Sample-to-answer salivary miRNA testing: New frontiers in point-of-care diagnostic technologies

Abstract

MicroRNA (miRNA), crucial non-coding RNAs, have emerged as key biomarkers in molecular diagnostics, prognosis, and personalized medicine due to their significant role in gene expression regulation. Salivary miRNA, in particular, stands out for its non-invasive collection method and ease of accessibility, offering promising avenues for the development of point-of-care diagnostics for a spectrum of diseases, including cancer, neurodegenerative disorders, and infectious diseases. Such development promises rapid and precise diagnosis, enabling timely treatment. Despite significant advancements in salivary miRNA-based testing, challenges persist in the quantification, multiplexing, sensitivity, and specificity, particularly for miRNA at low concentrations in complex biological mixtures. This work delves into these challenges, focusing on the development and application of salivary miRNA tests for point-of-care use. We explore the biogenesis of salivary miRNA and analyze their quantitative expression and their disease relevance in cancer, infection, and neurodegenerative disorders. We also examined recent progress in miRNA extraction, amplification, and multiplexed detection methods. This study offers a comprehensive view of the development of salivary miRNA-based point-of-care testing (POCT). Its successful advancement could revolutionize the early detection, monitoring, and management of various conditions, enhancing healthcare outcomes. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > Diagnostic Nanodevices.

Keywords: cancer; infectious disease; microRNA; multiplexed detection; neurodegenerative disease; point of care testing (POCT); saliva.

FIGURE 1

The biogenesis and regulatory mechanisms of miRNA and their relevance to various diseases (a) Schematic of miRNA biogenesis, processing, and mechanism of action in the network of gene expression by translational repression and mRNA degradation (b) salivary miRNA as biomarkers to its relevance to diseases for early disease diagnosis, including cancers, parkinson’s disease, concussion, and infectious disease.

FIGURE 2

Schematic illustration of detection of salivary miRNAs through traditional techniques and POCT. Traditional methods involve four steps with expensive equipment for high sensitivity and specificity with a long detection time. POCT is conducted on-site by integrated devices to isolate and detect miRNAs to rapidly get results with simple operation.

FIGURE 3

Isothermal methods for sensitive and multiplexed detection of miRNAs by in situ hybridization, enzyme-free amplification, and enzyme-based amplification.

FIGURE 4

In situ hybridization-based miRNA detection. (Adapted from J. Wu et al., 2021). a) sandwich structure-based multiplexed miRNA detection. b) DNA tetrahedron nanotag for the multiplexed detection of miRNA based on FRET from TOTO-1 to dyes (Adapted from H. Zhao et al., 2020).

FIGURE 5

Enzyme-free isothermal amplification for miRNA detection. a) HCR-TG-FRET amplification for duplexed miRNA (miR-20a and miR-21) detection. b) miRNA-initiated DNA molecular motor-based multiplexed miRNA detection.

FIGURE 6

Enzyme-assisted isothermal amplification a) the principle of exponential isothermal amplification, b) illustration of CAL-LAMP for imRNAs detection. c) the ligation/RPA-based multiplexed miRNA detection. d) digital multiplex miRNA detection procedure.

FIGURE 7

Enzyme-assisted cyclic amplification for miRNA detection. a) Exo I-based multiplexed miRNA detection based on the fluorescence enhancement of molecular beacon (MB) probes. b) DSN-assisted target recycling and multilayer core-satellite magnetic superstructures.

FIGURE 8

Microfluidic biosensor for miRNA detection. a) Schematic illustration of multiplexed miRNA detection based on shape-coded hydrogel microparticles integrated with HCR (Adapted from K. Zhao et al., 2022). b) miRNA assay scheme: target hybridization, universal linker ligation, gold nanoparticle labeling, and gold ion deposition-based signal amplification on chip (Adapted from H. Lee et al., 2020).

FIGURE 9

Paper-based miRNA extraction and detection on integrated devices. a) EXPAR amplification-based multiplexed exosomal miRNA assay through the on-chip (Adapted from H. Deng et al., 2017). b) Hairpin-EXPAR amplification-based multiplexed miRNA assay on the paper fluidic chip (Adapted from J. Qian et al., 2022).