Signal-amplified cell-free biosensing of antibiotics using tandem fluorescent aptamers

Transcription factor-based cell-free biosensors are attractive for rapid and portable antibiotic detection, yet their performance is often constrained by limited sensitivity and high background signals. Here, we developed enhanced cell-free biosensors using RNA fluorescent aptamers and evaluated the Pepper-HBC620 system as a low-background, high-signal-to-noise reporter compared with the widely used Broccoli aptamer. To overcome intrinsic sensitivity limitations, two complementary strategies were employed. First, the concentrations of DNA templates and transcription factors were simultaneously reduced while maintaining constant molar ratios, which effectively lowered the activation thresholds for erythromycin and tetracycline. Second, tandem fluorescent aptamer modules (4×Broccoli and 8×Pepper) were designed as enzyme-free signal amplifiers. RNA secondary structure prediction and in vitro transcription confirmed that the tandem constructs preserved proper folding and displayed fluorescence intensities proportional to aptamer copy number without altering excitation or emission wavelengths. By integrating these strategies, strong fluorescence outputs were obtained with substantially reduced amounts of DNA templates and regulatory proteins. The optimized mphO-4×Broccoli biosensor achieved a detection limit of 0.1 μM for erythromycin, while the tetO-8×Pepper system enabled tetracycline detection down to 0.05 μM with a shortened response time. Overall, this study establishes tandem RNA aptamer-based cell-free biosensors for sensitive antibiotic detection.