Smartphone-Based Fluorescent Profiling of Quaternary MicroRNAs in Urine for Rapid Diagnosis of Urological Cancers Using a Multiplexed Isothermal Exponential Amplification Reaction

Urological cancers such as bladder or prostate cancer represent one of the most malignant tumors that accounts for an extremely high mortality. However, conventionally standard diagnostics for urological cancers are hardly available in low-resource settings. We developed herein a hand-held fluorescent imaging platform by integrating a multiplexed isothermal exponential amplification reaction (EXPAR) with a microgel-enriched methodology for sensitive profiling of quaternary microRNAs (miRNAs) in urine and quick diagnosis of urological cancers at the early stage. The target miRNA mixtures in the urine underwent four parallel EXPARs without cross-reactivity, followed by surface concentration and hybridization by the encoded polyacrylamide microgels. This mix-and-read strategy allowed for one-pot analysis of several key miRNAs simultaneously and provided 5-fold enhancement in fluorescent detection sensitivities compared to the individual EXPAR-based assays. Four urinary miRNAs (let-7a, miRNA-155, -223, and -143) could be quantitatively determined in a wide linear range from 50 fM to 30 nM, with the limits of detection at femtomolar levels. Using a smartphone-based imaging microreader, healthy and cancerous cohorts with prostate, bladder, and renal cell cancers could be discriminated in 30 min with the accuracy >83% using linear discriminant analysis. The developed detection platform has proven to be a portable, noninvasive, and useful complement to the toolbox for miRNA-based liquid biopsies, which holds immense potential and advantage for regular and large-scale applications in early cancer diagnosis.