Numrin Thaitrong

Integrated microfluidic bioprocessor for single-cell gene expression analysis

An integrated microdevice is developed for the analysis of gene expression in single cells. The system captures a single cell, transcribes and amplifies the mRNA, and quantitatively analyzes the products of interest. The key components of the microdevice include integrated nanoliter metering pumps, a 200-nL RT-PCR reactor with a single-cell capture pad, and an affinity capture matrix for the purification and concentration of products that is coupled to a microfabricated capillary electrophoresis separation channel for product analysis. Efficient microchip integration of these processes enables the sensitive and quantitative examination of gene expression variation at the single-cell level. This microdevice is used to measure siRNA knockdown of the GAPDH gene in individual Jurkat cells. Single-cell measurements suggests the presence of 2 distinct populations of cells with moderate (≈50%) or complete (≈0%) silencing. This stochastic variation in gene expression and silencing within single cells is masked by conventional bulk measurements.

Polymerase chain reaction-capillary electrophoresis genetic analysis microdevice with in-line affinity capture sample injection.

An integrated polymerase chain reaction (PCR)-capillary electrophoresis (CE) microdevice with an efficient in-line affinity-based injector has been developed for genetic analysis. Double stranded DNA PCR amplicons generated in an integrated 250 nL PCR reactor are captured, purified, and preconcentrated by an oligonucleotide probe immobilized in an in situ polymerized gel matrix followed by thermal release and injection into the CE-separation channel. This in-column injector employs a photopolymerized oligonucleotide-modified acrylamide capture gel to eliminate band broadening and increase the injection efficiency to 100%. The on-chip generated PCR amplicons processed on this microdevice exhibit a 3-5 fold increase in signal intensities and improved resolution compared to our previous T-shaped injector. Multiplex analysis of 191-bp amplicons from Escherichia coli O157 and 256-bp amplicons from E. coli K12 is achieved with a 6-fold increase in resolution. These advances are exploited to successfully detect E. coli O157 in a 500-fold higher background of E. coli K12. This microdevice with in-line affinity capture gel injection provides an improved platform for low-volume, high sensitivity, fully integrated genetic analysis.

Integrated Capillary Electrophoresis Microsystem for Multiplex Analysis of Human Respiratory Viruses

We developed a two-layer, four-channel polymerase chain reaction (PCR)-capillary electrophoresis microdevice that integrates nucleic acid amplification, sample cleanup and concentration, capillary electrophoretic separation, and detection for multiplex analysis of four human respiratory viral pathogens, influenza A, influenza B, coronavirus OC43, and human metapneumovirus. Biotinylated and fluorescently labeled double-stranded (ds) deoxyribonucleic acid (DNA) amplification products are generated in a 100 nL PCR reactor incorporating an integrated heater and a temperature sensor. After amplification, the products are captured and concentrated in a cross-linked acrylamide gel capture matrix copolymerized with acrydite-functionalized streptavidin-capture agents. Thermal dehybridization releases the fluorescently labeled DNA strand for capillary electrophoresis injection, separation, and detection. Using plasmid standards containing the viral genes of interest, each target can be detected starting from as few as 10 copies/reactor. When a two-step reverse transcription PCR amplification is employed, the device can detect ribonucleic acid (RNA) analogues of all four viral targets with detection limits in the range of 25-100 copies/reactor. The utility of the microdevice for analyzing samples from nasopharyngeal swabs is demonstrated. When size-based separation is combined with four-color detection, this platform provides excellent product discrimination, making it readily extendable to higher-order multiplex assays. This portable microsystem is also suitable for performing automated assays in point-of-care diagnostic applications.

Microchip electrophoresis of DNA following preconcentration at photopatterned gel membranes

Rapid separation of nucleic acids by microchip electrophoresis could streamline many biological applications, but conventional chip injection strategies offer limited sample stacking, and thus limited sensitivity of detection. We demonstrate the use of photopatterned polyacrylamide membranes in a glass microfluidic device, with or without fixed negative charges, for preconcentration of double‐stranded DNA prior to electrophoretic separation to enhance detection limits. We compared performance of the two membrane formulations (neutral or negatively charged) as a function of DNA fragment size, preconcentration time, and preconcentration field strength, with the intent of optimizing preconcentration performance without degrading the subsequent electrophoretic separation. Little size‐dependent bias was observed for either membrane formulation when concentrating dsDNA > 100 bp in length, while the negatively charged membrane more effectively blocks passage of single‐stranded oligonucleotide DNA (20‐mer ssDNA). Baseline resolution of a six‐band dye‐labeled ladder with fragments 100–2000 bp in size was obtained in <120 s of separation time, with peak efficiencies in the range of 2000–15 000 plates/cm, and detection limits as low as 1 pM per single dye‐labeled fragment. The degree of preconcentration is tunable by at least 49‐fold, although the efficiency of preconcentration was found to have diminishing returns at high field and/or long times. The neutral membrane was found to be more robust than the negatively charged membrane, with approximately 2.5‐fold larger peak area during the subsequent separation, and less decrease in resolution upon increasing the preconcentration field strength.