Ultrasensitive electrochemical detection for thrombin using hybridization chain reaction with enzyme-amplification

• Ultrasensitive electrochemical detection for thrombin was obtained with aptasensor.
• Hybridization chain reaction was employed for amplifying signal.
• The proposed strategy is successfully applied in real sample assay.
• Scanning electrochemical microscopy was adopted to visualize captured thrombin.

In this work, a new electrochemical aptasensor using hybridization chain reaction (HCR) for signal amplification was developed for highly sensitive detection of thrombin. The sandwich system of aptamer/thrombin/aptamer–primer complex was fabricated as the sensing platform. As the initiator strands, aptamer–primer complex could propagate a chain reaction of hybridization events between the two hairpin probes, and whether long nicked DNA polymers could be formed on the modified electrode. Then the biotin-labeled dsDNA polymers could introduce numerous avidin-labeled horseradish peroxidase (HRP), resulting in significantly amplified electrochemical signal through the electrocatalysis of HRP. On the basis of the enzymatic oxidization of Fe2+ by H2O2 to yield Fe3+, the imaging of thrombin was detected by the reduction current of Fe3+ with the scanning electrochemical microscopic tip. The electrochemical signals had a good linear with logarithm of thrombin concentration in the range from 1.0 fM to 100 fM, reaching a detection limit of thrombin as low as 0.04 fM. In addition, the proposed strategy exhibited excellent specificity and was successfully applied in real sample assay which demonstrated the potential application in clinical diagnostics.

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