Yoji Saito

In this study, we developed a sensing device that can detect deoxyribonuclease (DNase) based on the electrical properties of deoxyribonucleic acid (DNA). We estimated the equivalent circuit between the electrodes with immobilized DNA and investigated whether the characteristics of the electrodes change before and after the DNase reaction. This method detects DNase by simply evaluating the electrical properties of DNA without using a fluorescent reagent. Therefore, inexpensive and highly accurate measurements can be performed with simple operations. However, detection sensitivity must be increased for practical feasibility. Hence, we investigated whether DNA immobilization is restricted by changing the shape of the electrode to a triangle with sharp edges, which may improve the sensitivity of DNase. Additionally, we attempted to detect DNase from an extremely small amount of sample solution using a microchannel. The device was able to quantitatively analyze DNase I activity with a detection limit of 5.5 × 10-5unit/μL. The results demonstrate the effectiveness of the proposed sensing device for various medical applications.

In this study, we developed a sensing device that can detect deoxyribonuclease (DNase) based on the electrical properties of deoxyribonucleic acid (DNA). We estimated the equivalent circuit between the electrodes with immobilized DNA and investigated whether the characteristics of the electrodes change before and after the DNase reaction. This method detects DNase by simply evaluating the electrical properties of DNA without using a fluorescent reagent. Therefore, inexpensive and highly accurate measurements can be performed with simple operations. However, detection sensitivity must be increased for practical feasibility. Hence, we investigated whether DNA immobilization is restricted by changing the shape of the electrode to a triangle with sharp edges, which may improve the sensitivity of DNase. Additionally, we attempted to detect DNase from an extremely small amount of sample solution using a microchannel. The device was able to quantitatively analyze DNase I activity with a detection limit of 5.5 x 10(-5) unit/mu L. The results demonstrate the effectiveness of the proposed sensing device for various medical applications.

Serum deoxyribonuclease I (DNase I) can serve as a functional biomarker for the therapeutic monitoring of acute myocardial infarction and other diseases. Here, we demonstrate that the electrical properties of DNA molecules can be exploited to monitor enzymatic activity. A label-free DNA biosensor for the detection of DNase I activity was devised based on electrochemical impedance spectroscopy (EIS). Multiple lambda phage DNA molecules were immobilized between two electrodes in a polydimethylsiloxane reservoir. An equivalent circuit estimated from the EIS measurement was used to calculate the impedance of DNA molecules between the electrodes. DNase detection was then achieved by measuring the increase in impedance, after DNA cleavage by DNase I. This was assessed by the impedance-increase ratio, defined as R-after/R-before (where R-before and R-after represent the resistance between the electrode-immobilized DNA molecules before and after DNase I treatment, respectively). After treatment with DNase I at a concentration 10(-2) unit/mu L, a reproducible impedance-increase ratio of approximately 3.3 times was obtained, with a standard deviation of less than 20%. When DNase solutions of various concentrations were introduced, we succeeded in obtaining a definite correlation between DNase concentration and impedance-increase rate, within the range of 10(-4) unit/mu L to 10(-1) unit/mu L.

As a step toward applications for biosensors, we characterized the electrical properties of lambda DNA molecules via their current-voltage characteristics and complex impedance plots. lambda DNA molecules were introduced to a microfluidic device using a microchannel (depth, 50 mu m; width, 500 mu m; length, 10 mm) and electrostatically stretched and immobilized in the 14-mu m gap between two triangular-shaped microlithographed aluminum electrodes by applying an alternating voltage of 1 MHz and 20 Vp-p. The aligned lambda DNA showed nonlinear current-voltage characteristics. From the complex impedance plots of the lambda DNA molecules, an equivalent circuit was obtained as a series connection of two resistance-capacitance parallel circuits. Finally, we demonstrated that the electrical characteristics of the lambda DNA between the electrodes varied with the number of immobilized lambda DNA molecules.

This paper presents a novel microdevice which detects deoxyribonuclease (DNase) electrically. The present device can be applied to diagnoses of diseases such as acute myocardial infarction and monitoring of DNase-free water used in genetic research. Multiple DNA molecules were immobilized between two electrodes in a microchannel, and DNase detection was achieved by measuring the increase in impedance between the electrodes after DNA cleavage by DNase. When DNase solutions of various concentrations were introduced, we succeeded to obtain a definite correlation between impedance increase rate and DNase concentration from 10-5 to 10-1 unit/μl.