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Photoelectrochemical (PEC) methods have recently witnessed ever expanding application in bioanalysis, but it is still desirable to further simplify the sensing procedures and develop simple and reliable PEC biosensing approaches. Herein, we proposed a truly label-free and immobilization-free PEC sensing platform, utilizing solution-phase methylene blue (MB) as the signal probe, and bare indium tin oxide (ITO) glass as the photoelectrode. Based on the diffusivity difference between free MB molecules and MB intercalated in DNA G-quadruplex, the activity and inhibition of DNA adenine methyltransferase (Dam), a proof-of-concept methyltransferase (MTase), is quantitatively analyzed. By taking advantage of the endonuclease-catalyzed cleavage of the Dam-methylated hairpin DNA probe, as well as the KF polymerase/Nt.AlwI endonuclease-aided signal amplification, highly sensitive and specific PEC detection of Dam activity has been achieved. Moreover, this approach can be easily extended to assay other types of MTase by choosing the appropriate methylation-sensitive endonucleases. The as-proposed strategy has also been successfully applied to analyze Dam spiked in human serum samples and to assess the inhibitory effects of antibiotics on Dam activity. More importantly, this label-free and truly immobilization-free PEC biosensing strategy shows additional merits of simplicity and satisfactory repeatability, due to the elimination of both labelling and immobilization procedures, making it a promising candidate for the application in highly sensitive, facile and reliable bioanalysis and drug screening.
This article was published in the following journal.
Name: Biosensors & bioelectronics
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