Hydrogel capsule-based digital quantitative polymerase chain reaction

Droplet digital PCR (ddPCR) is accurate in nucleic acid quantification owing to its linearity and high sensitivity. Amplification of nucleic acid in droplets, however, is limited by the stability of droplets against thermal cycling. While the use of fluorinated oil or supplementation of surfactant could improve the stability of droplets, this process has also greatly increased the cost of ddPCR and limited post-PCR analysis. Here, we report a novel method known as gel capsule-based digital PCR (gc-dPCR) which includes a method to prepare hydrogel capsules encapsulating the PCR reaction mix, conducting PCR reaction, and readout by either quantitative PCR (qPCR) system or fluorescence microplate reader. We have compared the developed method to vortex ddPCR. Our approach results in higher fluorescence intensity compared to ddPCR suggesting higher sensitivity of the system. As hydrogel capsules are more stable than droplets in fluorinated oil throughout thermal cycling, all partitions can be quantified, thus preventing loss of information from low-concentration samples. The new approach should extend to all droplet-based PCR methods. It has greatly improved ddPCR by increasing droplets stability and sensitivity, and reducing the cost of ddPCR, which help to remove the barrier of ddPCR in settings with limited resources. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00604-023-05827-7.