Hydrogel capsule-based digital quantitative polymerase chain reaction

Abstract

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.

Keywords: Droplet digital polymerase chain reaction; Gel capsule digital polymerase chain reaction; Fluorescence microplate reader; Hydrogel; Nucleic acid quantification.

Fig. 1

Mechanical stability of barium alginate hydrogel capsules. a Barium alginate hydrogel capsules generated with soybean oil. Black arrows show the positions of gel capsules. Mechanical stability of gel capsules against mechanical stress was evaluated by micrographs of hydrogel capsules encapsulating red dye b before and c during compression. Expansion of droplets due to compression was compared with black dashed line. The scale bars are 10 μm

Fig. 2

Gellification of droplets retain the efficiency of vddPCR. a qPCR readout of vddPCR and gc-dPCR suggested that both vddPCR and gc-dPCR are more sensitive than qPCR. b Endpoint measurement of each assay. vddPCR and gc-dPCR produced stronger fluorescence intensity compared to qPCR. The boxplots represent statistical analysis of data obtained from 6 independent tests. Statistical significance was evaluated by one-way analysis of variance followed by post-hoc Tukey honest significance difference test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns indicates not significant

Fig. 3

gc-dPCR in fluorinated oil and soybean oil. a The effect of oil on gc-dPCR. The boxplots represent statistical analysis of data obtained from 11 independent tests. Statistical significance was evaluated by Student’s t-test. * p < 0.05, ** p < 0.01. b Micrographs of hydrogel capsules after gc-dPCR. The hydrogel capsules from each condition was observed under fluorescence filter and merged image of brightfield and fluorescence micrographs. Green in merge channel was signal originated from fluorescein in fluorescence channel. The scale bars are 100 μm