Gold-Nanoparticle-Coated Magnetic Beads for ALP-Enzyme-Based Electrochemical Immunosensing in Human Plasma

Abstract

A simple and sensitive AuNP-coated magnetic beads (AMB)-based electrochemical biosensor platform was fabricated for bioassay. In this study, AuNP-conjugated magnetic particles were successfully prepared using biotin-streptavidin conjugation. The morphology and structure of the nanocomplex were characterized by scanning electron microscopy (SEM) with energy-dispersive X-ray analysis (EDX) and UV-visible spectroscopy. Moreover, cyclic voltammetry (CV) was used to investigate the effect of AuNP-MB on alkaline phosphatase (ALP) for electrochemical signal enhancement. An ALP-based electrochemical (EC) immunoassay was performed on the developed AuNP-MB complex with indium tin oxide (ITO) electrodes. Subsequently, the concentration of capture antibodies was well-optimized on the AMB complex via biotin-avidin conjugation. Lastly, the developed AuNP-MB immunoassay platform was verified with extracellular vesicle (EV) detection via immune response by showing the existence of EGFR proteins on glioblastoma multiforme (GBM)-derived EVs (10⁸ particle/mL) spiked in human plasma. Therefore, the signal-enhanced ALP-based EC biosensor on AuNP-MB was favorably utilized as an immunoassay platform, revealing the potential application of biosensors in immunoassays in biological environments.

Keywords: electrochemical immunoassay; gold nanoparticle; human plasma; indium tin oxide; magnetic bead.

Figure 1

Schematics for AuNP-coated MB complex (AMB) preparation and electrochemical immunoassay on AMB.

Figure 2

SEM/EDX analysis and UV–Vis absorption spectrum of AuNP-MB complex (AMB): (a) SEM image of AMB; (b) SEM image of MB; (c) EDX data of AMB; (d) UV–Vis absorption spectrum of the AMB, MB, and AuNP.

Figure 3

Analysis of signal enhancement of AMB: cyclic voltammetry (ranging from $-$0.2 V to 0.8 V) results of (a) AMB or MB labeled with ALP (red and blue lines, respectively). AMB or MB without ALP labeling were controls (green and dotted black lines, respectively); (b) current values for the tested samples at 0.6 V were recorded from Figure 3a; data = mean ± standard deviation, n = 3.

Figure 4

Cyclic voltammetry (CV) recorded (from $-$0.2 to 0.8 V) after an incubation period of 2 min at immunosensing electrodes treated with various concentrations of biotinylated anti-EpCAM mouse IgG antibody in PBS (a) and in 90% human plasma (c) in the developed AMB-based ITO platform. Anti-mouse IgG ab labeled with ALP was utilized for signaling. (b,d) Current values for the tested samples at 0.6 V were recorded in Figure 4a,c, respectively. Data = mean ± standard deviation, n = 3. As shown in the insets, all the data could be fitted using a linear line when plotting the X-axis in log-scale. The data were well-fitted with R² of 0.99 in PBS for (b) and with R² of 0.98 in 90% of human plasma for (d).

Figure 5

Chronocoulogram recorded (at 0.6 V) (a,c) and charge values at 50 s from Figure 5a,c are shown at 50 s after an incubation period of 2 min at immunosensing electrodes treated with different concentrations of EV and different capture antibodies (anti- EGFR or - EpCAM) in PBS (a,b) or in 90% of human plasma (c,d); Data = mean ± standard deviation, n = 3; * indicates a significant difference (p ≤ 0.05) between 0 and 10⁸ ptcl/mL of EVs with anti-EGFR antibodies.