Poly(hydroquinone)-coated electrode for immobilizing of 5′-amine functioned capture probe DNA and electrochemical response to DNA hybridization

Abstract

Enzymatically polymerized hydroquinone, PHQ, was applied for polymer-coated electrode whose surface was further modified with 5 0 -amine dodecamer DNAs (capture probe DNA, CP-DNA) by taking advantage of Michael reaction. The film-forming property of PHQ on graphite substrate surfaces was confirmed to be satisfactory by AFM imaging. Cyclic voltammetric (CV) measurements showed that the PHQ-modified graphite electrode gave well-defined redox waves showing characteristics for surface process. The pH dependence of the formal potential, E 1/2 , suggested that the electrode reaction occurred by two-proton and two-electron mechanism (À59 mV per a pH decade). CVs also gave the specific amount of PHQ adsorption of 0.52 or 0.83 nmol cm À2 (monomer unit) for the different electrode preparations. This was indicative of two-or three-monolayer adsorption of PHQ. For applications of gene detection, the immobilization reaction including the CP-DNA hybridization was studied by microgravimetric analysis using a quartz-crystal microbalance (QCM). Summaries for the two different runs were 1.01 and 0.69 nmol cm À2 (monomer unit) for PHQ adsorption on gold surfaces, 0.19 and 0.15 nmol cm À2 for CP-DNA attachment on the PHQ/Au, and 0.14 and 0.11 nmol cm À2 for hybridization with the complementary DNA on the CP-DNA/PHQ/Au, respectively. Importantly, monitoring of the series of the experiments were possible by measuring the voltammetric properties of the electrode; the distinct redox waves due to PHQ-electrode reaction are suppressed upon immobilizing of the CP-DNA, and its hybridization further suppressed the redox activity. Neither treating of the PHQ-electrode with native DNAs nor treating of CP-DNA/PHQ-electrode with non-target DNA gave no noticeable responses. A possible mechanism for the electrode response was discussed briefly based on electrochemical QCM measurements. We think these observations are important as the basis of DNA hybridization sensor that enables totally, label-free electrochemical detection of the target DNA. r

Positive-feedback-mode scanning electrochemical microscopy imaging of redox-active DNA–poly(1,4-benzoquinone) conjugate film deposited on carbon fiber electrode for micrometer-sized hybridization biosensor applications

“…The formal potential of the surface adlayer was confirmed to be +245 mV, and the particular surface concentration of the redox species was determined to be typically 1.9 Â 10 -9 unit mol cm -2 : the surface coverage was more than twice compared with the previous data (0.85 nmol cm À2 vide infra). On the other hand, AFM imaging witnessed that the quinone polymer adsorb on carbon substrate surfaces to form defect-free, uniform polymer membrane [13]. These results seem to provide the evidence that the quinone polymer coating occurs almost completely at the CF electrode surface.…”

“…After dialysis, deglycosylation of the poly(arbutin) was made to give the product, poly(1,4-hydroquinone) (n = 10) as a dark brown powder [13]. CF microelectrodes (diameter, 33 lm) were obtained from BAS Inc., Japan and were used as the substrate electrodes.…”

“…We have recently reported the surface-grafting immobilization of capture probe (CP) DNA sequences on carbon electrodes coated with poly(1,4-benzoquinone), PQ [13]. The DNA-polymer conjugate film was found to be electrochemically responsive, allowing the label-free detection of hybridization.…”

3 Scanning Electrochemical Microscopy Imaging of DNA Arrays for High Throughput Analysis Applications

“…46 Previously, we reported the surface-grafting immobilization of capture-probe (CP) DNA sequences on carbon electrodes coated with poly(1,4-benzoquinone), PQ, that were oxidatively polymerized using peroxidase. 47 The DNA-polymer conjugate film was found to be electrochemically responsive, allowing the label-free detection of hybridization. Furthermore, the potentially electroconductive nature of the covalently linked, redox-active conjugate molecule was expected to be suitable for the robust, positive-feedback detection in SECM.…”

Application of dynamic impedance spectroscopy to atomic force microscopy

“…Since its development [1], atomic force microscopy (AFM) has found application in many scientific fields such as life sciences [2][3][4][5], materials engineering [6][7][8][9][10] and physics [11]. On the other hand, impedance spectroscopy is one of most important techniques for determining the electrical properties of materials [12][13][14][15][16][17][18].…”