Acryloyl Gelatin as Polymer Additive to Droplet Digital LAMP for DNA Quantification

Abstract

Absolute quantification of target nucleic acids is essential for life analysis and can be achieved through droplet digital microfluidic nucleic acid quantification (droplet digital NAQ). Recent advancements in this field have incorporated polymers into droplets to form microgel particles, which add new functionalities to the droplets and offer opportunities for tandem analysis. However, the range of materials available for microgel-based droplet digital NAQ remains limited. In this study, we introduce acryloyl gelatin (GelA) as an additive polymer for droplet digital NAQ. GelA is photopolymerizable, biocompatible, and biodegradable, making it an ideal candidate for droplet digital NAQ applications. Our findings demonstrate that GelA exhibits minimal thermal variation in viscosity, which is favorable for droplet microfluidic production and handling. Additionally, GelA does not significantly affect the efficiency of droplet digital loop-mediated amplification (droplet digital LAMP) under the optimized conditions. Upon the addition of GelA, the droplet digital LAMP assay for the detection of the N gene in SARS-CoV-2 can be realized within the range of 25 to 250,000 copies/test, and the amplification is completed within 30 min. Following droplet digital LAMP, GelA droplets can be transformed into either physical or chemical microgel particles. In the microgel form, the particles are stable through transfer cycles, can prevent leakage of amplification products, and can facilitate controlled release of the products for further applications. This work broadens the range of materials available for droplet-based digital nucleic acid detection, increases the versatility of droplet digital LAMP, and opens new possibilities for advancements in this field.