[Show abstract][Hide abstract] ABSTRACT: Genetic alternations can serve as highly specific biomarkers to distinguish fatal bacteria or cancer cells from their normal counterparts. However, these mutations normally exist in very rare amount in the presence of a large excess of non-mutated analogs. Taking the notorious pathogen E. coli O157:H7 as the target analyte, we have developed an agarose droplet-based microfluidic ePCR method for highly sensitive, specific and quantitative detection of rare pathogens in the high background of normal bacteria. Massively parallel singleplex and multiplex PCR at the single-cell level in agarose droplets have been successfully established. Moreover, we challenged the system with rare pathogen detection and realized the sensitive and quantitative analysis of a single E. coli O157:H7 cell in the high background of 100 000 excess normal K12 cells. For the first time, we demonstrated rare pathogen detection through agarose droplet microfluidic ePCR. Such a multiplex single-cell agarose droplet amplification method enables ultra-high throughput and multi-parameter genetic analysis of large population of cells at the single-cell level to uncover the stochastic variations in biological systems.
[Show abstract][Hide abstract] ABSTRACT: The application of microfluidic droplet PCR for single-molecule amplification and analysis has recently been extensively studied. Microfluidic droplet technology has the advantages of compartmentalizing reactions into discrete volumes, performing highly parallel reactions in monodisperse droplets, reducing cross-contamination between droplets, eliminating PCR bias and nonspecific amplification, as well as enabling fast amplification with rapid thermocycling. Here, we have reviewed the important technical breakthroughs of microfluidic droplet PCR in the past five years and their applications to single-molecule amplification and analysis, such as high-throughput screening, next generation DNA sequencing, and quantitative detection of rare mutations. Although the utilization of microfluidic droplet single-molecule PCR is still in the early stages, its great potential has already been demonstrated and will provide novel solutions to today's biomedical engineering challenges in single-molecule amplification and analysis.
Full-text · Article · Mar 2012 · Analytical and Bioanalytical Chemistry
[Show abstract][Hide abstract] ABSTRACT: We have developed a novel method for efficiently screening affinity ligands (aptamers) from a complex single-stranded DNA (ssDNA) library by employing single-molecule emulsion polymerase chain reaction (PCR) based on the agarose droplet microfluidic technology. In a typical systematic evolution of ligands by exponential enrichment (SELEX) process, the enriched library is sequenced first, and tens to hundreds of aptamer candidates are analyzed via a bioinformatic approach. Possible candidates are then chemically synthesized, and their binding affinities are measured individually. Such a process is time-consuming, labor-intensive, inefficient, and expensive. To address these problems, we have developed a highly efficient single-molecule approach for aptamer screening using our agarose droplet microfluidic technology. Statistically diluted ssDNA of the pre-enriched library evolved through conventional SELEX against cancer biomarker Shp2 protein was encapsulated into individual uniform agarose droplets for droplet PCR to generate clonal agarose beads. The binding capacity of amplified ssDNA from each clonal bead was then screened via high-throughput fluorescence cytometry. DNA clones with high binding capacity and low K(d) were chosen as the aptamer and can be directly used for downstream biomedical applications. We have identified an ssDNA aptamer that selectively recognizes Shp2 with a K(d) of 24.9 nM. Compared to a conventional sequencing-chemical synthesis-screening work flow, our approach avoids large-scale DNA sequencing and expensive, time-consuming DNA synthesis of large populations of DNA candidates. The agarose droplet microfluidic approach is thus highly efficient and cost-effective for molecular evolution approaches and will find wide application in molecular evolution technologies, including mRNA display, phage display, and so on.
Full-text · Article · Nov 2011 · Analytical Chemistry
[Show abstract][Hide abstract] ABSTRACT: An agarose droplet method was developed for highly parallel and efficient single molecule emulsion PCR. The method capitalizes on the unique thermoresponsive sol-gel switching property of agarose for highly efficient DNA amplification and amplicon trapping. Uniform agarose solution droplets generated via a microfluidic chip serve as robust and inert nanolitre PCR reactors for single copy DNA molecule amplification. After PCR, agarose droplets are gelated to form agarose beads, trapping all amplicons in each reactor to maintain the monoclonality of each droplet. This method does not require cocapsulation of primer labeled microbeads, allows high throughput generation of uniform droplets and enables high PCR efficiency, making it a promising platform for many single copy genetic studies.
[Show abstract][Hide abstract] ABSTRACT: We have developed a microfluidic method to produce monodisperse, size-controllable silica microspheres via hydrolysis and polymerization of TEOS at the interface of water-in-oil droplets. By altering the concentration of TEOS in oil phase and CTAB in aqueous phase, we achieved control over morphology of the microspheres from hollow, to partly hollow, to solid. The hollow silica microsphere can be used for rapid waste removal and detoxification extraction with a very simple procedure.
[Show abstract][Hide abstract] ABSTRACT: Poly(methyl methacrylate) (PMMA) is gaining in popularity in microfluidic devices because of its low cost, excellent optical transparency, attractive mechanical/chemical properties, and simple fabrication procedures. It has been used to fabricate micromixers, PCR reactors, CE and many other microdevices. Here we present the design, fabrication, characterization and application of pneumatic microvalves and micropumps based on PMMA. Valves and pumps are fabricated by sandwiching a PDMS membrane between PMMA fluidic channel and manifold wafers. Valve closing or opening can be controlled by adjusting the pressure in a displacement chamber on the pneumatic layer via a computer regulated solenoid. The valve provides up to 15.4 microL s(-1) at 60 kPa fluid pressure and seals reliably against forward fluid pressure as high as 60 kPa. A PMMA diaphragm pump can be assembled by simply connecting three valves in series. By varying valve volume or opening time, pumping rates ranging from nL to microL per second can be accurately achieved. The PMMA based valves and pumps were further tested in a disposable automatic nucleic acid extraction microchip to extract DNA from human whole blood. The DNA extraction efficiency was about 25% and the 260 nm/280 nm UV absorption ratio for extracted DNA was 1.72. Because of its advantages of inexpensive, facile fabrication, robust and easy integration, the PMMA valve and pump will find their wide application for fluidic manipulation in portable and disposable microfluidic devices.