A reusable ratiometric electrochemical biosensor on the basis of the binding of methylene blue to DNA with alternating AT base sequence for sensitive detection of adenosine.

We develop a reusable ratiometric electrochemical biosensor on the basis of the binding of methylene blue (MB) to DNA with alternating AT base sequence for sensitive detection of adenosine. We design a strand 1 with MB-modified thymine (T) base in the proximal 3' termini as the capture probe for its immobilization on the gold electrode and a 3' termini ferrocene (Fc)-modified aptamer for the recognition of adenosine. The hybridization of strand 1 with the aptamer leads to the formation of a double-stranded DNA (dsDNA) and consequently the away of MB from the electrode surface and the close of Fc to the electrode surface, generating a small value of IMB/IFc (IMB and IFc are the peak currents of MB and Fc, respectively). In the presence of adenosine, its binding with the aptamer induces the release of Fc from the electrode surface and the close of MB to the electrode surface, generating a large value of IMB/IFc. As a result, adenosine may be accurately quantified by the measurement of ratiometric signal (IMB/IFc). This ratiometric electrochemical biosensor can be simply fabricated and exhibits high sensitivity with a limit of detection of as low as 90.8pM and a large dynamic range from 0.1nM to 100μM. Moreover, this biosensor demonstrates good performance with excellent selectivity, regeneration capability, high reliability and good reproducibility, and may become a universal platform for the detection of various biomolecules which can be recognized by aptamers, holding great potential for further applications in biomedical research and clinical diagnosis.