Real-Time Monitoring of Chemisorption of Antibodies onto Self-Assembled Monolayers Deposited on Gold Electrodes Using Electrochemical Impedance Spectroscopy

Abstract

Understanding protein binding to biosensing surfaces is paramount to the design and performance of biosensing devices in fields such as point-of-care testing and bioanalytics. Here, we systematically demonstrated the use of electrical impedance spectroscopy (EIS) and equivalent circuit modeling for real-time tracking of chemisorption of IgG antibody to large-area circular gold electrodes (1.3 mm²) functionalized with a self-assembled monolayer (SAM). Using 1 μg/mL IgG and 5 mM of [Fe(CN)₆]^3-/4-, the measured low-frequency impedance proved sensitive to both equilibrium and kinetics of antibody binding, with a slope of ∼74 kΩ/h for the first 2 h and taking approximately 4 h to reach equilibrium in a standard 6 mm-diameter well. Changes in impedance were found to be proportional to the reciprocal of the change in capacitance up to half-to-full IgG monolayer bound to the SAM. Further experiments with a flat microchannel confirmed that the low-frequency impedance and equivalent charge-transfer resistance (R_ct) depend not only on antibody diffusion but also on the surface-to-volume ratio, which can represent a major challenge previously unreported for the miniaturization of EIS in microfluidic devices. This challenge arises as it requires a higher concentration of [Fe(CN)₆]^3-/4-, of 50 mM or above, which was found to interfere with R_ct during chemisorption at low IgG concentrations. Chemisorption of IgG to SAM was confirmed with fluorescence microscopy and FTIR. This study marks, to the best of our knowledge, the first experimental demonstration of EIS as a real-time technique for quantitation of Langmuir isotherms during chemisorption of antibodies to SAM, with the potential to improve the design of EIS-based biosensors, especially those integrated into microfluidic devices.

1

Schematic view of the sensor fabrication: (a) sensor representation of one of the wells with two circular electrodes, each with 1.3 mm² of area. (b) Cross-sectional view of the sensing apparatus, comprising the following layers: SiO₂ (blue), gold electrodes (yellow), silicone gasket (light gray) and aluminum wall (dark gray). (c) Sensor representation with a PVC microchannel 6 mm (width) × 6 mm (length) × 0.3 mm (height). (d) COOH-terminated self-assembled monolayer (SAM) of mercaptohexadecanoic acid, activation of the surface, antibody incubation, and blocking.

2

EIS characteristics for SAM-activated electrodes (1.3 mm² area) before and after 2 h of incubation of various concentrations of IgG antibody (0.0625, 0.125, 0.1875, 0.25, 0.5, 0.75, 1, 5, 10, and 15 μg/mL). Experimental (symbols) and fitted (lines) data, measured between 1 Hz and 10⁵ Hz: (a) Impedance (Ω) as a function of frequency (Hz) in logarithmic scale. The Randles equivalent circuit model is represented in the inset. (b) Phase angle (θ) as a function of frequency (Hz) in semilog scale. (c) Nyquist plot, where Z’’ and Z’ are the real and imaginary parts of the impedance (Ω), respectively. (d) Capacitance (F) and dielectric loss, Gp/ω (F) over frequency (Hz) in semilog scale. The Maxwell–Wagner relaxation frequency is shown as f _r.

3

EIS characteristics in semilog scale, for functionalized electrodes, before and after 2 h of incubation of various concentrations of IgG (0.0625, 0.125, 0.1875, 0.25, 0.5, 0.75, 1, 5, 10, and 15 μg/mL). Relationship between (a) %Δ|Z| and (b) %ΔR _ct and antibody concentrations, with the continuous lines showing the best-fit Langmuir isotherms, calculated using eq . (c) Maxwell–Wagner relaxation frequency (f _r) over IgG concentration. All error bars shown in (a) and (b) indicate the standard deviation (±1 SD) based on at least 3 experimental replicas, reflecting the reproducibility of the measured impedance parameters.

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EIS results for the incubation of 1 μg/mL of IgG over 20 h of incubation. (a) Impedance (|Z|) and (b) Phase angle as a function of time. (c) Impedance (|Z|) as a function of time^1/2, suggesting the process becomes diffusion-limited beyond the initial 2 h, due to the long distance of diffusion of IgG in the well. The light gray area in (a) represents the standard deviation (±1 SD). This shaded area was used for visual clarity and representation of at least 3 experimental replicas, indicating experimental variability; nonetheless, the general trend remained robust.

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EIS measurements with redox probe concentrations between 1 and 100 mM [Fe­(CN)₆]^3–/4– in 1× PBS in a 6 mm internal diameter well. (a) Impedance values for various redox probe concentrations over 2 h each (at 1 Hz) in semilog scale. (b) Charge transfer resistance as a function of the concentration of redox probe (mM), at t = 0, presented in log scale. (c) Maxwell–Wagner relaxation frequency (f _r) over redox probe concentration (t = 2 h).

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Amide bond formation on gold electrodes. (a) SAM activation, with the surface modified to form pentafluorophenyl ester as a reactive intermediate. (b) Surface after chemisorption of IgG antibody.

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EIS measurements for concentrations between 5 and 500 mM of [Fe­(CN)₆]^3–/4– in 1× PBS (at 1 Hz). Measurements were performed using a flat microchannel with 0.3 mm in height. (a) Impedance values for various redox probe concentrations over 2 h on a bare gold surface, in semilog scale. (b) Impedance values for various redox probe concentrations on bare gold (squares) and activated SAM (triangles). (c) Charge transfer resistance as a function of the concentration of redox probe (mM), presented in log scale, for bare gold (squares) the activated SAM on surface (triangle). All error bars shown in (a) represent the standard deviation (±1 SD) based on at least 3 experimental replicas. Dashlines in (c) show a power regression fitting to the experimental data.

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EIS measurements for various IgG concentrations (1, 10, 20, 40, and 100 μg/mL) using 50 mM of [Fe­(CN)₆]^3–/4– in 1× PBS, presented in semilog scale. Measurements were performed using a flat microchannel, height of 0.3 mm. R _ct for IgG incubated in the presence of redox probe (triangle, red) and R _ct for IgG incubations in 1× PBS (star, black).

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Amide bond formation on gold electrodes and interaction between the charged peptides and the ions in the solution. (a) SAM activation, showing the surface is modified to form pentafluorophenyl ester as a reactive intermediate, interacting with the ions in the solution. (b) Surface showing competitive binding between proteins and ions on buffered solution.

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Fluorescence microscopy and FTIR analysis confirming IgG antibodies adhesion to gold electrodes surface. (a) Control for nonspecific binding of antirabbit IgG in the absence of capture IgG antibody. (b) Gold electrode’s surface functionalized with IgG antibody and probed with selective antirabbit IgG. (c) Fourier-transform infrared spectra of chemically activated gold surface, before (red) and after the immobilization of the IgG antibodies (black).