A microfluidic liquid phase nucleic acid purification chip to selectively isolate DNA or RNA from low copy/single bacterial cells in minute sample volume followed by direct on-chip quantitative PCR assay.

Purification of nucleic acids from a low quantity of bacterial cells in minute volume is important in many clinical and biological applications. We developed a novel microfluidic liquid phase nucleic acid purification chip to selectively isolate DNA or RNA from bacterial cells in the range of 5000 down to a single cell in the sample volume of 1 µl or 125 nl, which can be directly put through on-chip quantitative PCR assay. The aqueous phase bacterial lysate was isolated in an array of microwells, after which an immiscible organic (phenol-chloroform) phase was introduced in a headspace channel connecting the microwell array. Continuous flow of the organic phase increases the interfacial contact with the aqueous phase to achieve purification of target nucleic acid through phase partitioning. Significantly enhanced nucleic acid recovery yield, up to 10 fold higher, was achieved using the chip-based liquid phase nucleic acid purification technique compared to that obtained by the conventional column-based solid phase nucleic acid extraction method. One step vacuum-driven microfluidics allowed an on-chip quantitative PCR assay to be carried out in the same microwells within which bacterial nucleic acids were isolated, avoiding sample loss during liquid transfer. Using this nucleic acid purification device set in a two-dimensional (2D) array format of 900 microwells, it was demonstrated for the first time that high-throughput extraction of RNA couple with direct on-chip PCR analysis from single bacterial cells could be achieved. Our microfluidic platform offered a simple and effective solution for nucleic acid preparation, which can be integrated for automated bacterial pathogen detection and high throughput transcriptional profiling.

A high-flux isopore micro-fabricated membrane for effective concentration and recovering of waterborne pathogens.

A high-flux metallic micro/nano-filtration membrane has been fabricated and validated for isolation of waterborne pathogens from drinking water. Obtained membrane with smooth surface and perfectly ordered pores was achieved by a high yield and cost effective multilevel lithography and electroplating technique. The micro-fabricated membrane was also strengthened with an integrated back-support, which can withstand a high pressure during filtration. The results of microfiltration tests with model particles revealed the superior performance of the micro-fabricated filter than current commercial filters in sample throughput, recovery ratio, and reusability. This study highlighted the potential application of micro-fabricated filer in rapid filtration and recovery of C. parvum oocysts for downstream analysis.

Fabrication of multi-layer polymeric micro-sieve having narrow slot pores with conventional ultraviolet-lithography and micro-fabrication techniques.

Fast detection of waterborne pathogens is important for securing the hygiene of drinking water. Detection of pathogens in water at low concentrations and minute quantities demands rapid and efficient enrichment methods in order to improve the signal-to-noise ratio of bio-sensors. We propose and demonstrate a low cost and rapid method to fabricate a multi-layer polymeric micro-sieve using conventional lithography techniques. The micro-fabricated micro-sieves are made of several layers of SU-8 photoresist using multiple coating and exposure steps and a single developing process. The obtained micro-sieves have good mechanical properties, smooth surfaces, high porosity (≈40%), and narrow pore size distribution (coefficient of variation < 3.33%). Sample loading and back-flushing using the multi-layer micro-sieve resulted in more than 90% recovery of pathogens, which showed improved performance than current commercial filters.