Absolute Q digital PCR system and microfluidic array partitioning (MAP) plates

The Combinati Absolute Q Digital PCR System consists of Microfluidic Array Partitioning (MAP) consumable plates and a fully integrated instrument that automates partitioning of reagents in the plate, PCR thermocycling and 5-color fluorescence image acquisition. The MAP plate has a standard microplate footprint capable of running up to 16 samples per dPCR run²⁹. Each unit is loaded with 10 µL of sample mixture and more than 90% of the loaded sample is partitioned and analyzed in ~ 20,000 pico-scale partitions. The partition volume in MAP plates is defined by the physical dimensions of the microarray chambers and not by a stochastic process such as fluid emulsions, which is important because consistent partition volume is a critical component of the dPCR statistical correction model. The physical array ensures that all samples across all plates yield ~ 20,000 analyzed partitions and minimal sample is lost to dead volume or compromised partitions.

The Absolute Q digital PCR System has a walk-away workflow identical to traditional qPCR. The MAP plate is loaded via pipette with 10 μL of PCR mix and an overlay of 10 μL Isolation Buffer in each well. The wells are then capped with specialized gaskets. The plate is then placed into the Absolute Q tray and retracted into the system. The Absolute Q uses positive pressure from an on-board compressor to partition the sample within the MAP plate without the need for microfluidic valves, sealing films or other moving parts. The MAP slides are constructed of a cyclo olefin polymer (COP; 80 μm) film that seals the microfluidic features that are molded into a separate piece of thicker material. Four identical slides are bonded to a rigid, microtiter format plate frame that includes the loading wells to complete the plate assembly. The thin film becomes gas permeable when positive pressure is applied to a well containing reagents. As the reagent enters the microfluidic features, air is passed out of the partition through the film. This allows reagent to completely fill dead-ended partitions and prevents any bubbles from forming inside of the microfluidic features. The Isolation Buffer overlay follows the reagent and physically separates the reagent reaction volumes to complete the partitioning. Positive pressure applied to the consumable during the PCR thermocycling prevents any evaporation and ensures that bubbles will not form and disrupt the isolated micro-reactions.

Before and after PCR thermocycling, entire arrays are imaged with up to five optical channels configured for the most commonly used dyes, including a ubiquitous quality control dye (ROX) used to verify proper partition filling and finding. The images taken before PCR are subtracted from the after-PCR images to remove background noise. Combinati Analysis software automatically applies optical crosstalk compensation and classifies the partitions using a convoluted neural network algorithm to eliminate false positives/ negatives and ensure robust quantification results. The full dPCR process occurs within the single benchtop instrument without operator’s interaction after setting up the protocol parameters (Fig. 1). The streamlined workflow reduces the potential for contamination, minimizes human handling errors, and reduces the time to result. No fluids ever contact instrument components, so minimal system maintenance is required.