Abstract
To harness the applicability of microribonucleic acid (miRNA) as a cancer biomarker, the detection sensitivity of serum miRNA needs to be improved. This study evaluated the detection sensitivity of miRNA hybridization using cyclic voltammograms (CVs) and microelectrode array chips modified with peptide nucleic acid (PNA) probes and 6-hydroxy-1-hexanethiol. We investigated the PNA probe modification pattern on array chips using fluorescently labeled cDNA. The pattern was not uniformly spread over the working electrode (WE) and had a one-dimensional swirl-like pattern. Accordingly, we established a new ion-channel sensor model wherein the WE is negatively biased through the conductive π–π stacks of the PNA/DNA duplexes. This paper discusses the mechanism underlying the voltage shift in the CV curves based on the electric double-layer capacitance. Additionally, the novel hybridization evaluation parameter ΔE is introduced. Compared to conventional evaluation using oxidation current changes, ΔE was more sensitive. Using ΔE and a new hybridization system for ultrasmall amounts of aqueous solutions (as low as 35 pL), 140 zeptomol label-free miRNA were detected without polymerase chain reaction (PCR) amplification at an adequate sensitivity. Herein, the differences in the target molar amount and molar concentration are elucidated from the viewpoint of hybridization sensitivity.
Highlights
Since the initial discovery of microribonucleic acid (miRNA) [1], its functions have been investigated worldwide, with regard to cancer
With regard to electrochemical gene sensor array chips based on ion-channel sensing, Aoki et al [17,18] have discussed the working principle of a sensor with oligonucleotide targets and peptide nucleic acid (PNA) probes
This working model was established based on the assumption that PNA/DNA duplexes are uniformly spread over the working electrode (WE)
Summary
Since the initial discovery of miRNA [1], its functions have been investigated worldwide, with regard to cancer. MiRNA contributes to tumorigenesis through numerous genetic mechanisms, and its expression profiles in serum or plasma serve as potential noninvasive diagnostic and prognostic biomarkers for cancer [2,3,4,5,6,7,8]. To harness the applicability of miRNA as a cancer biomarker, we must establish appropriate methods for its detection. Among the many requirements that must be satisfied to accomplish this, a high miRNA detection sensitivity is paramount because miRNA levels in the human blood are very low. The levels of circulating miRNA in cancer patients depend on the miRNA type, of which mir-21-5p is present at relatively high levels.
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