Nanoliter Multiplex PCR Arrays on a SlipChip

Abstract

The SlipChip platform was tested to perform high throughput nanoliter multiplex PCR. The advantages of using the SlipChip platform for multiplex PCR include the ability to preload arrays of dry primers, instrument-free sample manipulation, small sample volume, and high throughput capacity. The SlipChip was designed to preload one primer pair per reaction compartment, and to screen up to 384 different primer pairs with less than 30 nanoliters of sample per reaction compartment. Both a 40-well and a 384-well design of the SlipChip were tested for multiplex PCR. In the geometries used here, the sample fluid was spontaneously compartmentalized into discrete volumes even before slipping of the two plates of the SlipChip, but slipping introduced additional capabilities that made devices more robust and versatile. The wells of this SlipChip were designed to overcome potential problems associated with thermal expansion. By using circular wells filled with oil and overlapping them with square wells filled with the aqueous PCR mixture, a droplet of aqueous PCR mixture was always surrounded by the lubricating fluid. In this design, during heating and thermal expansion, only oil was expelled from the compartment and leaking of the aqueous solution was prevented. Both 40-well and 384-well devices were found to be free from cross-contamination, and end point fluorescence detection provided reliable readout. Multiple samples could also be screened on the same SlipChip simultaneously. Multiplex PCR was validated on the 384-well SlipChip with 20 different primer pairs to identify 16 bacterial and fungal species commonly presented in blood infections. The SlipChip correctly identified five different bacterial or fungal species in separate experiments. In addition, the presence of the resistance gene mecA in methicillin resistant Staphylococcus aureus (MRSA) was identified. The SlipChip will be useful for applications involving PCR arrays, and lays the foundation for new strategies for diagnostics, point-of-care devices, and immobilization based arrays.

Gene-targeted microfluidic cultivation validated by isolation of a gut bacterium listed in Human Microbiome Project's Most Wanted taxa

“…It can be used in the limited space of an anaerobic chamber, which is widely used to cultivate anaerobes that dominate the human gut microbiota. Second, SlipChip is compatible with PCR (24) and enzymatic assays (25). Third, compartmentalization on SlipChip is reversible and the microcolonies can be spatially indexed as described in an accompanying paper (22), which facilitates the retrieval of reagents and organisms from the device (24,26).…”

Recent developments in nucleic acid identification using solid-phase enzymatic assays

“…The rapidly growing field of soft-lithography driven microfluidic technology has resulted in the development a plethora of micro-devices for extraction [75] and amplification and analysis [31,[76][77][78][79][80][81] of nucleic acids. The main idea of microarray technology, namely spatial separation of microarray elements and their simultaneous subjection to one sample of interest can be easily realized using highly parallel architectures of micrometresized channels available/affordable due to microfluidics.…”

“…Following amplification and counting of number of compartments showed positive amplification result reveals the initial number of the targeted molecules in the sample allowing quantitative analysis of ultimate precision and accuracy [85]. This compartmentalization can be realized in form of water in oil emulsion [86,87] where each water droplet is considered as unique and independent reaction volume; or by distribution of the reaction mixture across hundreds and thousands of individual microfluidic vessels [88][89][90]. The approach was successfully shown both for PCR [87,88,91,92] and isothermal amplification techniques (LAMP) [89].…”

“…This compartmentalization can be realized in form of water in oil emulsion [86,87] where each water droplet is considered as unique and independent reaction volume; or by distribution of the reaction mixture across hundreds and thousands of individual microfluidic vessels [88][89][90]. The approach was successfully shown both for PCR [87,88,91,92] and isothermal amplification techniques (LAMP) [89]. Zhang et al [90] showed implementation of the microfluidic arrays full cycle of nucleic acids analysis including DNA/RNA extraction and their real-time amplification (Fig.…”

Real-time microfluidic recombinase polymerase amplification for the toxin B gene of Clostridium difficile on a SlipChip platform

“…A high-throughput nL multiplex PCR platform was demonstrated for up to 16 pathogens, including E. coli and MRSA, in 40 and 384 wells. 17 In the latter, 384 primer pairs were dispensed in the wells and individual PCR reactions were run in parallel from a single 10 mL sample of genomic DNA. Total reaction time was $75 minutes and end-point uorescence was measured with an epi-uorescence microscope.…”