Showing papers on "Biochip published in 2009"

Microfluidic Systems for Pathogen Sensing: A Review

Abstract: Rapid pathogen sensing remains a pressing issue today since conventional identification methodsare tedious, cost intensive and time consuming, typically requiring from 48 to 72 h. In turn, chip based technologies, such as microarrays and microfluidic biochips, offer real alternatives capable of filling this technological gap. In particular microfluidic biochips make the development of fast, sensitive and portable diagnostic tools possible, thus promising rapid and accurate detection of a variety of pathogens. This paper will provide a broad overview of the novel achievements in the field of pathogen sensing by focusing on methods and devices that compliment microfluidics.

Overview of Electrochemical DNA Biosensors: New Approaches to Detect the Expression of Life

Abstract: DNA microarrays are an important tool with a variety of applications in gene expression studies, genotyping, pharmacogenomics, pathogen classification, drug discovery, sequencing and molecular diagnostics. They are having a strong impact in medical diagnostics for cancer, toxicology and infectious disease applications. A series of papers have been published describing DNA biochips as alternative to conventional microarray platforms to facilitate and ameliorate the signal readout. In this review, we will consider the different methods proposed for biochip construction, focusing on electrochemical detection of DNA. We also introduce a novel single-stranded DNA platform performing high-throughput SNP detection and gene expression profiling.

A portable cell-based impedance sensor for toxicity testing of drinking water.

Abstract: A major limitation to using mammalian cell-based biosensors for field testing of drinking water samples is the difficulty of maintaining cell viability and sterility without an on-site cell culture facility. This paper describes a portable automated bench-top mammalian cell-based toxicity sensor that incorporates enclosed fluidic biochips containing endothelial cells monitored by Electric Cell-substrate Impedance Sensing (ECIS) technology. Long-term maintenance of cells on the biochips is made possible by using a compact, self-contained disposable media delivery system. The toxicity sensor monitors changes in impedance of cell monolayers on the biochips after the introduction of water samples. The fluidic biochip includes an ECIS electronic layer and a polycarbonate channel layer, which together reduce initial impedance disturbances seen in commercially available open well ECIS chips caused by the mechanics of pipetting while maintaining the ability of the cells to respond to toxicants. A curve discrimination program was developed that compares impedance values over time between the control and treatment channels on the fluidic biochip and determines if they are significantly different. Toxicant responses of bovine pulmonary artery endothelial cells grown on fluidic biochips are similar to cells on commercially-available open well chips, and these cells can be maintained in the toxicity sensor device for at least nine days using an automated media delivery system. Longer-term cell storage is possible; bovine lung microvessel endothelial cells survive for up to four months on the fluidic biochips and remain responsive to a model toxicant. This is the first demonstration of a portable bench top system capable of both supporting cell health over extended periods of time and obtaining impedance measurements from endothelial cell monolayers after toxicant exposure.

Detection of viruses with molecularly imprinted polymers integrated on a microfluidic biochip using contact-less dielectric microsensors.

Abstract: Rapid detection of viral contamination remains a pressing issue in various fields related to human health including clinical diagnostics, the monitoring of food-borne pathogens, the detection of biological warfare agents as well as in viral clearance studies for biopharmaceutical products. The majority of currently available assays for virus detection are expensive, time-consuming, and labor-intensive. In the present work we report the creation of a novel micro total analysis system (µTAS) capable of continuously monitoring viral contamination with high sensitivity and selectivity. The specific interaction between shape and surface chemistry between molecular imprinted polymer (MIP) and virus resulted in the elimination of non-specific interaction in the present sensor configuration. The additional integration of the blank (non-imprinted) polymer further allowed for the identification of non-specific adsorption events. The novel combination of microfluidics containing integrated native polymer and MIP with contact-less dielectric microsensors is evaluated using the Tobacco Mosaic Virus (TMV) and the Human Rhinovirus serotype 2 (HRV2). Results show that viral binding and dissociation events can be readily detected using contact-less bioimpedance spectroscopy optimized for specific frequencies. In the present study optimum sensor performance was achieved at 203 kHz within the applied frequency range of 5–500 kHz. Complete removal of the virus from the MIP and device reusability is successfully demonstrated following a 50-fold increase in fluid velocity. Evaluation of the microfluidic biochip revealed that microchip technology is ideally suited to detect a broader range of viral contaminations with high sensitivity by selectively adjusting microfluidic conditions, sensor geometries and choice of MIP polymeric material.

Invited Review Article: Review of centrifugal microfluidic and bio-optical disks

Abstract: Spinning biodisks have advantages that make them attractive for specialized biochip applications. The two main classes of spinning biodisks are microfluidic disks and bio-optical compact disks (BioCD). Microfluidic biodisks take advantage of noninertial pumping for lab-on-a-chip devices using noninertial valves and switches under centrifugal and Coriolis forces to distribute fluids about the disks. BioCDs use spinning-disk interferometry, under the condition of common-path phase quadrature, to perform interferometric label-free detection of molecular recognition and binding. The optical detection of bound molecules on a disk is facilitated by rapid spinning that enables high-speed repetitive sampling to eliminate 1/f noise through common-mode rejection of intensity fluctuations and extensive signal averaging. Multiple quadrature classes have been developed, such as microdiffraction, in-line, phase contrast, and holographic adaptive optics. Thin molecular films are detected through the surface dipole density with a surface height sensitivity for the detection of protein spots that is approximately 1 pm. This sensitivity easily resolves a submonolayer of solid-support immobilized antibodies and their antigen targets. Fluorescence and light scattering provide additional optical detection techniques on spinning disks. Immunoassays have been applied to haptoglobin using protein A/G immobilization of antibodies and to prostate specific antigen. Small protein spots enable scalability to many spots per disk for high-throughput and highly multiplexed immonoassays.

Electrical Detection Of Biomarkers Using Bioactivated Microfluidic Channels

Abstract: The present disclosure encompasses the manufacture and use of rapid and inexpensive electrical biosensors comprising microelectrodes in a micro-channel. The devices of the disclosure can be used to detect and quantify target cells, protein biomarkers, and nucleic acid biomarkers, and the like, by measuring instantaneous changes in ionic impedance. The micro-channel devices of the disclosure are also suitable for the detection of target protein and oligonucleotide, and small molecule target biomarkers using protein-functionalized micro-channels for the rapid electrical detection and quantification of any type of target protein biomarker in a sample. The biochip microfluidic devices may be combined with an integrated circuitry into a portable handheld device for multiplex high throughput analysis using an array of micro-channels for probing clinically relevant samples, such as the human serum, for multiple protein and nucleic acid biomarkers for disease diagnosis, and the detection of potentially pathogenic organisms.

Fast multiplexed polymerase chain reaction for conventional and microfluidic short tandem repeat analysis.

Abstract: The time required for short tandem repeat (STR) amplification is determined by the temperature ramp rates of the thermal cycler,the components of the reaction mix, and the properties of the reaction vessel. Multiplex amplifications in microfluidic biochip-based and conventionaltube-based thermal cyclers have been demonstrated in 17.3 and 19 min, respectively. Optimized 28-cycle amplification protocols generated alleleswith signal strengths above calling thresholds, heterozygous peak height ratios of greater than 0.65, and incomplete nontemplate nucleotide additionand stutter of less than 15%. Full CODIS-compatible profiles were generated using the Profiler Plus ID, COfiler and Identifiler primer sets. PCR per-formance over a wide range of DNA template levels from 0.006 to 4 ng was characterized by separation and detection on a microfluidic electropho-resis system, Genebench-FXTM. The fast multiplex PCR approach has the potential to reduce process time and cost for STR analysis and enablesdevelopment of a fully integrated microfluidic forensic DNA analysis system

Detection of pathogenic E. coli O157:H7 by a hybrid microfluidic SPR and molecular imaging cytometry device

Abstract: Current methods to screen for bacterial contamination involve using costly reagents such as antibodies or PCR reagents or time-costly growth in cultures There is need for portable, real-time, multiplex pathogen detection technology that can predict the safety of food Surface plasmon resonance (SPR) imaging is a sensitive, label-free method that can detect the binding of an analyte to a surface by the changes in refractive index that occur upon binding We have designed a hybrid microfluidic biochip to perform multiplexed detection of single-celled pathogens using a combination of SPR and fluorescence imaging The device consists of an array of gold spots, each functionalized with a capture biomolecule targeting a specific pathogen This biosensor array is enclosed by a polydimethylsiloxane microfluidic flow chamber that delivers a magnetically concentrated sample to be tested The sample is imaged by SPR on the bottom of the biochip and epi-fluorescence on the top The prototype instrument was successfully able to image antibody-captured E coli O157:H7 bacteria by SPR and fluorescence imaging The efficiency of capture of these bacteria by the magnetic particles was determined using spectrophotometric ferric oxide absorbance measurements The binding of the E coli to each spot was quantified by measuring the percent of the gold spot area upon which the bacteria was bound and analyzed using NIH ImageJ software This hybrid imaging approach of pathogenic E coli detection coupled with an estimate of relative infectivity is shown to be a working example of a testing device for potential foodborne pathogens

Design and testing of a microfluidic biochip for cytokine enzyme-linked immunosorbent assay

Abstract: Enzyme-linked immunosorbent assay (ELISA) has been widely used in medical diagnostics, environmental analyses, and biochemical studies. To reduce assay time and lower consumption of reagents in cytokine ELISA analysis, a polymeric microfluidic biochip has been designed and fabricated via several new techniques: Polyaniline-based surface modification for superhydrophobic capillary valving and oxygen plasma-poly(ethyleneimine)-tyrosinase-protein A modification for high sensitivity protein detection. The proper flow sequencing was achieved using the superhydrophobic capillary valves. The burst frequency of each valve was experimentally determined and compared with two capillary force equations and the fluent finite element simulation. This fully automated microfluidic biochip with an analyzer is able to provide high fluorescence signal of ELISA with a wider linear detection range and a much shorter assay time than 96-well microtiter plates. It is applicable to a variety of nonclinic research and clinically relevant disease conditions. The modification technologies in this study can be implemented in other lab-on-a-chip systems, drug/gene delivery carriers, and other immunoassay biosensor applications.

Fault Modeling and Functional Test Methods for Digital Microfluidic Biochips

Abstract: Dependability is an important attribute for microfluidic biochips that are used for safety-critical applications, such as point-of-care health assessment, air-quality monitoring, and food-safety testing. Therefore, these devices must be adequately tested after manufacture and during bioassay operations. Known techniques for biochip testing are all function oblivious (i.e., while they can detect and locate defect sites on a microfluidic array, they cannot be used to ensure correct operation of functional units). In this paper, we introduce the concept of functional testing of microfluidic biochips. We address fundamental biochip operations, such as droplet dispensing, droplet transportation, mixing, splitting, and capacitive sensing. Long electrode actuation times are avoided to ensure that there is no electrode degradation during testing. The functional testing of pin-constrained biochips is also studied. We evaluate the proposed test methods using simulations as well as experiments for a fabricated biochip.

Tabu search-based synthesis of dynamically reconfigurable digital microfluidic biochips

Abstract: Microfluidic biochips are replacing the conventional biochemical analyzers, and are able to integrate on-chip all the necessary functions for biochemical analysis. The "digital" microfluidic biochips are manipulating liquids not as a continuous flow, but as discrete droplets, and hence they are highly reconfigurable and scalable. A digital biochip is composed of a two-dimensional array of cells, together with reservoirs for storing the samples and reagents. Several adjacent cells are dynamically grouped to form a virtual device, on which operations are executed. During the execution of an operation, the virtual device can be reconfigured to occupy a different group of cells on the array. In this paper, we present a Tabu Search metaheuristic for the synthesis of digital microfluidic biochips, which, starting from a biochemical application and a given biochip architecture, determines the allocation, resource binding, scheduling and placement of the operations in the application. In our approach, we consider moving the modules during their operation, in order to improve the completion time of the biochemical application. The proposed heuristic has been evaluated using three real-life case studies and ten synthetic benchmarks.

DNA as a Force Sensor in an Aptamer-Based Biochip for Adenosine

Abstract: Without prior signal amplification, small molecules are difficult to detect by current label-free biochip approaches. In the present study, we developed a label-free capture biochip based on the comparative measurement of unbinding forces allowing for direct detection of small-molecule−aptamer interactions. The principle of this assay relies on increased unbinding forces of bipartite aptamers due to complex formation with their cognate ligands. The bipartite aptamers are immobilized on glass support via short DNA duplexes that serve as references to which unbinding forces can be compared. In a simple model system, adenosine is captured from solution by an adenosine-selective aptamer. Linking the molecular chains, each consisting of a short DNA reference duplex and a bipartite aptamer, between glass and a poly(dimethylsiloxane) (PDMS) surface and subsequently separating the surfaces compares the unbinding forces of the two bonds directly. Fluorescence readout allows for quantification of the fractions of b...

Monitoring of cell growth in vitro using biochips packaged with indium tin oxide sensors

Abstract: Cell-based biosensors, which treat living cells as sensing elements, are able to detect the functional information of biologically active analytes and also provide quantitative analysis. In general, they maintain living cells and observe the cellular physiological response after subjecting cells to stimulus and verify the presence and the concentration of these stimuli. This paper describes the use of indium tin oxide (ITO) as a bioimpedance sensor. ITO was chosen as the sensor material due to its optical transparency, electrical characteristics and biocompatibility. These features enabled cells to be visualised and cell signalling to be assessed thus enabling real time non-invasive in vitro analysis of the physiological state of biological cells. The developed biochip integrates optical and electronic devices for monitoring cell behaviour and would facilitate long-term measurement of cytotoxicity. It is envisaged that the developed technology together with proprietary assays will address markets including the pharmaceutical industry, environmental monitoring, health care and security/defence sectors.

ILP-based pin-count aware design methodology for microfluidic biochips

Abstract: Digital microfluidic biochips have emerged as a popular alternative for laboratory experiments. To make the biochip feasible for practical applications, pin-count reduction is a key problem to higher-level integration of reactions on a biochip. Most previous works approach the problem by post-processing the placement and routing solutions to share compatible control signals; however, the quality of such sharing algorithms is inevitably limited by the placement and routing solutions. We present in this paper a comprehensive pin-constrained biochip design flow that addresses the pin-count issue at all design stages. The proposed flow consists of three major stages: (1) pin-count aware stage assignment that partitions the reactions in the given bioassay into execution stages, (2) pin-count aware device assignment that determines a specific device used for each reaction, and (3) guided placement, routing, and pin assignment that utilize the pin-count saving properties from the stage and device assignments to optimize the assay time and pin count. For both the stage and device assignments, exact ILP formulations and effective solution-space reduction schemes are proposed to minimize the assay time and pin count. Experimental results show the efficiency of our algorithms/flow and a 55--57% pin-count reduction over the state-of-the-art algorithms/flow.

Real-time detection of the early event of cytotoxicity of herbal ingredients on single leukemia cells studied in a microfluidic biochip

Abstract: A microfluidic approach has been developed for the real-time detection of drug effects, based on the quantitative measurement of calibrated cytosolic calcium ([Ca2+]i) on single cancer cells. This microfluidic method is rapid by detecting the early event of cytotoxicity of drug candidates on cancer cells, without waiting for a couple of days needed for cell seeding and drug treatment by conventional assays. The miniaturized biochip consists of a V-shaped structure for the single-cell selection and retention. Various test reagents such as the chemotherapy drug (daunorubicin), an ionophore (ionomycin), and herbal ingredients from licorice (isoliquiritigenin or IQ) were investigated for their abilities to stimulate sustained cellular [Ca2+]i elevations. The microfluidic results obtained in hours have been confirmed by conventional cytotoxicity assays which take days to complete. Moreover, any color or chemical interference problems found in the conventional assays of herbal compounds could be resolved. Using the microfluidic approach, IQ (50 μM) has been found to cause a sustained [Ca2+]i elevation and cytotoxic effects on leukemia cells. The microfluidic single-cell analysis not only reduces reagent cost, and demands less cells, but also reveals some phenomena due to cellular heterogeneity that cannot be observed in bulk analysis.

Single step patterning of molecularly imprinted polymers for large scale fabrication of microbiochips.

Abstract: We use photolithography to pattern molecularly imprinted polymers for the wafer-scale production of biochips. We are able to produce multiplexed, spatially resolved micrometer-sized features of functional materials capable of molecular recognition. Using a fluorescent probe, dansyl-L-Phe, we show specific analyte binding to MIP patterns imprinted with boc-L-Phe, by fluorescence microscopy. Advantages of this technique are the control of shape and size of the patterns with a resolution of 1.5 µm, and the possibility of depositing a number of different MIPs on the same chip (parallelization). Multiplexing chips on the same substrate paves the road to their mass-production. Because of the simplicity of the method and the low cost of chip fabrication, we believe that mass production of portable microbiochips based on stable MIPs is now in close reach. Their combination with integrated transducers fabricated by micromachining techniques appears also possible.

Cross-contamination avoidance for droplet routing in digital microfluidic biochips

Abstract: Recent advances in droplet-based digital microfluidics have enabled biochip devices for DNA sequencing, immunoassays, clinical chemistry, and protein crystallization. Since cross-contamination between droplets of different biomolecules can lead to erroneous outcomes for bioassays, the avoidance of cross-contamination during droplet routing is a key design challenge for biochips. We propose a droplet-routing method that avoids cross-contamination in the optimization of droplet flow paths. The proposed approach targets disjoint droplet routes and minimizes the number of cells used for droplet routing. We also minimize the number of wash operations that must be used between successive routing steps that share unit cells in the microfluidic array. Two real-life biochemical applications are used to evaluate the proposed droplet-routing methods.

Protein-diazonium adduct direct electrografting onto SPRi-biochip.

Abstract: A direct protein immobilization method for surface plasmon resonance imaging (SPRi) gold chip arraying is exposed. The biomolecule electroaddressing strategy, previously demonstrated by our team on carbon surfaces, is here valuably involved and adapted to create a straightforward and efficient protein immobilization process onto SPRi-biochips. The proteins, modified with an aryl-diazonium adduct, are addressed to the SPRi chip surface through the electroreduction of the aryl-diazonium. The biomolecule deposition was followed through SPRi live measurements during the electrografting process. A specially designed setup enabled us to directly observe the mass increasing at the sensor surface while the proteins were electrografted. A pin electrospotting method, allowing the achievement of distinct sensing layers on gold SPRi-biochips, was used to generate microarray biochips. The integrity of the immobilized proteins and the specificity of the detection, based on antigen/antibody interactions, were demonstrated for the detection of specific antibodies and ovalbumin. The SPRi detection limit of ovalbumin using the electroaddressing of anti-ovalbumin IgG was compared with two other immobilization procedures, cystamine-glutaraldehyde self-assembled monolayer and pyrrole, and was found to be a decade lower than these ones (100 ng/mL, i.e., 2 nM).

A conventional route to scalable morphology-controlled regular structures and their superhydrophobic/hydrophilic properties for biochips application.

Abstract: We use a conventional and straightforward route to fabricate scalable morphology-controlled regular structures. This route is based on the etching of PDMS microlens array in CF4 and CF4/O2 plasma. PDMS microlens array can be changed to regularly isolated microdot structures array in CF4 plasma. Microbowl shaped structures array can be reached in CF4/O2 plasma. Moreover, a set of structures after CF4 plasma treatment display superhydrophobicity, while a set of structures after CF4/O2 plasma treatment present hydrophilicity. DNA molecules can be readily enriched on the hydrophilic surface. We believe that the regular structure array surfaces provide a useful inspiration towards biomolecular detection and transportation in biochips.

"Print-n-Shrink" technology for the rapid production of microfluidic chips and protein microarrays.

Abstract: An innovative method for the production of microfluidic chips integrating protein spots is described. The technology, called “Print-n-Shrink”, is based on the screen-printing of a microfluidic design (using a dielectric ink) onto Polyshrink™ polystyrene sheets. The initial print which has a minimum size of 15 µm (height) × 230 µm (width) is thermally treated (30 seconds, 163 °C) to shrink and generate features of 85 µm (height) × 100 µm (width). Concomitantly, proteins such as monoclonal antibodies or cellular adhesion proteins are spotted onto the Polyshrink™ sheets and shrunk together with the microfluidic design, creating a complete biochip integrating both complex microfluidic designs and protein spots for bioanalytical applications.

New cysteamine based functionalization for biochip applications

Abstract: A new versatile chemistry on gold surface has been developed and studied with a Surface Plasmon Resonance Imaging system. It is based on a monolayer of homobifunctional cross-linker 1,4 phenylenediisothiocyanate (PDC) modified with neutravidin bounded onto a gold film coated with a self-assembled monolayer of β-mercaptoethylamine (cysteamine). Using this surface chemistry, a DNA microarray affinity sensor has been developed. Specificity, regenerability and surface homogeneity were quantified and successfully applied to both synthetic oligonucleotides and PCR-amplified products DNA–DNA interactions.

A biochip reader using super critical angle fluorescence

Abstract: We present fluorescence chip reader for rapid, multiplexed assay measurements on plastic biochips. The system is designed as a low-cost, compact and robust instrument for point of care testing. Fluorescence imaging of biochip arrays is accomplished with a custom designed piezo-motor-driven scanning stage coupled to an optical element based on supercritical angle fluorescence (SAF). The use of SAF not only provides substantial enhancement of the fluorescence collection efficiency but also confines the fluorescence detection volume strictly to the close proximity of the biochip surface, thereby discriminating against fluorescence background from the analyte solution. The high imaging resolution of the SAF scanner is demonstrated by measuring the point spread function of 200 nm fluorescence beads. The sensitivity of the system is determined by measuring an array of low fluorophore concentrations and by real-time monitoring of a model bioassay.