PEG-mediated viscous crowding controls dynamics and quantitative range in recombinase polymerase amplification nucleation site analysis.

Quantitative nucleic acid amplification tests (NAATs), such as qPCR or digital PCR, are essential diagnostic tools in the fields of medicine, environmental monitoring, and food safety. Recent efforts to bring nucleic acid quantification to the point-of-need using rapid isothermal amplification assays struggle to provide quantitative precision and suitable quantitative range. Here we investigate a digital isothermal NAAT in a membrane-free microfluidic chip by systematically tuning recombinase polymerase amplification (RPA) chemistry with a polyethylene glycol crowding agent (PEG 35 000) that controls the dynamics of individual amplification nucleation site development. Amplification nucleation site analysis (ANSA) using real-time epifluorescence imaging reveals that individual nucleation sites exhibit Gaussian spatial intensity profiles whose amplitude, width, and total fluorescence evolve in time. Site diameter increases linearly during active amplification, while peak intensity follows an exponential growth curve before plateauing. Increasing PEG concentration results in smaller reaction site diameters and enables reliable discrimination of closely spaced amplification events with 8% PEG providing the best balance between amplification robustness and site separation. We observe quantitative detection from 10