Amperometric sensors with nucleic acid amplification are expected to be useful for sensitive RNA detection. Nucleic acid amplification reaction involves current changes associated with accelerated redox reactions. Among the nucleic acid amplification methods, we have attempted to apply the signal amplification by ternary initiation complexes (SATIC) system to amperometric sensors1,2. Amperometric sensor with SATIC system detected target RNA by measuring the reduction of hydrogen peroxide by DNAzyme via ferrocene methyl alcohol (FMA). Although linear sweep voltammetry (LSV) was used in previous study, the current was included reactions other than reduction by DNAzyme, such as the charge of the electrical double layer and mass diffusion of redox species. Pulsed electrochemical measurement removes the other current by refreshing the diffusion layer of redox species with each pulse voltage. Therefore, in this study, we focused on pulsed electrochemical measurement methods to further increase the signal-to-noise ratio of amperometric sensors with SATIC system by removing the noise. Current values derived from nucleic acid amplification with the SATIC system were compared by LSV, differential pulse voltammetry (DPV), normal pulse voltammetry (NPV) and square wave voltammetry (SWV.)DNA primers and 6-merchapto-1-hexanol (MCH) were immobilized on gold electrode via gold-thiol bond. Then, the SATIC reaction proceeded (37 ºC, 2 h) on the electrode by target RNA, a circular DNA template, and φ29 DNA polymerase. After that, the electrode was placed in phosphate buffer containing hemin which iron-containing porphyrin. DNAzyme was formed by the G-quadruplex (G4) in the amplified nucleic acid by binding to hemin. The current value changes resulting from the catalytic reaction of DNAzyme before and after the SATIC reaction were measured by LSV, DPV, NPV, and SWV. The ranges of voltage scan were -0.15〜-0.4 V vs. Hg/Hg2SO4 and -0.05〜-0.4 V vs. Hg/Hg2SO4 in LSV and other pulsed electrochemical measurement, respectively. The step potential was 5 mV, pulse wide was 50 ms, and pulse periode was 100 ms. Additionally, the pulse amplitudes of DPV and SWV were 50 mV and 25 mV, respectively. The measuring points each were acquired after 40 s from applying the pulse signals.First, each electrochemical measurement methods in a simple redox reaction were compared. The current density corresponding to the reduction reaction of FMA was measured using a polished bare gold electrode. The results showed an increase in current density depending on FMA concentration in all electrochemical measurement methods. Furthermore, the slope of the current value change relative to the FMA concentration was found to be greater in the order LSV < SWV ≦ DPV ≦ NPV. As the pulse eliminated the contribution of the charging current due to the electrical double layer, the quantitative detectability of DPV, NPV and SWV was higher than LSV. Based on the above results, the signal-to-noise ratio of amperometric sensors with SATIC system was compared using each electrochemical measurement method. The amount of current density changes with the progression of the SATIC reaction to 1 µM Target RNA was measured. As a result, current density changes of LSV: 1.98 μA/cm2, DPV: 12.2 μA/cm2, NPV: 3.41 μA/cm2 and SWV: 9.04 μA/cm2 were observed (Figure 1). In NPV measurement, the variation in current density change was more varied than that using other electrochemical measurements. As the pulse signal were impressed from the open circuit potential in NPV, the current included not only electron transfer but also mass diffusion of FMA mediator, resulting in the variation of repeatability in each measurement. Moreover, in DPV and SWV measurements, the changing in current density were greater than that of LSV measurement. This result could be attributed to the fact that the contribution of the current due to the charging electrical double layer and mass diffusion of FMA mediator was removed by the impressed pulse signal. However, the signals obtained from measurements using DPV and SWV represent the slope of the current value change relative to the potential scan. Therefore, it is not advisable to compare each electrochemical measurement at a single RNA concentration point. In the presentation, we will further discuss the signal-to-noise ratio of measurement methods of amperometric sensors using the SATIC system by comparing the concentration dependence of the target RNA concentration obtained from each electrochemical measurement.Reference:[1] H.Fujita et al., Anal. Chem. 88, 7137−7144 (2016).[2] H.Saze et al., 2023 ECSJ Fall Metting, Abstr, S12_2_04, [in Japanese] Figure 1
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Jan 13, 2025
Panting Wang + 4
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