Digital-to-Droplet Microfluidic Platform Powered Digital Isothermal Nucleic Acid Amplification for On-Demand Detection of Post-Transplantation Infection-Causing Viruses

The etiological diagnosis of severe infectious diseases is complicated by variability in the timing of sample submissions, heterogeneity in detection targets, and stringent requirements for rapid turnaround times, thereby necessitating the development of on-demand detection methodologies. To address these challenges, we introduce an automated digital isothermal nucleic acid amplification (dINA) assay facilitated by a digital-to-droplet (D2D) microfluidic platform. This D2D system permits the continuous introduction and extraction of water-in-oil droplets, enabling the digital microfluidics (DMF) module to alternately interface with multiple droplet microfluidic units. Within this framework, samples or reagents encapsulated in droplets are sequentially delivered to the DMF module, where nucleic acid extraction and subsequent mixing with loop-mediated isothermal amplification (LAMP) reagents are accomplished through a series of precise droplet manipulations. The resultant microliter-scale LAMP reaction droplets are then dispensed into the sample inlets of individual droplet microfluidic modules, where they are partitioned into picoliter-scale droplets for dINA analysis. The principal advantages of this system include (1) the ability to process samples immediately upon receipt, (2) substantial scalability in throughput, and (3) absolute quantification independent of calibration standards. As a proof of concept, this platform was employed to detect viruses responsible for post-transplantation infections, demonstrating a markedly reduced turnaround time for specimens submitted at variable intervals. These findings underscore the potential of this system as an efficient and adaptable diagnostic tool for the on-demand detection of pathogens implicated in severe infectious diseases.