A Dual-Port, Smartphone-Linked, Pocket-Size Fluorimeter for Rapid Molecular Diagnostic Assays at Point of Care

We present the design, testing, and demonstration of a portable, pocket-size, smartphone-linked fluorimeter called the "VPodDuo." The instrument is capable of reading the output of several fluorescence-generating biomolecular detection assays with sensitivity that is similar to larger and more expensive laboratory-based instruments. In this work, we focus on demonstrating the capability for readout of assays used to detect target nucleic acid sequences associated with infectious pathogens and cancer with incubation times of approximately 10 min. The VPodDuo features a dual-port configuration that allows simultaneous measurements of a negative experimental control (CTRL) in parallel with the test sample. We demonstrated compatibility with several assay protocols, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), recombinase polymerase amplification (RPA), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, and the target recycling amplification process (TRAP). Benchmarking against three commercially available fluorimeters showed comparable detection limits for clinically relevant nucleic acid sequences, including Zika virus (ZIKV, 10⁴ copies/μL), methicillin-susceptible Staphylococcus aureus (MSSA, 10² copies/μL), human immunodeficiency virus (HIV, 6.83 pM), a lung cancer-associated circulating tumor DNA sequence [L858R point-mutated epidermal growth factor receptor (EGFR) gene, 29.2 pM], and a microRNA biomarker associated with lung cancer (miR-375-3p, 500 pM). We further validated the VPodDuo's performance under varying ambient temperatures through in-lab simulations and real-world outdoor testing using the TRAP assay for miR-375-3p detection, demonstrating cancer-associated biomarker detection at point-of-care (POC) settings. With its compact form-factor, low cost, portability, and real-time data transmission and analytical capabilities, the VPodDuo represents a promising solution for expanding access to rapid, on-site molecular diagnostics in diverse clinical and field settings.