Enzyme-free dual amplification strategy for protein assay by coupling toehold-mediated DNA strand displacement reaction with hybridization chain reaction

• An electrochemical method has been developed for ultra-sensitive protein detection.
• The assay can realize low detection limit through the dual amplification strategy.
• The assay is relatively simple and cheap due to the enzyme-free process.
• Excellent performance in serum sample shows high selectivity of this method.

In this work, we present an enzyme-free electrochemical assay method that can detect target protein with ultra-high sensitivity through dual amplification. Firstly, target protein is specifically captured by its aptamer, and then is released due to toehold-mediated click chemical ligation (the first round of recycling) via DNA strand displacement reaction. Secondly, the overhang of aptamer on the electrode surface can hybridize with RP DNA and trigger hybridization chain reaction (the second round of recycling). Consequently, large amount of electrochemical species hexaammineruthenium (III) chloride ([Ru(NH3)6]3 +) can be embedded into double-stranded DNA to produce a remarkable electrochemical signal, thus the target protein can be quantified with ultra-high sensitivity. Taken thrombin as a model analyte, a wide linear dynamic range from 100 fM to 10 nM and a detection limit of 30 fM (S/N = 3) can be obtained. Meanwhile, since no enzyme is required for the measurement, the assay is relatively simple and inexpensive. Therefore, the protein assay method proposed in this work may have a great potential for clinical diagnosis and biomedical research in the future.

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