Fluorescence Lifetime Decoupling Enables Absolute Quantification of MicroRNA at Dense DNA-Gold Interfaces

Abstract Accurate quantification of microRNAs in complex biological fluids remains a challenge due to two intertwined analytical artifacts: false-positive signals from sterically bulky interferents and systematic signal underestimation from nanometal surface energy transfer (NSET) quenching. Here, we report a fluorescence lifetime (FL)-based decoupling strategy that mathematically isolates the NSET quenching bias and retrieves the true catalytic turnover number (TON ≈42.2) from conventionally quenched plasmonic readouts. Integrated with a dense DNA polymer brush that enforces strict size-exclusion selectivity (σ ≈ 0.22 chains/nm2), this approach enables absolute, calibration-free quantification of microRNA-21 directly in untreated human serum. The method achieves a limit of detection of 0.4 pM, excellent correlation with RT-qPCR (R2 = 0.978), and high diagnostic accuracy for colorectal cancer (AUC = 0.945 and threshold 1.55 pM). This FL decoupling protocol provides a standardized calibration method for eliminating optical artifacts in surface-tethered biosensing across different nanoprobe batches.