Real-Time and Absolute Quantification of Infectious Pathogens using Wide-Field Imaging-Based Droplet Analysis System

Droplet-based microfluidic (DMF) system has been widely used as molecular diagnostic tool for digital analysis with absolute quantification and high-resolution. Especially, droplet digital analysis is useful and powerful diagnostic tool because droplets are worked as chemical and biological containers to partition biomarkers, such as virus, bacteria and nucleic acid, in single molecule manner. Due to coronavirus disease 2019 (COVID-19) pandemic, digital analysis is becoming more important as a next-generation diagnostic platform to monitor infectious pathogens affecting human health. For example, droplet digital PCR (ddPCR), the third-generation PCR, has been used because it can provide more accurate and sensitive results for monitoring infectious pathogens at a single-molecule manner comparing real-time PCR, the second-generation PCR. However, commercialized-digital analytical systems (ddPCR, digital ELISA and digital Cell Analyzer) require expensive and complex optical systems to detect droplets containing target molecule such as DNA, RNA, protein and bacteria. Even commercialized imaging systems also have a small field of view because of high-magnification lens. Examples include high-performance optical, fluorescence and confocal microscopes. To address these issues, we have developed an analytical system that combines a wide-field imaging system with DMF system to capture and quantify target droplets (we called the DropVIST, Droplet analysis system with Vast Imaging and Statistical Tools). In this approach, first of all, droplets are encapsulated with microfluidic devices and incubate for chemical reaction. Then, reacted droplets are imaged and quantified with the DropVIST for absolute quantification and high-throughput assay. In this work, droplets were encapsulated within flow-focusing structure of microfluidic devices made of polydimethylsiloxane (PDMS) material using soft-lithography technology. To setup the DropVIST, this system was composed of a simple optical system using a 50.2-megapixel CMOS image sensor-based DSLR camera and a 100mm macro lens to obtain wide-field images with high-resolution. A MATLAB-based software, called the dFinder software, was also made for absolute quantification of target droplets. In this study, the DropVIST was identified with the dFInder software that simultaneously analyzes eight individual colors for multiplexed and absolute quantification from a single image. Then, a size of detectable droplet was characterized to be over 30 μm in diameter. Also, it can be analyzed using the dFinder software that a droplet image with a minimum size of 32 pixels in a field of view of 201.8 cm²(The focal length of camera was 65 cm). The DropVIST allowed a field of view from a minimum of 11.48 cm² to a maximum of 201.8 cm2 and theoretical total sample volume of droplets were calculated from 69.65 μL to 1224 μL when droplets are encapsulated at 91 μm in a diameter. We demonstrated with the DropVIST that digital proliferation assay and digital colony forming unit (CFU) assay were performed to identify more rapid and accurate analysis than conventional assays (microbroth dilution and CFU assays). In these cases, bacterial cells were used at concentrations ranging from 10⁶ cells/μL to 0.1 cells/μL and a limit of detection (LOD) was 0.1 cells/μL when used the DropVIST. In the future, this system will be utilized as a diagnostic tool for rapid and accurate droplet digital analysis such as digital antimicrobial susceptibility test and real-time digital nucleic acid amplification test.