Truly Immobilization-Free Diffusivity-Mediated Photoelectrochemical Biosensing Strategy for Facile and Highly Sensitive MicroRNA Assay

• Published in: Analytical chemistry (Washington) 2018-08, Vol.90 (15), p.9591-9597
• Main Authors: Hou, Ting, Xu, Ningning, Wang, Wenxiao, Ge, Lei, Li, Feng
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Assaying; Biosensing Techniques - instrumentation; Biosensing Techniques - methods; Biosensors; Chemistry; Deoxyribonucleic acid; Diffusion; Diffusivity; DNA; DNA methylation; DNA Probes - chemistry; DNA, Single-Stranded - chemistry; Electrochemical Techniques - instrumentation; Electrochemical Techniques - methods; Electrodes; Equipment Design; Humans; Hybridization; Immobilization; Indium tin oxides; Methylene blue; Methylene Blue - chemistry; MicroRNAs; MicroRNAs - analysis; MicroRNAs - blood; Photoelectric effect; Photoelectric emission; Reproducibility; Reproducibility of Results; Ribonucleic acid; Ribonucleotide reductase; RNA; Strategy; Tin Compounds - chemistry
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.8b02523

In conventional photoelectrochemical (PEC) analysis, photoactive materials are usually immobilized on electrode surfaces, and such immobilization procedures are tedious and time-consuming, and it is also difficult to prepare electrodes with good reproducibility. To circumvent such limitations, we propose here a truly immobilization-free diffusivity-mediated PEC bionsensing strategy for microRNA assay, using methylene blue (MB) in solution as the photoactive probe, and nonmodified indium tin oxide (ITO) glass as the working electrode. The hybridization between the target microRNA and the MB-labeled single-stranded DNA probe (MB-DNA) triggers the digestion of MB-DNA by T7 exonuclease (T7 Exo), thus to generate MB-labeled mononucleotide, and then the released target microRNA initiates the subsequent cycling processes and generates a large amount of MB-labeled mononucleotides. Due to the diffusivity difference between MB-DNAs and MB-labeled mononucleotides, significantly increased photocurrent signal is observed for MB-labeled mononucleotides as compared to that of MB-DNAs. Therefore, via this “signal-on” mode and the T7 Exo facilitated signal amplification, a facile and highly sensitive immobilization-free PEC microRNA assay is readily realized, with a detection limit down to 27 aM. Moreover, this strategy exhibits excellent specificity and is successfully applied in detecting microRNA spiked in serum samples. Since all the reactions take place in homogeneous solutions and no electrode modification is needed, this PEC biosensing strategy exhibits the advantages of simplicity, rapidness, and good reproducibility. More significantly, it provides a novel concept to design truly immobilization-free PEC biosensing systems, and shows potential to be applied in bioanalysis and biochemical research.