Microarray Needle Sensor Composite Conducting Polymer Coated on Au/Zn Oxide Layer for Continuous Glucose Detection in Cells

Abstract

Diabetes mellitus is a serious metabolic disease that may pose a threat to life owing to various complications. Therefore, patients are required the tight and accurate monitoring of blood glucose level to prevent and reduce complications [1]. In this regard, the continuous glucose monitoring system is most desirable to the efficient management of diabetes, which especially provides painless, and accuracy glucose level. Hence, we report for the development of a robust and simple enzymatic glucose sensor using Au coated microneedle arrays.The AuZn layer was electrochemically grown on Au coated microneedle arrays in Na2SO4 containing HAuCl4·4H2O and ZnCl2 using normal pulse voltammetry method. Electrochemical oxidation of the AuZn was performed in 0.1 M PBS (pH 7.4) using chronoamperometry method. Then, glucose oxidase bound on a conductive polymer precursor monomer (GOx-TCA) was formed on the AuZnOx through cyclic voltammetry method in TCA-GOx solution [2]. To prepare the TCA-GOx, TCA monomer was dissolved in CH3CN containing 0.1 M TBAP. GOx was added in distilled water containing 10 mM EDC/NHS. After that, the solutions were mixed with stirring and incubated at 40oC for 3 h that result in GOx couple on TCA through the amide bond formation. Finally, GOx-pTCA/AuZnOx probe was dipped into Nafion solution and dried for 4 hours at CaCl2 atmosphere. The polymer/enzyme composite stably produced H2O2 by the enzyme reaction. The produced H2O2 was catalytically reduced by the AuZnOx layer. We oxidized AuZn alloy nanostructured layer, which increased the surface area, and the catalytic activity towards hydrogen peroxide [3]. The sensitivity of the AuZnOx probe shows four times higher than AuZn layer. This indicates that AuZnOx layer improves the sensitivity for glucose detection. The reproducibility was examined using ten different sensors, and a relative standard deviation of 1.89 % at 10mM glucose was achieved. The sensitivity was 0.43 μA mM-1with a linearity of 0.998, and within 1 sec, in glucose concentrations ranging from 0.1 to 50 mM. The calibration plots for the glucose were obtained between 0.1 to 5.0 mM and 5.0 to 50.0 mM. The glucose sensor achieved a low detection limit of 90.0 ± 1.6 μM. In order to assess the possibility of interference, the chronoamperometric response of the modified micro needle sensor was measured for ascorbic acid (AA), uric acid (UA), acetaminophen (AP) and dopamine at a concentration of 0.1 mM. Amperometric responses show that, approximately 5% response for AA, UA, AP, and dopamine compared with the glucose signal. The sensitivity of the sensors retained 95% of its initial sensitivity for up to seven days of continuous monitoring at 10 mM glucose concentration. For the determination of glucose concentration inside and outside a single cell we have used PC - 12 and Vero cell lines and isolated and grown a single cell of each in a 60 × 15 mm petri dish for ease of access. Then at first we have measured the voltammogramms for glucose by placing the microneedle adjacent to the cell in culture, followed by insertion of the needle electrode into the cell by piercing the plasma membrane. We have observed the glucose oxidation peaks both inside and outside the single cell using a needle electrode. The result showed that the glucose concentration inside the cells is comparatively higher than the external environment and the cancer cells show almost double the concentration of intracellular glucose as compared to normal cells. Conclusively, the response of the modified micro needle type sensor was very selective, and it could be extended for use in glucose detection in medical diagnosis.[1] Wang, J.; et al., Chem. Rev., 2008, 108, 814.[2] Lee, T.-K.; Shim, Y.-B.; et al., Anal.Chem., 2001, 73, 5629-5632[3] Kim, D.-M.; Shim, Y.-B.; et al., Biosens.Bioelectron., 2016 , 79, 165–172.