Detection of E. coli using a microfluidics-based antibody biochip detection system

This work demonstrates the detection of E. coli using a 2-dimensional photosensor array biochip which is efficiently equipped with a microfluidics sample/reagent delivery system for on-chip monitoring of bioassays. The biochip features a 4 × 4 array of independently operating photodiodes that are integrated along with amplifiers, discriminators and logic circuitry on a single platform. The microfluidics system includes a single 0.4 mL reaction chamber which houses a sampling platform that selectively captures detection probes from a sample through the use of immobilized bioreceptors. The independently operating photodiodes allow simultaneous monitoring of multiple samples. In this study the sampling platform is a cellulosic membrane that is exposed to E. coli organisms and subsequently analyzed using a sandwich immunoassay involving a Cy5-labeled antibody probe. The combined effectiveness of the integrated circuit (IC) biochip and the immunoassay is evaluated for assays performed both by conventional laboratory means followed by detection with the IC biochip, and through the use of the microfluidics system for on-chip detection. Highlights of the studies show that the biochip has a linear dynamic range of three orders of magnitude observed for conventional assays, and can detect 20 E. coli organisms. Selective detection of E. coli in a complex medium, milk diluent, is also reported for both off-chip and on-chip assays. Received: 13 October 2000 / Revised: 13 November 2000 / Accepted: 13 November 2000     

Multi-analyte analysis system using an antibody-based biochip

A multi-analyte detection system using a unique antibody (Ab) biochip is described. The Ab-based biochip, also referred to as the protein biochip, uses a sensor array based on a complementary metal oxide silicon (CMOS) integrated circuit. The Ab-biochip has a sampling platform of four-by-four microarrays of antibodies deposited onto a Nylon membrane substrate. The micro-arrayed antibodies can be interrogated simultaneously or sequentially using the biochip sensing array detector with the use of a diffractive optical element illuminating each antibody spot individually. The usefulness of the Ab biochip is illustrated by the measurements of immunoglobulin G (IgG) used as the model analyte system. The detection limit for Cy5-labeled IgG molecules was 13 pg.     

Miniature biochip system for detection of Escherichia coli O157:H7 based on antibody-immobilized capillary reactors and enzyme-linked immunosorbent assay

In this work, we report Escherichia coli O157:H7 detection using antibody-immobilized capillary reactors, enzyme-linked immunosorbent assay (ELISA), and a biochip system. ELISA selective immunological method to detect pathogenic bacteria. ELISA is also directly adaptable to a miniature biochip system that utilizes conventional sample platforms such as polymer membranes and glass. The antibody-immobilized capillary reactor is a very attractive sample platform for ELISA because of its low cost, compactness, reuse, and ease of regeneration. Moreover, an array of capillary reactors can provide high-throughput ELISA. In this report, we describe the use of an array of antibody-immobilized capillary reactors for multiplex detection of E. coli O157:H7 in our miniature biochip system. Side-entry laser beam irradiation to an array of capillary reactors contributes significantly to miniaturized optical configuration for this biochip system. The detection limits of E. coli O157:H7 using the ELISA and Cy5 label-based immunoassays were determined to be 3 and 230 cells, respectively. This system shows capability to simultaneously monitor multifunctional immunoassay and high sensitive detection of E. coli O157:H7.     

A high-throughput microfluidic biochip to quantify bacterial adhesion to single host cells by real-time PCR assay

A high-throughput microfluidic poly-(dimethylsiloxane) biochip was developed to quantify bacterial adhesion to single host cells by real-time PCR assay. The biochip is simply structured with a two-dimensional array of 900 micro-wells, one inlet, and one outlet micro-channels. Isolation of single infected host cells into the individual micro-wells of the biochip was achieved by one-step vacuum-driven microfluidics. The adhered bacterial cells were then quantified by direct on-chip real-time PCR assay with single-bacterium-detection sensitivity. The performance of this microfluidic platform was demonstrated through profiling of the association of a common bacterial pathogen, Pseudomonas aeruginosa, to single host human lung epithelial A549 cells, revealing an adherence distribution that has not been previously reported. This microfluidic platform offers a simple and effective tool for biologists to analyze pathogen–host interaction at the single-cell level without the necessities of fluorescence labeling. The chip can similarly be used for other PCR-based applications requiring single-cell analysis.     

A polymer lab-on-a-chip for magnetic immunoassay with on-chip sampling and detection capabilities

This paper presents a new polymer lab-on-a-chip for magnetic bead-based immunoassay with fully on-chip sampling and detection capabilities, which provides a smart platform of magnetic immunoassay-based lab-on-a-chip for point-of-care testing (POCT) toward biochemical hazardous agent detection, food inspection or clinical diagnostics. In this new approach, the polymer lab-on-a-chip for magnetic bead-based immunoassay consists of a magnetic bead-based separator, an interdigitated array (IDA) micro electrode, and a microfluidic system, which are fully incorporated into a lab-on-a-chip on cyclic olefin copolymer (COC). Since the polymer lab-on-a-chip was realized using low cost, high throughput polymer microfabrication techniques such as micro injection molding and hot embossing method, a disposable polymer lab-on-a-chip for the magnetic bead-based immunoassay can be successfully realized in a disposable platform. With this newly developed polymer lab-on-a-chip, an enzyme-labelled electrochemical immunoassay (ECIA) was performed using magnetic beads as the mobile solid support, and the final enzyme product produced from the ECIA was measured using chronoamperometry. A sampling and detection of as low as 16.4 ng mL(-1) of mouse IgG has been successfully performed in 35 min for the entire procedure.     

Demonstration of a surface plasmon-coupled emission (SPCE)-based immunoassay in the absence of a spacer layer

The technique of surface plasmon-coupled emission (SPCE) involves the coupling of light which is emitted from a fluorophore into the surface plasmon of an adjacent thin metal film, giving rise to highly directional emission. We have combined the advantages of SPCE with the high light collection efficiency of supercritical angle fluorescence by carrying out an immunoassay on a paraboloid array biochip in the absence of the conventional SPCE spacer layer normally used to minimize metal quenching of the fluorescence. In this work, we have successfully demonstrated an SPCE-based assay by utilizing the protein assay layer as the spacer layer. A novel 3 × 3 injection molded polymer biochip with paraboloid elements was used. The paraboloid elements served to enhance the light collection efficiency while the top surface was coated with a gold layer to use excitation of surface plasmons and detection of SPCE emission. Theoretical modeling of the gold-protein layer structure showed that the surface plasmon resonance angles were located in the detection range of the paraboloid biochip. The polarization dependence of SPCE emission was also demonstrated. Finally, a human IgG sandwich immunoassay was carried out which exhibited a limit of detection of ~10 ng/ml using 3σ. The results demonstrate the potential of the SPCE-based paraboloid array biochip as a novel platform for high-throughput analysis of biomolecular interactions.     

1-Million droplet array with wide-field fluorescence imaging for digital PCR

Digital droplet reactors are useful as chemical and biological containers to discretize reagents into picolitre or nanolitre volumes for analysis of single cells, organisms, or molecules. However, most DNA based assays require processing of samples on the order of tens of microlitres and contain as few as one to as many as millions of fragments to be detected. Presented in this work is a droplet microfluidic platform and fluorescence imaging setup designed to better meet the needs of the high-throughput and high-dynamic-range by integrating multiple high-throughput droplet processing schemes on the chip. The design is capable of generating over 1-million, monodisperse, 50 picolitre droplets in 2-7 minutes that then self-assemble into high density 3-dimensional sphere packing configurations in a large viewing chamber for visualization and analysis. This device then undergoes on-chip polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the sample's nucleic acid contents. Wide-field fluorescence images are captured using a low cost 21-megapixel digital camera and macro-lens with an 8-12 cm(2) field-of-view at 1× to 0.85× magnification, respectively. We demonstrate both end-point and real-time imaging ability to perform on-chip quantitative digital PCR analysis of the entire droplet array. Compared to previous work, this highly integrated design yields a 100-fold increase in the number of on-chip digitized reactors with simultaneous fluorescence imaging for digital PCR based assays.     

Multiplex detection platform for tumor markers and glucose in serum based on a microfluidic microparticle array

We present a multiplex detection platform based on a microfluidic microparticle array to detect proteins and glucose in serum simultaneously. Multiplex detection of proteins and glucose was performed using biofunctionalized microparticles arrayed on gel-based microstructures integrated in microfluidics. The microparticles immobilized on these microstructures showed high stability under microfluidic flow conditions. With arrays of antibody-coated microbeads, microfluidic quantitative immunoassays for two protein tumor markers, human chorionic gonadotropin (hCG) and prostate specific antigen (PSA) were performed in serum samples with detection limits bellow the cut-off values for cancer diagnosis. Parallel to the immunoassays, quantitative enzymatic assays for glucose in the physiological concentration range were performed. Multiplex detection was achieved by using a spatially encoded microarray. By patterning antibody-coated microbeads and enzyme-containing microparticles on a novel mixed structure array, we successfully demonstrated simultaneous immunoassays (binding based assay) for proteins and an enzymatic assay (reaction kinetic based assay) for glucose. Our microparticle arrays could be potentially used for the detection of multiple categories of biomolecules (proteins, small metabolites and DNA) for clinical diagnostics and other biological applications.     

Nanophotonic lab-on-a-chip platforms including novel bimodal interferometers, microfluidics and grating couplers

One of the main limitations for achieving truly lab-on-a-chip (LOC) devices for point-of-care diagnosis is the incorporation of the "on-chip" detection. Indeed, most of the state-of-the-art LOC devices usually require complex read-out instrumentation, losing the main advantages of portability and simplicity. In this context, we present our last advances towards the achievement of a portable and label-free LOC platform with highly sensitive "on-chip" detection by using nanophotonic biosensors. Bimodal waveguide interferometers fabricated by standard silicon processes have been integrated with sub-micronic grating couplers for efficient light in-coupling, showing a phase resolution of 6.6 × 10(-4)× 2π rad and a limit of detection of 3.3 × 10(-7) refractive index unit (RIU) in bulk. A 3D network of SU-8 polymer microfluidics monolithically assembled at the wafer-level was included, ensuring perfect sealing and compact packaging. To overcome some of the drawbacks inherent to interferometric read-outs, a novel all-optical wavelength modulation system has been implemented, providing a linear response and a direct read-out of the phase variation. Sensitivity, specificity and reproducibility of the wavelength modulated BiMW sensor has been demonstrated through the label-free immunodetection of the human hormone hTSH at picomolar level using a reliable biofunctionalization process.     

Integrated microfluidic devices

“With the fundamentals of microscale flow and species transport well developed, the recent trend in microfluidics has been to work towards the development of integrated devices which incorporate multiple fluidic, electronic and mechanical components or chemical processes onto a single chip sized substrate. Along with this has been a major push towards portability and therefore a decreased reliance on external infrastructure (such as detection sensors, heaters or voltage sources).” In this review we provide an in-depth look at the “state-of-the-art” in integrated microfludic devices for a broad range of application areas from on-chip DNA analysis, immunoassays and cytometry to advances in integrated detection technologies for and miniaturized fuel processing devices. In each area a few representative devices are examined with the intent of introducing the operating procedure, construction materials and manufacturing technique, as well as any unique and interesting features.     

Integrated circuit/microfluidic chip to programmably trap and move cells and droplets with dielectrophoresis

We present an integrated circuit/microfluidic chip that traps and moves individual living biological cells and chemical droplets along programmable paths using dielectrophoresis (DEP). Our chip combines the biocompatibility of microfluidics with the programmability and complexity of integrated circuits (ICs). The chip is capable of simultaneously and independently controlling the location of thousands of dielectric objects, such as cells and chemical droplets. The chip consists of an array of 128 x 256 pixels, 11 x 11 microm(2) in size, controlled by built-in SRAM memory; each pixel can be energized by a radio frequency (RF) voltage of up to 5 V(pp). The IC was built in a commercial foundry and the microfluidic chamber was fabricated on its top surface at Harvard. Using this hybrid chip, we have moved yeast and mammalian cells through a microfluidic chamber at speeds up to 30 microm sec(-1). Thousands of cells can be individually trapped and simultaneously positioned in controlled patterns. The chip can trap and move pL droplets of water in oil, split one droplet into two, and mix two droplets into one. Our IC/microfluidic chip provides a versatile platform to trap and move large numbers of cells and fluid droplets individually for lab-on-a-chip applications.     

Gold patterned biochips for on-chip immuno-MALDI-TOF MS: SPR imaging coupled multi-protein MS analysis

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of immuno-captured target protein efficiently complements conventional immunoassays by offering rich molecular information such as protein isoforms or modifications. Direct immobilization of antibodies on MALDI solid support enables both target enrichment and MS analysis on the same plate, allowing simplified and potentially multiplexing protein MS analysis. Reliable on-chip immuno-MALDI-TOF MS for multiple biomarkers requires successful adaptation of antibody array biochips, which also must accommodate consistent reaction conditions on antibody arrays during immuno-capture and MS analysis. Here we developed a facile fabrication process of versatile antibody array biochips for reliable on-chip MALDI-TOF-MS analysis of multiple immuno-captured proteins. Hydrophilic gold arrays surrounded by super-hydrophobic surfaces were formed on a gold patterned biochip via spontaneous chemical or protein layer deposition. From antibody immobilization to MALDI matrix treatment, this hydrophilic/phobic pattern allowed highly consistent surface reactions on each gold spot. Various antibodies were immobilized on these gold spots both by covalent coupling or protein G binding. Four different protein markers were successfully analyzed on the present immuno-MALDI biochip from complex protein mixtures including serum samples. Tryptic digests of captured PSA protein were also effectively detected by on-chip MALDI-TOF-MS. Moreover, the present MALDI biochip can be directly applied to the SPR imaging system, by which antibody and subsequent antigen immobilization were successfully monitored.     

Integrated cell manipulation system--CMOS/microfluidic hybrid

Manipulation of biological cells using a CMOS/microfluidic hybrid system is demonstrated. The hybrid system starts with a custom-designed CMOS (complementary metal-oxide semiconductor) chip fabricated in a semiconductor foundry. A microfluidic channel is post-fabricated on top of the CMOS chip to provide biocompatible environments. The motion of individual biological cells that are tagged with magnetic beads is directly controlled by the CMOS chip that generates microscopic magnetic field patterns using an on-chip array of micro-electromagnets. Furthermore, the CMOS chip allows high-speed and programmable reconfiguration of the magnetic fields, substantially increasing the manipulation capability of the hybrid system. Extending from previous work that verified the concept of the hybrid system, this paper reports a set of manipulation experiments with biological cells, which further confirms the advantage of the hybrid approach. To enhance the biocompatibility of the system, the microfluidic channel is redesigned and the temperature of the device is monitored by on-chip sensors. Combining microelectronics and microfluidics, the CMOS/microfluidic hybrid system presents a new model for a cell manipulation platform in biological and biomedical applications.     

Probing chemical induced cellular stress by non-Faradaic electrochemical impedance spectroscopy using an Escherichia coli capacitive biochip

A new capacitive biochip was developed using carboxy-CNT activated gold interdigitated (GID) capacitors immobilized with E. coli cells for the detection of cellular stress caused by chemicals. Here, acetic acid, H(2)O(2) and NaCl were employed as model chemicals to test the biochip and monitored the responses under AC electrical field by non-Faradaic electrochemical impedance spectroscopy (nFEIS). The electrical properties of E. coli cells under different stresses were studied based on the change in surface capacitance as a function of applied frequency (300-600 MHz) in a label-free and noninvasive manner. The capacitive response of the E. coli biochip under normal conditions exhibited characteristic dispersion peaks at 463 and 582 MHz frequencies. Deformation of these signature peaks determined the toxicity of chemicals to E. coli on the capacitive biochip. The E. coli cells were sensitive to, and severely affected by 166-498 mM (1-3%) acetic acid with declined capacitance responses. The E. coli biochip exposed to H(2)O(2) exhibited adaptive responses at lower concentrations (<2%), while at a higher level (882 mM, 3%), the capacitance response declined due to oxidative toxicity in cells. However, E. coli cells were not severely affected by high NaCl levels (513-684 mM, 3-4%) as the cells tend to resist the salt stress. Our results demonstrated that the biochip response at a particular frequency enabled the determination of the severity of the stress imposed by chemicals and it can be potentially applied for monitoring unknown chemicals as an indicator of cytotoxicity.     

Integrated circuit microchip system with multiplex capillary electrophoresis module for DNA analysis

In this paper, we describe the use of an integrated circuit (IC) microchip system as a detector in multiplex capillary electrophoresis (CE). This combination of multiplex capillary gel electrophoresis and the IC microchip technology represents a novel approach to DNA analysis on the microchip platform. Separation of DNA ladders using a multiplex CE microsystem of four capillaries was monitored simultaneously using the IC microchip system. The IC microchip-CE system has advantages such as low cost, rapid analysis, compactness, and multiplex capability, and has great potential as an alternative system to conventional capillary array gel electrophoresis systems based on charge-coupled device (CCD) detection.     

Optofluidic detection for cellular phenotyping

Quantitative analysis of the output of processes and molecular interactions within a single cell is highly critical to the advancement of accurate disease screening and personalized medicine. Optical detection is one of the most broadly adapted measurement methods in biological and clinical assays and serves cellular phenotyping. Recently, microfluidics has obtained increasing attention due to several advantages, such as small sample and reagent volumes, very high throughput, and accurate flow control in the spatial and temporal domains. Optofluidics, which is the attempt to integrate optics with microfluidics, shows great promise to enable on-chip phenotypic measurements with high precision, sensitivity, specificity, and simplicity. This paper reviews the most recent developments of optofluidic technologies for cellular phenotyping optical detection.     

Denaturing gradient-based two-dimensional gene mutation scanning in a polymer microfluidic network

An integrated two-dimensional (2-D) DNA separation platform, combining standard gel electrophoresis with temperature gradient gel electrophoresis (TGGE) on a polymer microfluidic chip, is reported. Rather than sequentially sampling DNA fragments eluted from standard gel electrophoresis, size-resolved fragments are simultaneously electrokinetically transferred into an array of orthogonal microchannels and screened for the presence of sequence heterogeneity by TGGE in a parallel and high throughput format. A bulk heater assembly is designed and employed to externally generate a temporal temperature gradient along an array of TGGE channels. Extensive finite element modeling is performed to determine the optimal geometries of the microfluidic network for minimizing analyte band dispersion caused by interconnected channels in the network. A pH-mediated on-chip analyte stacking strategy is employed prior to the parallel TGGE separations to further reduce additional band broadening acquired during the electrokinetic transfer of DNA fragments between the first and second separation dimensions. A comprehensive 2-D DNA separation is completed in less than 5 min for positive detection of single-nucleotide polymorphisms in multiplex PCR products that vary in size and sequence.     

One-step pathogen specific DNA extraction from whole blood on a centrifugal microfluidic device

We report a fully integrated, pathogen-specific DNA extraction device utilizing centrifugal microfluidics on a polymer based CD platform. By use of the innovative laser irradiated Ferrowax microvalve (LIFM) together with the rapid cell lysis method using laser irradiation on magnetic particles, we could, for the first time, demonstrate a fully integrated pathogen specific DNA extraction from whole blood on a CD. As a model study, DNA extraction experiments from whole blood spiked with Hepatitis B virus (HBV) and E.coli were conducted. The total process of the plasma separation, mixing with magnetic beads conjugated with target specific antibodies, removal of plasma residual, washing and DNA extraction was finished within 12 min with only one manual step, the loading of 100 microL of whole blood. Real-time PCR results showed that the concentration of DNA prepared on a CD using a portable sample preparation device was as good as those by conventional bench top protocol. It demonstrates that our novel centrifugal microfluidics platform enables a full integration of complex biological reactions that require multi-step fluidic control.     

Patterning chemical stimulation of reconstructed neuronal networks

A spatially resolved delivery of substances integrated with cell culture substrates shows promise for application in pharmacological assays, bioanalytical studies on cell signaling pathways and cell-based biosensors, where control over the extracellular biochemical environment with a cellular resolution is desirable. In this work, we studied a biohybrid system where rat embryonic cortical neuronal networks are reconstructed on microstructured silicon chips and interfaced to microfluidics. The design of cell-cell and cell-medium interactions in confined geometries is presented. We developed an aligned microcontact printing technique (AμCP) for poly(lysine)-extracellular matrix proteins on microstructured chips, which allows a high degree of geometrical control over the network architecture and alignment of the neuronal network with the microfluidic features of a substrate. Spatially resolved on-chip delivery of compounds with a cellular resolution is demonstrated by chemical stimulation of patterned rat cortical neurons within a network with a number of solutions of excitatory neurotransmitter glutamate delivered via microfluidics. The combination of the system described with a patch-clamp technique allowed both modulation of the biochemical environment on a cellular level and the monitoring of electrophysiological properties in the reconstructed rat embryonic cortical networks changed by this microenvironment.     

Development of an Evidence biochip array kit for the multiplex screening of more than 20 anthelmintic drugs

Anthelmintic drugs are used in clinical and veterinary practice for the treatment of infections caused by parasitic worms. Their extensive use in food-producing animals can cause the presence of residues in food. For consumer protection it is necessary to monitor the levels of anthelmintic residues to ensure that they remain within the legally permitted maximum acceptable concentrations. For this purpose, the use of multiplex screening methods is advantageous. Biochip array technology allows the simultaneous determination of multiple analytes from a single sample at a single point in time. This study reports the development of an Evidence biochip array for the multiplex screening of anthelmintic drugs. Simultaneous competitive chemiluminescent immunoassays are employed. The solid support and vessel is the biochip, which contains an array of discrete test sites. The assays were applied to the semiautomated bench-top analyser Evidence Investigator. The aminobenzimidazoles assay detected aminomebendazole, albendazole 2-aminosulphone and aminoflubendazole, the avermectins assay detected emamectin benzoate, eprinomectin, abamectin, ivermectin and doramectin, the benzimidazoles assay detected albendazole sulphone, albendazole, albendazole sulphoxide, oxibendazole, oxfendazole and flubendazole, the thiabendazole assay detected cambendazole, thiabendazole and 5-hydroxythiabendazole and the triclabendazole assay detected ketotriclabendazole, triclabendazole and triclabendazole sulphoxide. The limits of detection ranged from 0.3 ppb (aminobenzimidazoles) to 2.0 ppb (levamisole) in milk and from 0.15 ppb (aminobenzimidazoles) to 6.5 ppb (levamisole) in tissue. The average recovery range was 71-135 %. This multianalytical approach on a biochip platform is applicable to the screening of more than 20 anthelmintic drugs in different food matrices, leading to consolidation of tests and enhancement of the test result output.