Abstract
The qualitative and quantitative determination of marker protein is of great significance in the life sciences and in medicine. Here, we developed an electrochemical DNA biosensor for protein detection based on DNA self-assembly and the terminal protecting effects of small-molecule-linked DNA. This strategy is demonstrated using the small molecule biotin and its receptor protein streptavidin (SA). We immobilized DNA with a designed structure and sequence on the surface of the gold electrode, and we named it M1-Biotin DNA. M1-Biotin DNA selectively combines with SA to generate M1-Biotin-SA DNA and protects M1-Biotin DNA from digestion by EXO III; therefore, M1-Biotin DNA remains intact on the electrode surface. M1-Biotin-SA DNA was modified with methylene blue (MB); the MB reporter molecule is located near the surface of the gold electrode, which generates a substantial electrochemical signal during the detection of SA. Through this strategy, we can exploit the presence or absence of an electrochemical signal to provide qualitative target protein determination as well as the strength of the electrochemical signal to quantitatively analyze the target protein concentration. This strategy has been proven to be used for the quantitative analysis of the interaction between biotin and streptavidin (SA). Under optimal conditions, the detection limit of the proposed biosensor is as low as 18.8 pM, and the linear range is from 0.5 nM to 5 μM, showing high sensitivity. The detection ability of this DNA biosensor in complex serum samples has also been studied. At the same time, we detected the folate receptor (FR) to confirm that this strategy can be used to detect other proteins. Therefore, this electrochemical DNA biosensor provides a sensitive, low-cost, and fast target protein detection platform, which may provide a reliable and powerful tool for early disease diagnosis.
Highlights
Protein is one of the typical biomarkers [1]
B1-DNA is modified with a sulfhydryl group ((−CH2 )6 SH) at the 50 end and self-assembled on a gold electrode via gold–thiol bonds, whereas the 30 end of B1-DNA is modified with a small molecular recognition compound biotin
Using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), we have shown that SA protects the 3’ end of M1Biotin DNA from EXO III hydrolysis
Summary
Protein is one of the typical biomarkers [1]. We used the biotinstreptavidin system as a template system to develop an electrochemical DNA biosensor that uses small molecule–protein molecule interactions to detect target proteins. Research and development of the interaction between small molecules, proteins, and other biological macromolecules has attracted increasing attention, which is the basis of signal transduction, energy transfer, metabolism, and functional regulation [3]. Exploiting the interaction of small molecules with proteins and other biological macromolecules to enable the specific binding of small molecules to proteins with strong affinity is essential in the fields of biogenetics and macromolecular detection [4,5]. The specific binding of some small molecules to proteins destroys some of the functions of the protein receptor
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