Antibody-Protein-Aptamer Electrochemical Biosensor based on Highly Efficient Proximity-Induced DNA Hybridization on Tetrahedral DNA Nanostructure for Sensitive Detection of Insulin-like Growth Factor-1.

Herein, an antibody-protein-aptamer electrochemical biosensor was designed by highly efficient proximity-induced DNA hybridization on a tetrahedral DNA nanostructure (TDN) for ultrasensitive detection of human insulin-like growth factor-1 (IGF-1). Impressively, the IGF-1 antibody immobilized on the top vertex of the TDN could effectively capture the target protein with less steric effect, and the ferrocene-labeled signal probe (SP) bound on the bottom vertex of the TDN was close to the electrode surface for generating a strong initial signal. In the presence of target protein IGF-1 and an aptamer strand, an antibody-protein-aptamer sandwich could be formed on the top vertex of TDN, which would trigger proximity-induced DNA hybridization to release the SP on the bottom vertex of TDN; therefore, the signal response would decrease dramatically, enhancing the sensitivity of the biosensor. As a result, the linear range of the proposed biosensor for target IGF-1 was 1 fM to 1 nM with the limit of detection down to 0.47 fM, which was much lower than that of the traditional TDN designs on electrochemical biosensors. Surprisingly, the use of this approach offered an innovative approach for the sensitive detection of biomarkers and illness diagnosis.

Ultrasensitive Electrochemical Biosensor with Powerful Triple Cascade Signal Amplification for Detection of MicroRNA.

In this work, by ingeniously integrating catalytic hairpin assembly (CHA), double-end Mg2+-dependent DNAzyme, and hybridization chain reaction (HCR) as a triple cascade signal amplifier, an efficient concatenated CHA-DNAzyme-HCR (CDH) system was constructed to develop an ultrasensitive electrochemical biosensor with a low-background signal for the detection of microRNA-221 (miRNA-221). In the presence of the target miRNA-221, the CHA cycle was initiated by reacting with hairpins H1 and H2 to form DNAzyme structure H1-H2, which catalyzed the cleavage of the substrate hairpin H0 to release two output DNAs (output 1 and output 2). Subsequently, the double-loop hairpin H fixed on the electrode plate was opened by the output DNAs, to trigger the HCR with the assistance of hairpins Ha and Hb. Finally, methylene blue was intercalated into the long dsDNA polymer of the HCR product, resulting in a significant electrochemical signal. Surprisingly, the double-loop structure of the hairpin H could prominently reduce the background signal for enhancing the signal-to-noise ratio (S/N). As a proof of concept, an ultrasensitive electrochemical biosensor was developed using the CDH system with a detection limit as low as 9.25 aM, achieving favorable application for the detection of miRNA-221 in various cancer cell lysates. Benefiting from its enzyme-free, label-free, low-background, and highly sensitive characteristics, the CDH system showed widespread application potential for analyzing trace amounts of biomarkers in various clinical research studies.