EIS and Amperometric Analysis of Gold Nanoparticle Probes on the Immunosensor Surface

Abstract

[Introduction]Biosensing systems with integrated nanomaterials have revolutionized technological advancements in recent decades. Gold nanoparticles (AuNPs) in particular have been widely used in electrochemical sensors owing to their stability and unique characteristics. Previously, we developed biosensors that utilize the redox activity of AuNPs. Briefly, antibody-modified AuNPs not only hold secondary antibodies to react with antigens and primary antibodies on the electrode but also supply redox signals. We specially call this biosensing method Gold-Linked Electrochemical ImmunoAssay (GLEIA). In recent years, GLEIA has evolved uniquely in combination with other nanomaterials. However, information regarding the optimal electrochemical measurement conditions and the relationship between the electrode surface and AuNPs remain unknown. In this study, we quantitatively determined kinetic parameters such as the diffusion coefficient and electron transfer rate in the redox reaction of AuNPs on disposable screen-printed electrodes. Furthermore, the relationship between the addition of antibodies and the decrease in the determined parameters could be deduced. This is useful for the determination of the fine balance between the sensitivity and specificity.[Experimental]Electrochemical characteristics of AuNPs were performed using the following method, where 2 µL of AuNPs solution was dropped onto the working electrode and allowed to dry spontaneously. The number of AuNPs on each electrode was calculated from the datasheet (2.6 x 1010 particles/mL, O.D. 520 =1.0), and all nanoparticles were assumed to have dried on the working electrode. To characterize the reduction current, the AuNPs on the electrode underwent a pre-oxidation process, followed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) to evaluate the reduction current. The pre-oxidation condition was set at 1.2 V for 40 s, which was considered optimal for immunoassays. To evaluate the concentration response, 2 µL of the AuNPs solution was diluted with ultrapure water and poured and allowed to dry on the working electrode, pre-oxidized in 2 M KCl solution, and DPV measurements were performed. This experiment was also performed on an antibody-modified electrode, and the differences between the modified and unmodified electrodes were established. The same DPV measurements were performed using antibody-modified AuNPs (Ab-AuNPs).For immunoassay, IgA was reacted with a secondary antibody and incubated dropwise on an electrode. After washing, electrochemical measurements were performed[Results and Discussion]We investigated the influence of the DPV parameters and electrode surface conditions on a biosensor for the redox reaction of AuNPs. CV and classical theory were used to investigate the reduction kinetics of the AuNPs’ reduction after oxidation. The diffusion coefficient and electron transfer rate, which are related to sensor performance, were significantly reduced in the presence of antibodies and blocking agents on the electrode surface. This was due to the interaction between proteins and gold ions, and the effect of the electrode surface on sensitivity was evaluated using electrochemical reaction rates. Next, the influence of the biosensor on sensitivity was examined by focusing on the pulse amplitude of the DPV, and the increase in signal current coupled to increase in pulse amplitude was confirmed. The signal of AuNPs relative to the blank sample was of particular interest. Theoretically, changing the pulse potential above 100 mV does not change the current values obtained; however, in this study, a significant difference was observed between 150 mV and 100 mV. This was presumably due to the detection of nonspecific adsorption of antibodies that could not be detected under conventional conditions, suggesting the use of a more sensitive biosensor. This study provides evidence for fabrication of an IgA sensor with an improved Limit of detection (LOD) by optimizing pulse amplitude.Limits of detection (LOD) were obtained as 5.0 fmol/L (M) for AuNPs on a bare electrode, 0.1 pM for AuNPs on an antibody-modified electrodes, and 9.3 pM for AuNPs after immunoassay, allowing discussion of the given principle limits and actual biosensor performance (Figure). Under optimal conditions, the electrochemical biosensor achieved nearly two-fold signal amplification using a reduction current after AuNPs oxidation. These findings will allow the investigation of the kinetics of AuNPs on electrodes consisting of different materials to further elucidate the framework for improved sensitivity. Figure 1