Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip.

The emergence of pathogens resistant to existing antimicrobial drugs is a growing worldwide health crisis that threatens a return to the pre-antibiotic era. To decrease the overuse of antibiotics, molecular diagnostics systems are needed that can rapidly identify pathogens in a clinical sample and determine the presence of mutations that confer drug resistance at the point of care. We developed a fully integrated, miniaturized semiconductor biochip and closed-tube detection chemistry that performs multiplex nucleic acid amplification and sequence analysis.

Multiplexed target detection using DNA-binding dye chemistry in droplet digital PCR.

Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries.

High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ~2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow.

Automated immunoassay system for AFP-L3% using on-chip electrokinetic reaction and separation by affinity electrophoresis.

Implementation of the on-chip immunoassay for alpha-fetoprotein (AFP)-L3% was achieved using a fully automated microfluidic instrument platform that will prepare the chip and run the assay with a total assay time of less than 10min. Reagent/sample mixing, concentration, and reaction in microfluidic channels occur by the electrokinetic analyte transport assay (EATA) technique, enabling the integration of all assay steps on-chip. The determination of AFP-L3%, a biomarker for hepatocellular carcinoma, was achieved by the presence of Lens culinaris agglutinin in the separation channel, causing separation of the fucosylated isoform, AFP-L3, from the nonfucosylated AFP-L1 by lectin affinity electrophoresis.

Analysis of plasmid samples on a microchip.

We have developed a LabChip-based plasmid assay that runs on the Agilent 2100 Bioanalyzer. The assay determines the sizes and relative concentrations of the multiple forms of plasmid samples. Twelve samples can be analyzed on each chip in an automated run lasting approximately 30min.

Electroosmotic pumping in microchips with nonhomogeneous distribution of electrolytes.

A general equation to calculate the node pressure at a junction in a microfluidic network is presented. The node pressure is generated from both the hydrodynamic flow due to the external applied hydraulic pressures and the electrokinetic flow resulted from the applied electric field. Pure electroosmotic flow has a plug-flow profile and pressure flow has a parabolic flow profile.

Synchronized cyclic capillary electrophoresis using channels arranged in a triangle and low voltages.

Synchronized cyclic capillary electrophoresis (SCCE) makes use of a closed loop separation channel by which the same sample can be separated during many cycles. This enables the repeated use of the same voltage for separations such that a high total voltage, and thus high efficiency, is obtained for the synchronized components. This can be accomplished by using any type of polygon geometry for the separation channel; and calculations of the available field and number of connections needed for polygons from 3 to 5 sides are presented.

Protein sizing on a microchip.

We have developed a microfabricated analytical device on a glass chip that performs a protein sizing assay, by integrating the required separation, staining, virtual destaining, and detection steps. To obtain a universal noncovalent fluorescent labeling method, we have combined on-chip dye staining with a novel electrophoretic dilution step. Denatured protein-sodium dodecyl sulfate (SDS) complexes are loaded on a chip and bind a fluorescent dye as the separation begins.

Electrokinetically controlled microfluidic analysis systems.

Electrokinetic forces are emerging as a powerful means to drive microfluidic systems with flow channel cross-sectional dimensions in the tens of micrometers and flow rates in the nanoliter per second range. These systems provide many advantages such as improved analysis speed, improved reproducibility, greatly reduced reagent consumption, and the ability to perform multiple operations in an integrated fashion. Planar microfabrication methods are used to make these analysis chips in materials such as glass or polymers.

Microfabrication in silicon microphysiometry.

Over the past 5 years, microphysiometry has proved an effective means for detecting physiological changes in cultured cells, particularly as a functional assay for the activation of many cellular receptors. To demonstrate the clinical relevance of this method, we have used it to detect bacterial antibiotic sensitivity and to discriminate between bacteriostatic and bacteriocidal concentrations. The light-addressable potentiometric sensor, upon which microphysiometry is based, is well suited for structural manipulations based on photolithography and micromachining, and we have begun to take advantage of this capability.

Detection of cell-affecting agents with a silicon biosensor.

Cellular metabolism is affected by many factors in a cell's environment. Given a sufficiently sensitive method for measuring cellular metabolic rates, it should be possible to detect a wide variety of chemical and physical stimuli. A biosensor has been constructed in which living cells are confined to a flow chamber in which a potentiometric sensor continually measures the rate of production of acidic metabolites.