1-Million droplet array with wide-field fluorescence imaging for digital PCR

Abstract

Digital droplet reactors are useful as chemical and biological containers to discretize reagents into picolitre or nanolitre volumes for analysis of single cells, organisms, or molecules. However, most DNA based assays require processing of samples on the order of tens of microlitres and contain as few as one to as many as millions of fragments to be detected. Presented in this work is a droplet microfluidic platform and fluorescence imaging setup designed to better meet the needs of the high-throughput and high-dynamic-range by integrating multiple high-throughput droplet processing schemes on the chip. The design is capable of generating over 1-million, monodisperse, 50 picolitre droplets in 2-7 minutes that then self-assemble into high density 3-dimensional sphere packing configurations in a large viewing chamber for visualization and analysis. This device then undergoes on-chip polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the sample's nucleic acid contents. Wide-field fluorescence images are captured using a low cost 21-megapixel digital camera and macro-lens with an 8-12 cm(2) field-of-view at 1× to 0.85× magnification, respectively. We demonstrate both end-point and real-time imaging ability to perform on-chip quantitative digital PCR analysis of the entire droplet array. Compared to previous work, this highly integrated design yields a 100-fold increase in the number of on-chip digitized reactors with simultaneous fluorescence imaging for digital PCR based assays.

Droplet microfluidics driven by gradients of confinement

“…In practice, they imply that a monodisperse emulsion is obtained without any losses from the initial sample. This is particularly important for cases when the emulsion is kept on chip for further analysis (28,29), because the complete initial sample can be analyzed.…”

PCR digitale en micro-compartiments

“…La méthode conventionnelle utilisée en clinique pour la détection de virus (hépatites B et C, VIH, virus de l'herpès) est la qPCR. Comme nous l'avons vu plus haut, cette technique n'est cependant pas standardisée, et de gouttes [29]. Dans ce système, le signal de chaque goutte peut être quantifié en dépit de la forte densité de gouttes dans le réservoir, permettant ainsi d'atteindre une sensibilité de moins de dix copies d'ADN cible de Chlamydia (ADN matrice synthétique de 150-300 bp) par échantillon de 50 μL.…”

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

“…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.…”