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.

Figure 1

Assembly and operation of the preloaded multiplex PCR SlipChip. A) Schematic drawing shows the PCR SlipChip after introducing two different samples (blue and green) and slipping to combine the sample with the preloaded primers. B) A bright field image of the experiment described in A) using food dyes instead of the samples. C) The top plate of the PCR SlipChip contained square wells, rectangular wells, sample inlets, and outlets (outlets not shown) and D) the bottom plate of the PCR SlipChip contained ducts for the samples and preloaded circular wells with different PCR primers. E) The SlipChip was assembled and the two plates were aligned such that the sample wells and sample ducts lined up to form a continuous fluidic path. F) The sample, the PCR master mixture, was flowed through the fluidic path to load the sample wells. G) The PCR SlipChip was slipped to align the square sample wells with the circular primer wells. When the PCR mixture touched the primer on the bottom of the primer well, the PCR primer dissolved in and mixed with the reaction mixture. After the SlipChip was slipped, thermal cycling was performed. H) Microphotograph shows the formation of droplets within the wells after the aqueous dye (representing the PCR mixture) was slipped to contact wells containing oil.

Figure 2

Control of thermal expansion during thermocycling on SlipChip by changing well geometry. A) Top and side view schematic drawings of a square well containing aqueous PCR reaction mixture (red) and mineral oil (green). The square well was completely filled when aqueous solution was introduced (left). After slipping to break the fluidic connections to other wells (not shown here), the aqueous solution filled the square well completely (middle, low T), and after an increase in temperature, the aqueous solution entered the gap between the two plates of the SlipChip (right, high T). B) Top and side view schematic drawings of a shallow circular well(bottom plate) containing oil (green) next to a deep square well (top plate) filled with aqueous PCR reaction mixture (red) (left). After slipping, the aqueous solution would form a droplet surrounded by mineral oil within the hydrophobic well due to the surface tension (middle, low T). When the temperature was increased, the aqueous solution expanded inside the reaction compartment and the mineral oil expanded and moved out of the well through the gap between the top and bottom plates in the SlipChip, serving as a buffer material (right, high T). C) Top view of fluorescent microphotographs from experiments described as in B). Aqueous solution containing red quantum dots (red) in square well was slipped and overlaid with circular well containing mineral oil staining with green quantum dots (green). An aqueous droplet was formed in hydrophobic well due to the surface tension at 55 °C (left). After increase of temperature to 95 °C, the aqueous solution expanded but still stayed inside the reaction compartment (right). No leakage of the aqueous solution outside of the reaction compartment was observed.

Figure 3

PCR was successful and robust on the SlipChip platform. A) Microphotograph of wells containing S. aureus (MSSA) gDNA showed significant increase of fluorescence intensity after thermal cycling. B) Microphotograph of the control wells showed no increase of fluorescence. C) Analysis of the fluorescence intensity of the wells containing template vs. the control wells. The average fluorescent intensity of the wells containing template was significantly higher than the control wells (p < 0.0001, n = 20). The fluorescent intensity of the wells before thermal cycling was around 80 units. D) Gel electrophoresis of the DNA amplified in the SlipChip. Column M contained the 100 bp DNA ladder, Column 1 contained sample recovered from wells loaded with template, and Column 2 contained sample collected from control wells. Only one molecular weight of DNA (∼ 270 bp) was found in wells containing template, and no DNA was found in control wells without template.

Figure 4

No cross-contamination among adjacent wells was seen after PCR on the SlipChip. Primer pairs for the nuc gene in S. aureus and the mecA gene in Methicillin-resistant S. aureus (MRSA) were preloaded on the chip alternatively in the same row. PCR master mixture, containing 100 pg/μL of S. aureus (MSSA) genomic DNA, was injected into the SlipChip. A) Fluorescent microphotograph shows that only wells preloaded primers for the nuc gene (left in each pair of wells) showed fluorescent signal corresponding to DNA amplification. B) A line scan across the middle of the top row in (A) shows that fluorescence intensity of wells loaded with nuc primers were more than 5 fold higher than that of the wells loaded with mecA primers (p < 0.0001, n = 10) The blue dashed line in (B) represents the fluorescence intensity before thermal cycling in the wells.

Figure 5

Detection of pathogens by PCR using panels of preloaded primers on the 384-well SlipChip. A panel of 20 different primer pairs was preloaded onto the SlipChip to differentiate and genotype 16 different pathogens, with one additional primer pair for a positive internal control. A) Bright field image of the whole chip filled with red dye and slipped into position. B) A schematic drawing of the 384-well SlipChip designed for high-throughput multiplex PCR. Different colors indicate wells preloaded with different primer pairs (see Table 1). (C-G) Fluorescence microphotographs showed that the SlipChip correctly identified and genotyped different microbes by using PCR. In all experiments, regions for positive control (pBad primer pair, A1, B1, C1, and D1) increased in fluorescence intensity while regions for negative control (no primer pairs, A2, B2, C2, and D2) did not. All samples were loaded with pBad 331 bp template as positive internal controls. Fluorescent images were stitched after acquisition (See Experimental Section in Supporting Information). (C) When loaded with MSSA, only region C3 (S. aureus nuc primer) showed increase of fluorescence intensity; D) When loaded with MRSA, both regions C3 (S. aureus nuc primer) and C7 (MRSA mecA primer) showed increase of fluorescence intensity; E) When loaded with C. albicans, region D5 (C. albicans calb primer) showed increase of fluorescence intensity; F) When loaded with P. aeruginosa, regions B5 (P. aeruginosa vic primer) and B7 (Pseudomonas 16S rRNA primer) showed increase of fluorescence intensity; G) When loaded with E. coli, region A3 (E. coli nlp primer) wells showed increase of fluorescence intensity.