To verify the feasibility of the

To verify the feasibility of the proposed method, EIS is firstly adopted to study the modification processes of the electrode. As shown in Fig. 1A, the nyquist plot of bare gold electrode exhibits a straight line, which indicates a fast charge transfer process (curve a). After modified with probe a, a small semicircle appears, indicating the increase of interfacial charge transfer resistance by the attached DNA SP600125 synthesis (Rct: 2425 ± 11 Ω). After further hybridized with probe b, the diameter of the semicircle increases, demonstrating successful formation of double-stranded DNA (Rct: 6148 ± 23 Ω). However, hOGG1 specifically cleaves the DNA probes, which leads to the decrease of the diameter of the semicircle (Rct: 5201 ± 18 Ω). Finally, the incubation of the electrode with probe c, d and e results in the formation of long double-stranded HCR product, which repels the [Fe(CN)6]3−/4− significantly and the diameter of the semicircle increases remarkably (Rct: 19250 ± 49 Ω). EIS results demonstrate the electrode modifications successfully. We have then performed DPV measurements of Fc and [Ru(NH3)6]3+ respectively. Fig. 1B shows DPV curves reflecting Fc signals in the absence and presence of hOGG1. As expected, without hOGG1 catalyzed reaction, the peak current is quite large. After the introduction of the target, the peak current decreases due to the release of Fc labeled DNA probe b. On the other hand, Fig. 1C depicts DPV curves reflecting [Ru(NH3)6]3+ signals in the absence and presence of hOGG1. Since HCR occurs only after hOGG1 catalyzed reaction, the peak current is increased after the introduction of hOGG1. These results have well confirmed the feasibility of the proposed strategy. Before quantitative detection of hOGG1, optimized experimental conditions are determined by comparing the peak currents of Fc and [Ru(NH3)6]3+: probe b immobilization time, 60 min; HCR reaction time, 60 min; hOGG1 reaction time, 40 min; hOGG1 reaction temperature, 37 °C. The sensitivity of the sensing method for the detection of hOGG1 activity is then evaluated under the optimized experimental conditions. As shown in Fig. 2A and C, the elevated concentration of hOGG1 from 0 to 10 U mL−1 causes a gradual decrease in the Fc peak and a gradual increase in [Ru(NH3)6]3+ peak. Detailed relationships between the peak currents and the concentrations of hOGG1 are summarized in Fig. 2B and D. Two equations are fitted as follows with the linear range from 0.01 to 10 U mL−1: The limit of detections (LODs) of single Fc and [Ru(NH3)6]3+ signals are calculated to be 0.004 U mL−1 and 0.006 U mL−1, respectively. We have then calculated corresponding values of iRu/iFc and found a double-logarithmic relationship between the value and hOGG1 concentration in a wider range from 0.002 to 10 U mL−1 (Fig. 3A). In addition, an ultralow LOD is achieved to be 0.0008 U mL−1 (S/N = 3). The regression equation is as follows: After comparing with recently developed hOGG1 assay, this proposed ratiometic electrochemical method exhibits a lower LOD and a wider linear range, which shows great superiority (Table 2). In addition, the hOGG1 level in certain cancer cells is about 1 U mL−1, which lies in the detection range of this method, demonstrating its potential practical utility [25]. To further evaluate the specificity of the proposed biosensor, it has been challenged by three proteins as interference species, including EcoRI, Bst and TE. As shown in Fig. 3B, these proteins cannot produce significant electrochemical responses compared with target hOGG1, even with 10 times higher concentrations. To further explore the application of the method in complex samples, we have spiked different levels of hOGG1 in serum samples, which are used for hOGG1 analysis. Analogical electrochemical signals could be obtained, verifying that the proposed method has potential to be used in complex biological matrix.