Enzyme-Free DNA Logic Circuit with Single-Color Readout for Dual Biomarker Detection

Abstract

This study introduces an innovative enzyme-free DNA circuit system for the simultaneous detection of two distinct nucleic acid biomarkers using a single colorimetric output with four discrete intensity levels. The system integrates toehold-mediated strand displacement (TMSD) and catalytic hairpin assembly (CHA) to generate diagnostic signals based on G-quadruplex structures. Two logic gates, ABC and DE, are designed to respond differentially to inputs: the ABC gate disassembles two G-quadruplexes upon recognition of Input 1, while the DE gate forms a G-quadruplex in response to Input 2. This configuration yields four unique output states corresponding to distinct input combinations. The circuit achieves high sensitivity, with detection limits of 5 pM for Input 1 and 1 pM for Input 2, and displays strong specificity against non-target DNA sequences. Validation in complex biological matrices confirmed the robustness of the system: it maintained 85 ± 3% signal in 50% human serum, indicating high compatibility with clinical sample environments. By employing a single output signal format, the approach simplifies data interpretation, reduces processing time, and enhances cost-effectiveness compared to conventional multi-signal platforms. As an automated, enzyme-free, and one-step detection system, this platform offers a powerful tool for accurate and efficient multiplex biomarker analysis, with promising applications in early disease diagnosis and point-of-care testing.

Keywords: Biomarker detection; Colorimetric assay; DNA circuits; Enzyme-free diagnostics; G-quadruplex; Toehold-mediated strand displacement.