organisms and viruses is presented in this article. The microfluidic biosensor consists of a network of microchannels fabricated in polydimethylsiloxane (PDMS) substrate. The microchannels are sealed with a glass substrate and packed in a Plexiglas housing to provide connection to the macro-world and ensure leakage-free flow operation. Reversible sealing permits easy disassembly for cleaning and replacing the microfluidic channels. The fluidic flow is generated by an applied positive pressure gradient, and the module can be operated under continuous solution flow of up to 80 µL min−1. The biosensor recognition principle is based on DNA/RNA hybridization and liposome signal amplification. Superparamagnetic beads are incorporated into the system as a mobile solid support and are an essential part of the analysis scheme. In this study, the design, fabrication and the optimization of concentrations and amounts of the different biosensor components are carried out. The total time required for an assay is only 15 min including sample incubation time. The biosensor module is designed so that it can be easily integrated with a micro total analysis system, which will combine sample preparation and detection steps onto a single chip.
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paper_title: Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems

paper_content:

Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems will cover the up-to-date biosensor technologies used for the detection of bacteria. Written by the world's mos ...
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paper_title: Multi-analyte single-membrane biosensor for the serotype-specific detection of Dengue virus.

paper_content:

A multi-analyte biosensor based on nucleic acid hybridization and liposome signal amplification was developed for the rapid serotype-specific detection of Dengue virus. After RNA amplification, detection of Dengue virus specific serotypes can be accomplished using a single analysis within 25 min. The multi-analyte biosensor is based on single-analyte assays (see Baeumner et al (2002) Anal Chem 74:1442-1448) developed earlier in which four analyses were required for specific serotype identification of Dengue virus samples. The multi-analyte biosensor employs generic and serotype-specific DNA probes, which hybridize with Dengue RNA that is amplified by the isothermal nucleic acid sequence based amplification (NASBA) reaction. The generic probe (reporter probe) is coupled to dye-entrapping liposomes and can hybridize to all four Dengue serotypes, while the serotype-specific probes (capture probes) are immobilized through biotin-streptavidin interaction on the surface of a polyethersulfone membrane strip in separate locations. A mixture of amplified Dengue virus RNA sequences and liposomes is applied to the membrane and allowed to migrate up along the test strip. After the liposome-target sequence complexes hybridize to the specific probes immobilized in the capture zones of the membrane strip, the Dengue serotype present in the sample can be determined. The amount of liposomes immobilized in the various capture zones directly correlates to the amount of viral RNA in the sample and can be quantified by a portable reflectometer. The specific arrangement of the capture zones and the use of unlabeled oligonucleotides (cold probes) enabled us to dramatically reduce the cross-reactivity of Dengue virus serotypes. Therefore, a single biosensor can be used to detect the exact Dengue serotype present in the sample. In addition, the biosensor can simultaneously detect two serotypes and so it is useful for the identification of possible concurrent infections found in clinical samples. The various biosensor components have been optimized with respect to specificity and sensitivity, and the system has been ultimately tested using blind coded samples. The biosensor demonstrated 92% reliability in Dengue serotype determination. Following isothermal amplification of the target sequences, the biosensor had a detection limit of 50 RNA molecules for serotype 2, 500 RNA molecules for serotypes 3 and 4, and 50,000 molecules for serotype 1. The multi-analyte biosensor is portable, inexpensive, and very easy to use and represents an alternative to current detection methods coupled with nucleic acid amplification reactions such as electrochemiluminescence, or those based on more expensive and time consuming methods such as ELISA or tissue culture.
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paper_title: Nucleic Acid-based Detection of Bacterial Pathogens Using Integrated Microfluidic Platform Systems

paper_content:

The advent of nucleic acid-based pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques, driving the development of portable, integrated biosensors. The miniaturization and automation of integrated detection systems presents a significant advantage for rapid, portable field-based testing. In this review, we highlight current developments and directions in nucleic acid-based micro total analysis systems for the detection of bacterial pathogens. Recent progress in the miniaturization of microfluidic processing steps for cell capture, DNA extraction and purification, polymerase chain reaction, and product detection are detailed. Discussions include strategies and challenges for implementation of an integrated portable platform.
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paper_title: Esterase 2-oligodeoxynucleotide conjugates as sensitive reporter for electrochemical detection of nucleic acid hybridization.

paper_content:

A thermostable, single polypeptide chain enzyme, esterase 2 from Alicyclobacillus acidocaldarius, was covalently conjugated in a site specific manner with an oligodeoxynucleotide. The conjugate served as a reporter enzyme for electrochemical detection of DNA hybridization. Capture oligodeoxynucleotides were assembled on gold electrode via thiol-gold interaction. The esterase 2-oligodeoxynucleotide conjugates were brought to electrode surface by DNA hybridization. The p-aminophenol formed by esterase 2 catalyzed hydrolysis of p-aminophenylbutyrate was amperometrically determined. Esterase 2 reporters allows to detect approximately 1.5 x 10(-18)mol oligodeoxynucleotides/0.6 mm2 electrode, or 3 pM oligodeoxynucleotide in a volume of 0.5 microL. Chemically targeted, single site covalent attachment of esterase 2 to an oligodeoxynucleotide significantly increases the selectivity of the mismatch detection as compared to widely used, rather unspecific, streptavidin/biotin conjugated proteins. Artificial single nucleotide mismatches in a 510-nucleotide ssDNA could be reliably determined using esterase 2-oligodeoxynucleotide conjugates as a reporter.
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paper_title: Fungal pathogenic nucleic acid detection achieved with a microfluidic microarray device.

paper_content:

Detection of polymerase chain reaction (PCR) products obtained from cultured greenhouse fungal pathogens, Botrytis cinerea and Didymella bryoniae has been achieved using a previously developed microfluidic microarray assembly (MMA) device. The flexible probe construction and rapid DNA detection resulted from the use of centrifugal pumping in the steps of probe introduction and sample delivery, respectively. The line arrays of the oligonucleotide probes were "printed" on a CD-like glass chip using a polydimethylsiloxane (PDMS) polymer plate with radial microfluidic channels, and the sample hybridizations were conducted within the spiral channels on the second plate. The experimental conditions of probe immobilization and sample hybridization were optimized, and both complementary oligonucleotides and PCR products were tested. We were able to achieve adequate fluorescent signals with a sample load as small as 0.5 nM (1 microL) for oligonucleotide samples; for PCR products, we achieved detection at the level of 3 ng.
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paper_title: Detection of viable oocysts of Cryptosporidium parvum following nucleic acid sequence based amplification.

paper_content:

A reliable method using nucleic acid sequence based amplification (NASBA) with subsequent electrochemiluminescent detection for the specific and sensitive detection of viable oocysts of Cryptosporidium parvum in environmental samples was developed. The target molecule was a 121-nt sequence from the C. parvum heat shock protein hsp70 mRNA. Oocysts of C. parvum were isolated from environmental water via vortex flow filtration and immunomagnetic separation. A brief heat shock was applied to the oocysts and the nucleic acid purified using an optimized very simple but efficient nucleic acid extraction method. The nucleic acid was amplified in a water bath for 60-90 min with NASBA, an isothermal technique that specifically amplifies RNA molecules. Amplified RNA was hybridized with specific DNA probes and quantified with an electrochemiluminescence (ECL) detection system. We optimized the nucleic acid extraction and purification, the NASBA reaction, amplification, and detection probes. We were able to amplify and detect as few as 10 mRNA molecules. The NASBA primers as well as the ECL probes were highly specific for C. parvum in buffer and in environmental samples. Our detection limit was approximately 5 viable oocysts/sample for the assay procedure, including nucleic acid extraction, NASBA, and ECL detection. Nonviable oocysts were not detected.
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paper_title: Biomicrofluidics: Recent trends and future challenges

paper_content:

Biomicrofluidics is an active area of research at present, exploring the synergy of microfluidics with cellular and molecular biology, biotechnology, and biomedical engineering. The present article outlines the recent advancements in these areas, including the development of novel lab-on-a-chip based applications. Particular emphasis is given on the microfluidics-based handling of DNA, cells, and proteins, as well as fundamental microfluidic considerations for design of biomedical microdevices. Future directions of research on these topics are also discussed.
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paper_title: A nanoliter rotary device for polymerase chain reaction

paper_content:

Polymerase chain reaction (PCR) has revolutionized a variety of assays in biotechnology. The ability to implement PCR in disposable and reliable microfluidic chips will facilitate its use in applications such as rapid medical diagnostics, food control testing, and biological weapons detection. We fabricated a microfluidic chip with integrated heaters and plumbing in which various forms of PCR have been successfully demonstrated. The device uses only 12 nL of sample, one of the smallest sample volumes demonstrated to date. Minimizing the sample volume allows low power consumption, reduced reagent costs, and ultimately more rapid thermal cycling.
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paper_title: Integrated Microfluidic CustomArray Device for Bacterial Genotyping and Identification

paper_content:

The ongoing threat of the potential use of biothreat agents (such as Bacillus anthracis) as a biochemical weapon emphasizes the need for a rapid, miniature, fully automated, and highly specific detection assay. An integrated and self-contained microfluidic device has been developed to rapidly detect B. anthracis and many other bacteria. The device consists of a semiconductor-based DNA microarray chip with 12,000 features and a microfluidic cartridge that automates the fluid handling steps required to carry out a genotyping assay for pathogen identification. This fully integrated and disposable device consists of low-cost microfluidic pumps, mixers, valves, fluid channels, reagent storage chambers, and DNA microarray silicon chip. Microarray hybridization and subsequent fluid handling and reactions were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. The genotyping results showed that the device was able to identify and distinguish B. anthrac...
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paper_title: A rapid biosensor for viable B. anthracis spores.

paper_content:

A simple membrane-strip-based biosensor assay has been combined with a nucleic acid sequence-based amplification (NASBA) reaction for rapid (4 h) detection of a small number (ten) of viable B. anthracis spores. The biosensor is based on identification of a unique mRNA sequence from one of the anthrax toxin genes, the protective antigen (pag), encoded on the toxin plasmid, pXO1, and thus provides high specificity toward B. anthracis. Previously, the anthrax toxins activator (atxA) mRNA had been used in our laboratory for the development of a biosensor for the detection of a single B. anthracis spore within 12 h. Changing the target sequence to the pag mRNA provided the ability to shorten the overall assay time significantly. The vaccine strain of B. anthracis (Sterne strain) was used in all experiments. A 500-μL sample containing as few as ten spores was mixed with 500 μL growth medium and incubated for 30 min for spore germination and mRNA production. Thus, only spores that are viable were detected. Subsequently, RNA was extracted from lysed cells, selectively amplified using NASBA, and rapidly identified by the biosensor. While the biosensor assay requires only 15 min assay time, the overall process takes 4 h for detection of ten viable B. anthracis spores, and is shortened significantly if more spores are present. The biosensor is based on an oligonucleotide sandwich-hybridization assay format. It uses a membrane flow-through system with an immobilized DNA probe that hybridizes with the target sequence. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye sulforhodamine B. The amount of liposomes captured in the detection zone can be read visually or quantified with a hand-held reflectometer. The biosensor can detect as little as 1 fmol target mRNA (1 nmol L−1). Specificity analysis revealed no cross-reactivity with 11 organisms tested, among them closely related species such as B. cereus, B. megaterium, B. subtilis, B. thuringiensis, Lactococcus lactis, Lactobacillus plantarum, and Chlostridium butyricum. Also, no false positive signals were obtained from nonviable spores. We suggest that this inexpensive biosensor is a viable option for rapid, on-site analysis providing highly specific data on the presence of viable B. anthracis spores.
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paper_title: High-throughput SPR sensor for food safety.

paper_content:

High-throughput surface plasmon resonance (SPR) biosensor for rapid and parallelized detection of nucleic acids identifying specific bacterial pathogens is reported. The biosensor consists of a high-performance SPR imaging sensor with polarization contrast and internal referencing (refractive index resolution 2 x 10(-7) RIU) and an array of DNA probes microspotted on the surface of the SPR sensor. It is demonstrated that short sequences of nucleic acids (20-23 bases) characteristic for bacterial pathogens such as Brucella abortus, Escherichia coli, and Staphylococcus aureus can be detected at 100 pM levels. Detection of specific DNA or RNA sequences can be performed in less than 15 min by the reported SPR sensor.
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paper_title: Nucleic acid approaches for detection and identification of biological warfare and infectious disease agents.

paper_content:

Biological warfare agents are the most problematic of the weapons of mass destruction and terror. Both civilian and military sources predict that over the next decade the threat from proliferation of these agents will increase significantly. In this review we summarize the state of the art in detection and identification of biological threat agents based on PCR technology with emphasis on the new technology of microarrays. The advantages and limitations of real-time PCR technology and a review of the literature as it applies to pathogen and virus detection are presented. The paper covers a number of issues related to the challenges facing biological threat agent detection technologies and identifies critical components that must be overcome for the emergence of reliable PCR-based DNA technologies as bioterrorism countermeasures and for environmental applications. The review evaluates various system components developed for an integrated DNA microchip and the potential applications of the next generation of fully automated DNA analyzers with integrated sample preparation and biosensing elements. The article also reviews promising devices and technologies that are near to being, or have been, commercialized.
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paper_title: DNA sensor based on an Escherichia coli lac Z gene probe immobilization at self-assembled monolayers-modified gold electrodes

paper_content:

Abstract A novel approach to construct an electrochemical DNA sensor based on immobilization of a 25 base single-stranded probe, specific to E. coli lac Z gene, onto a gold disk electrode is described. The capture probe is covalently attached using a self-assembled monolayer of 3,3′-dithiodipropionic acid di( N -succinimidyl ester) (DTSP) and mercaptohexanol (MCH) as spacer. Hybridization of the immobilized probe with the target DNA at the electrode surface was monitored by square wave voltammetry (SWV), using methylene blue (MB) as electrochemical indicator. Variables involved in the sensor performance, such as the DTSP concentration in the modification solution, the self-assembled monolayers (SAM) formation time, the DNA probe drying time atop the electrode surface and the amount of probe immobilized, were optimized. A good stability of the single- and double-stranded oligonucleotides immobilized on the DTSP-modified electrode was demonstrated, and a target DNA detection limit of 45 nM was achieved without signal amplification. Hybridization specificity was checked with non-complementary and mismatch oligonucleotides. A single-base mismatch oligonucleotide gave a hybridization response only 7 ± 3%, higher than the signal obtained for the capture probe before hybridization. The possibility of reusing the electrochemical genosensor was also tested.
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paper_title: A DNA electrochemical sensor based on nanogold-modified poly-2,6-pyridinedicarboxylic acid film and detection of PAT gene fragment.

paper_content:

Abstract A new DNA electrochemical biosensor is described for electrochemical impedance spectroscopy (EIS) detection of the sequence-specific DNA related to PAT transgene in the transgenic plants. Poly-2,6-pyridinedicarboxylic acid film (PDC) was fabricated by electropolymerizing 2,6-pyridinedicarboxylic acid on the glassy carbon electrode (GCE). The gold nanoparticles (NG) were modified on the PDC/GCE to prepare NG/PDC/GCE, and then DNA probe (ssDNA) was immobilized on the NG/PDC/GCE by the interaction of NG with DNA. The immobilization of NG and the immobilization and hybridization of DNA probe were characterized with differential pulse voltammetry (DPV) and cyclic voltammetry (CV) using methylene blue (MB) as indicator and EIS. MB had a couple of well-defined CV peaks at the NG/PDC/GCE, and these redox peak currents increased after the immobilization of the DNA probe. After the hybridization of the DNA probe with the complementary single-stranded DNA (cDNA), the redox peak currents of MB decreased greatly. The electron transfer resistance ( R et ) of the electrode surface in EIS in [Fe(CN) 6 ] 3–/4– solution increased after the immobilization of the DNA probe on the NG/PDC/GCE. The hybridization of the DNA probe with cDNA made R et increase further. EIS was used for the label-free detection of the target DNA. The NG modified on the PDC dramatically enhanced the immobilization amount of the DNA probe and greatly improved the sensitivity of DNA detection. The difference between the R et value at the ssDNA/NG/PDC/GCE and that at hybridization DNA-modified electrode (dsDNA/NG/PDC/GCE) was used as the signal for detecting the PAT gene fragment with the dynamic range from 1.0 × 10 −10 to 1.0 × 10 −5 mol/L. A detection limit of 2.4 × 10 –11 mol/L could be estimated.
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paper_title: Two electrophoreses in different pH buffers to purify forest soil DNA contaminated with humic substances

paper_content:

Direct extraction of DNA from soils is a useful way to gain genetic information on the soil source. However, DNA extraction from soils, especially forest soils, may be contaminated by humic substances due to their similar physical and chemical characteristics to soil. Even commercial soil DNA extraction kits fail to retrieve DNA from these soils. Using the potential changes of specific charge of DNA and humic substances in a pH solution, we performed two electrophoreses in different pH buffers to eliminate the interfering substances. The method produced high quality soil DNA, which is applicable for PCR amplification.
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paper_title: DNA single-base mismatch study with an electrochemical enzymatic genosensor

paper_content:

Abstract A thorough selectivity study of DNA hybridization employing an electrochemical enzymatic genosensor is discussed here. After immobilizing on a gold film a 30-mer 3′-thiolated DNA strand, hybridization with a biotinylated complementary one takes place. Then, alkaline phosphatase is incorporated to the duplex through the interaction streptavidin–biotin. Enzymatic generation of indigo blue from 3-indoxyl phosphate and subsequent electrochemical detection was made. The influence of hybridization conditions was studied in order to better discern between fully complementary and mismatched strands. Detection of 3, 2 and 1 mismatch was possible. The type and location of the single-base mismatch, as well as the influence of the length of the strands was studied too. Mutations that suppose displacement of the reading frame were also considered. The effect of the concentration on the selectivity was tested, resulting a highly selective genosensor with an adequate sensitivity and stability.
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paper_title: Microfluidic device architecture for electrochemical patterning and detection of multiple DNA sequences.

paper_content:

Electrochemical biosensors pose an attractive solution for point-of-care diagnostics because they require minimal instrumentation and they are scalable and readily integrated with microelectronics. The integration of electrochemical biosensors with microscale devices has, however, proven to be challenging due to significant incompatibilities among biomolecular stability, operation conditions of electrochemical sensors, and microfabrication techniques. Toward a solution to this problem, we have demonstrated here an electrochemical array architecture that supports the following processes in situ, within a self-enclosed microfluidic device: (a) electrode cleaning and preparation, (b) electrochemical addressing, patterning, and immobilization of sensing biomolecules at selected sensor pixels, (c) sequence-specific electrochemical detection from multiple pixels, and (d) regeneration of the sensing pixels. The architecture we have developed is general, and it should be applicable to a wide range of biosensing schemes that utilize gold-thiol self-assembled monolayer chemistry. As a proof-of-principle, we demonstrate the detection and differentiation of polymerase chain reaction (PCR) amplicons diagnostic of human (H1N1) and avian (H5N1) influenza.
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paper_title: Optical study of DNA surface hybridization reveals DNA surface density as a key parameter for microarray hybridization kinetics

paper_content:

We investigate the kinetics of DNA hybridization reactions on glass substrates, where one 22 mer strand (bound-DNA) is immobilized via phenylene-diisothiocyanate linker molecule on the substrate, the dye-labeled (Cy3) complementary strand (free-DNA) is in solution in a reaction chamber. We use total internal reflection fluorescence for surface detection of hybridization. As a new feature we perform a simultaneous real-time measurement of the change of free-DNA concentration in bulk parallel to the total internal reflection fluorescence measurement. We observe that the free-DNA concentration decreases considerably during hybridization. We show how the standard Langmuir kinetics needs to be extended to take into account the change in bulk concentration and explain our experimental results. Connecting both measurements we can estimate the surface density of accessible, immobilized bound-DNA. We discuss the implications with respect to DNA microarray detection.
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paper_title: A nucleic acid biosensor for the detection of a short sequence related to the hepatitis B virus using bis(benzimidazole)cadmium(II) dinitrate as an electrochemical indicator

paper_content:

Abstract A novel hybridization indicator, bis(benzimidazole)cadmium(II) dinitrate (Cd(bzim) 2 (NO 3 ) 2 ), was utilized to develop an electrochemical DNA biosensor for the detection of a short DNA sequence related to the hepatitis B virus (HBV). The sensor relies on the immobilization and hybridization of the 21-mer single-stranded oligonucleotide from the HBV long repeat at the glassy carbon electrode (GCE). The hybridization between the probe and its complementary sequence as the target was studied by enhancement of the peak of the Cd ( bzim ) 2 2 + indicator using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Numerous factors affecting the probe immobilization, target hybridization, and indicator binding reactions were optimized to maximize the sensitivity and speed of the assay time. With this approach, a sequence of the HBV could be quantified over the range from 1.49 × 10 −7 M to 1.06 × 10 −6 M, with a linear correlation of r = 0.9973 and a detection limit of 8.4 × 10 −8 M. The Cd ( bzim ) 2 2 + signal observed from the probe sequence before and after hybridization with a four-base mismatch containing sequence was lower than that observed after hybridization with a complementary sequence, showing good selectivity. These results demonstrate that the Cd ( bzim ) 2 2 + indicator provides great promise for the rapid and specific measurement of the target DNA.
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paper_title: A DNA electrochemical sensor prepared by electrodepositing zirconia on composite films of single-walled carbon nanotubes and poly(2,6-pyridinedicarboxylic acid), and its application to detection of the PAT gene fragment.

paper_content:

Carboxyl group-functionalized single-walled carbon nanotubes (SWNTs) and 2,6-pyridinedicarboxylic acid (PDC) were electropolymerized by cyclic voltammetry on a glassy-carbon electrode (GCE) surface to form composite films (SWNTs/PDC). Zirconia was then electrodeposited on the SWNTs/PDC/GCE from an aqueous electrolyte containing ZrOCl2 and KCl by cycling the potential between -1.1 V and +0.7 V at a scan rate of 20 mV s(-1). DNA probes with a phosphate group at the 5' end were easily immobilized on the zirconia thin films, because of the strong affinity between zirconia and phosphate groups. The sensors were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). EIS was used for label-free detection of the target DNA by measuring the increase of the electron transfer resistance (R(et)) of the electrode surface after the hybridization of the probe DNA with the target DNA. The PAT gene fragment and polymerase chain reaction (PCR) amplification of the NOS gene from transgenically modified beans were satisfactorily detected by use of this DNA electrochemical sensor. The dynamic range of detection of the sensor for the PAT gene fragment was from 1.0 x 10(-11) to 1.0 x 10(-6) mol L(-1) and the detection limit was 1.38 x 10(-12) mol L(-1).
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paper_title: Characterization of insecticidal crystal proteincry gene ofBacillus thuringiensis from soil of Sichuan Basin, China and cloning of novel haplotypescry gene

paper_content:

Sichuan Basin situated in the west of China, the fourth-largest basin of China, is a special area with complicated geomorphology (mountain, pasture, gorge, virgin forest, highland, hurst, glacier, and plain), and contains a rich and unique biodiversity. In order to describe a systematic study ofcry gene resources fromBacillus thuringiensis (Bt) strains of different ecological regions in Sichuan Basin, a total of 791 Bt strains have been screened from 2650 soil samples. The analysis of thecry genes was based on the method of PCR-restriction fragment length polymorphism (PCR-RFLP).cry1, cry2, cry3, cry4/10, cry9, cry30, andcry40-type genes were found in this basin. Strains containingcry1 genes were the most abundant in our collection (66%), and twenty-one differentcry1-type gene combinations were found. Bt strains harboringcry2 genes were the second most abundant (39.5%), and the strains containingcry3, cry9, cry4/10, cry30, andcry40 genes were found in 2.5, 3.5, 4.2, 4.2, and < 1%, respectively. Furthermore, several novel haplotypescry genes were found, and the full-length sequences of three novelcry genes were obtained, which were designated ascry52Ba1, cry54Aa1, cry30Fa1 by theB. thuringiensis Pesticide Crystal Protein Nomenclature Committee, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) assay of 80 strains which did not produce any PCR products indicated that these strains may harbour potentially novel Cry proteins. All these researches mentioned above revealed that the diversity and particularity ofcry gene resources fromB. thuringiensis strains in Sichuan Basin.
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paper_title: Integrated Microfluidic Electrochemical DNA Sensor

paper_content:

Effective systems for rapid, sequence-specific nucleic acid detection at the point of care would be valuable for a wide variety of applications, including clinical diagnostics, food safety, forensics, and environmental monitoring. Electrochemical detection offers many advantages as a basis for such platforms, including portability and ready integration with electronics. Toward this end, we report the Integrated Microfluidic Electrochemical DNA (IMED) sensor, which combines three key biochemical functionalities--symmetric PCR, enzymatic single-stranded DNA generation, and sequence-specific electrochemical detection--in a disposable, monolithic chip. Using this platform, we demonstrate detection of genomic DNA from Salmonella enterica serovar Typhimurium LT2 with a limit of detection of <10 aM, which is approximately 2 orders of magnitude lower than that from previously reported electrochemical chip-based methods.
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paper_title: Tracking the influence of long-term chromium pollution on soil bacterial community structures by comparative analyses of 16S rRNA gene phylotypes.

paper_content:

Abstract Bacterial community structures of highly chromium-polluted industrial landfill sites (G1 and G2) and a nearby control site (G3) were assessed using cultivation-dependent and cultivation-independent analyses. Sequencing of 16S rRNA genes discerned a total of 141 distinct operational taxonomic units (OTUs). Twelve different bacterial phyla were represented amongst 35, 34 and 72 different bacterial genera retrieved from sites G1, G2 and G3, respectively. The bacterial community of site G1 consisted of Firmicutes (52.75%), Gammaproteobacteria (18%), Actinobacteria (14.5%), Bacteriodetes (9.5%) and Deinococcus-Thermus (5.25%) and that of site G2 consisted of Firmicutes (31.25%), Alphaproteobacteria (7%), Betaproteobacteria (8%), Gammaproteobacteria (19%), Deltaproteobacteria (9.5%), Epsilonproteobacteria (3%), Actinobacteria (13%), Bacteriodetes (7.75%) and Deinococcus-Thermus (1.5%). The bacterial community of site G3 consisted of Firmicutes (6.25%), Alphaproteobacteria (7.5%), Betaproteobacteria (17.25%), Gammaproteobacteria (29.75%), Deltaproteobacteria (7.5%), Epsilonproteobacteria (4%), Actinobacteria (9.5%), Bacteriodetes (11.25%), Gemmatimonadetes (2.5%), Deinococcus-Thermus (1.8%), Chloroflexi (1.5%) and Planctomycetes (1.2%). The phyla of Gemmatimonadetes , Chloroflexi and Planctomycetes were not detected in sites G1 and G2; likewise, Alpha , Beta , Delta and Epsilon subdivisions of Proteobacteria were not recovered from site G1. These findings reveal that long-term chromium-induced perturbation results in community shifts towards a dominance of Firmicutes from Proteobacteria in the soil environment.
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paper_title: Cylinder-shaped conducting polypyrrole for labelless electrochemical multidetection of DNA

paper_content:

Abstract A new multidetection biosensor has been developed using the electrochemical properties of cylinder-shaped conducting polypyrrole grown on miniaturized graphite electrodes. Our objective was to conceive a sensitive, labelless and real-time DNA sensor for biomedical diagnosis. In a first step, copolymers bearing both ferrocene redox markers and oligonucleotide probes were selectively electro-addressed on microchip electrodes. Then, the study of their voltammetric response upon the addition of DNA targets revealed that the hybridization was efficiently transduced through the variation of ferrocene oxidation intensity. Using this technique, a good selectivity between Human Immunodeficiency Virus and Hepatitis B Virus targets was obtained. It was indeed possible to directly follow the hybridization. Complementary DNA detection limit reached 100 pM (3 fmol in 30 μL), which represents a good performance for such a practical, labelless and real-time sensor.
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paper_title: Selective Photoelectrochemical Detection of DNA with High-Affinity Metallointercalator and Tin Oxide Nanoparticle Electrode

paper_content:

Selective detection of double-stranded DNA (ds-DNA) in solution was achieved by photoelectrochemistry using a high-affinity DNA intercalator, Ru(bpy)2dppz (bpy = 2,2‘-bipyridine, dppz = dipyrido[3,2-a:2‘,3‘-c]phenazine) as the signal indicator and tin oxide nanoparticle as electrode material. When Ru(bpy)2dppz alone was irradiated with 470-nm light, anodic photocurrent was detected on the semiconductor electrode due to electron injection from its excited state into the conduction band of the electrode. The current was sustained in the presence of oxalate in solution, which acted as a sacrificial electron donor to regenerate the ground-state metal complex. After addition of double-stranded calf thymus DNA into the solution, photocurrent dropped substantially. The drop was attributed to the intercalation of Ru(bpy)2dppz into DNA and, consequently, the reduced mass diffusion of the indicator to the electrode, as well as electrostatic repulsion between oxalate anion and negative charges on DNA. The degree of ...
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paper_title: Microfluidic device architecture for electrochemical patterning and detection of multiple DNA sequences.

paper_content:

Electrochemical biosensors pose an attractive solution for point-of-care diagnostics because they require minimal instrumentation and they are scalable and readily integrated with microelectronics. The integration of electrochemical biosensors with microscale devices has, however, proven to be challenging due to significant incompatibilities among biomolecular stability, operation conditions of electrochemical sensors, and microfabrication techniques. Toward a solution to this problem, we have demonstrated here an electrochemical array architecture that supports the following processes in situ, within a self-enclosed microfluidic device: (a) electrode cleaning and preparation, (b) electrochemical addressing, patterning, and immobilization of sensing biomolecules at selected sensor pixels, (c) sequence-specific electrochemical detection from multiple pixels, and (d) regeneration of the sensing pixels. The architecture we have developed is general, and it should be applicable to a wide range of biosensing schemes that utilize gold-thiol self-assembled monolayer chemistry. As a proof-of-principle, we demonstrate the detection and differentiation of polymerase chain reaction (PCR) amplicons diagnostic of human (H1N1) and avian (H5N1) influenza.
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paper_title: Integrated Microfluidic Electrochemical DNA Sensor

paper_content:

Effective systems for rapid, sequence-specific nucleic acid detection at the point of care would be valuable for a wide variety of applications, including clinical diagnostics, food safety, forensics, and environmental monitoring. Electrochemical detection offers many advantages as a basis for such platforms, including portability and ready integration with electronics. Toward this end, we report the Integrated Microfluidic Electrochemical DNA (IMED) sensor, which combines three key biochemical functionalities--symmetric PCR, enzymatic single-stranded DNA generation, and sequence-specific electrochemical detection--in a disposable, monolithic chip. Using this platform, we demonstrate detection of genomic DNA from Salmonella enterica serovar Typhimurium LT2 with a limit of detection of <10 aM, which is approximately 2 orders of magnitude lower than that from previously reported electrochemical chip-based methods.
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Title: Microfluidics-Based Lab-on-Chip Systems in DNA-Based Biosensing: An Overview

Section 1: Introduction
Description 1: Write an introductory overview of advances in microfluidics for nanotechnology-based sensing methods and the challenges faced in developing diagnostic devices for environmental monitoring.

Section 2: The Physics of Microfluidics
Description 2: Discuss the fundamental flow physics and interfacial phenomena in microfluidics, emphasizing design considerations and the critical importance of fluid control and flow stability.

Section 3: Dimensionless Numbers
Description 3: Explain the key dimensionless numbers relevant to fluid mechanics and species transport in microfluidics, including the Reynolds number and the Peclet number, and their significance in microfluidic device design.

Section 4: Droplet Flow
Description 4: Introduce the concept of droplet-based microfluidics (digital microfluidics), detailing the advantages of droplet flow over traditional continuous-flow systems.

Section 5: Microfluidics-Based Pathogen Detection
Description 5: Explore the application of microfluidic biochips for pathogen detection, covering different electrochemical techniques for DNA hybridization and pathogen identification.

Section 6: DNA-Based Biosensor
Description 6: Describe the functioning and types of DNA-based biosensors, focusing on electrochemical detection methods and advancements in the field.

Section 7: Conclusions
Description 7: Summarize the key points covered in the paper, highlighting the importance of microfluidics-based LOC systems in environmental pathogen detection and discussing future directions in the field.