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<p>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.</p> Graphical Abstract <p></p>

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

  • 期刊论文

  • 发表日期：

    2026年1月8日

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  • Fatemeh Jafari,
  • Hadi Ravan,
  • Moj Khaleghi

摘要

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.

Graphical Abstract