Early myocardial infarction diagnosis using a paper-based nano-biosensor and image-driven quantification of microRNA-499

Early detection of myocardial infarction (MI) is critical for improving patient outcomes. However, current protein-based biomarkers are limited by delayed elevation and insufficient specificity for MI. Recently, circulating microRNAs (miRNAs) have emerged as promising alternatives due to their rapid and specific expression profiles. Notably, miRNA-499 rises swiftly post-ischemia, but conventional detection methods demand nucleic acid amplification or costly instrumentation, restricting their use in point-of-care settings. We developed a low-cost, paper-based nano-biosensor by functionalizing 12 nm gold nanoparticles with thermodynamically optimized hairpin molecular beacons, yielding ∼18 probes per particle. Nanoprobes are directly applied to the paper substrates, where quenched fluorophores “turn on” upon miR-499 binding. Fluorescence signal quantification was performed using both a benchtop reader and standard desktop camera, with image analysis processed via a custom OpenCV-based pipeline. The nano-biosensor demonstrates a linear detection range from 10 fM to 100 pM (R² = 0.92) with a limit of detection of 10 fM based on image analysis. Specificity studies reveal clear discrimination from single- and multi-base mismatches. Reproducibility testing across three independently fabricated nano-biosensor batches showed no statistically significant variability. Stability assessments confirmed consistent nano-biosensor response over 50 days at room temperature, affirming robust batch-to-batch reproducibility and long-term shelf life. The designed and fabricated nano-biosensor offers 10 fM limit of detection and high specificity for miR-499 on an inexpensive, disposable paper platform. The rapid, amplification-free assay requires only basic imaging equipment, supporting its potential for early MI detection and decentralized screening, particularly in resource-limited environments.