Ultrasensitive Multiplexed Detection of Protein Biomarkers via Aptamer-Driven T7 RNA Polymerase Amplification

Accurate and multiplexed detection of protein biomarkers in complex biological fluids is essential for early diagnosis and precision medicine. However, existing technologies face critical trade-offs among sensitivity, specificity, multiplexing capability, and operational simplicity. Antibody-based assays like ELISA often lack the sensitivity for early detection, while nucleic acid amplification methods are poorly suited for protein targets and require complex workflows. This creates a pressing need for a simple, highly sensitive, and interference-free approach for simultaneous protein detection in real-world samples. Here, we report an aptamer-driven T7 RNA polymerase cascade amplification system for visual, dual-channel detection of protein biomarkers directly in serum. The platform converts target recognition into isothermal RNA transcription, producing red or green fluorescence via fluorogenic RNA-dye complexes. Using alpha-fetoprotein and human serum albumin as models, the system achieved femtogram-per-milliliter sensitivity, excellent specificity, and broad linear ranges. A red-green fluorescence logic enables intuitive clinical interpretation. Validation with spiked and clinical samples showed recovery rates between 90 and 110% and strong agreement with ELISA results. These findings demonstrate that protein-responsive nucleic acid circuits can achieve precise, multiplexed detection with instrument-free signal amplification. By integrating molecular recognition, signal amplification, and visual readout, this strategy offers a scalable, point-of-care diagnostic framework and contributes to advancing protein-regulated synthetic biology circuits.