Abstract
Nucleic acid technology (NAT) based detection is a popular technique to detect nucleic acid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which can be used for early diagnosis of cancer, confirmation of genetic disease, and many others. Usually, the copy number of the nucleic acid molecules in a sample is too less to be detected. As a result, the first step of nucleic acid detection is to amplify the nucleic acid molecules to a detectable level. Polymerase chain reaction (PCR) is one of the most commonly used methods for DNA or RNA amplification, in which nucleic acid samples go through thermal cycling between two or three different operating temperatures. The thermal cycling requires accurate and robust control of the operating temperature. To simplify the complicated processes of thermal cycling, isothermal amplification methods have been developed. Isothermal amplification reactions such as loop-mediated amplification (LAMP) can be implemented at a fixed temperature with no need for thermal cycling, which reduces the complexity of the temperature control module and consumes less energy, making them more suitable for microfluidics-based applications. Moreover, when compared with PCR, LAMP is more sensitive and specific. Furthermore, it takes less than one hour to finish the amplification process. These advantages make LAMP become a promising method for NAT based detection. In biomedical applications, it is often of great significance to quantify the accurate nucleic acid molecule copies in original samples. The original DNA copies can be measured by real-time PCR. However, the quantification relays on a reference or a standard curve. Besides, this bulk reaction fails to distinguish subtle difference of the target copy number. In an effort to overcome the disadvantages, digital DNA amplification has been developed. In this study, we reported a new method to implement array-based digital LAMP analysis, in which we integrated the emulsion droplet microfluidic device and hydrodynamic trapping techniques to form a droplet array which could be further applied digital LAMP assay. By generating monodisperse water-in-oil droplets which contain LAMP reaction mixture, the bulk LAMP reaction mixture could be partitioned into many separate compartments. Each droplet could function as a reaction chamber. Hydrodynamic trapping technique was further used for immobilizing the droplets to form a droplet array, which is good for later analysis of the result. In this work, we used our integrated microfluidic chip to demonstrate digital LAMP assay. The microfluidic chip could be able to generate uniform droplet with a size variation less than 3% and the droplet could be hydrodynamically immobilized to form a droplet array. Besides, the LAMP assay could be successfully implemented in water-in-oil droplet.