Gold nanoparticles-based electrochemical method for the detection of protein kinase with a peptide-like inhibitor as the bioreceptor

Abstract

This article presents a general method for the detection of protein kinase with a peptide-like kinase inhibitor as the bioreceptor, and it was done by converting gold nanoparticles (AuNPs)-based colorimetric assay into sensitive electrochemical analysis. In the colorimetric assay, the kinase-specific aptameric peptide triggered the aggregation of AuNPs in solution. However, the specific binding of peptide to the target protein (kinase) inhibited its ability to trigger the assembly of AuNPs. In the electrochemical analysis, peptides immobilized on a gold electrode and presented as solution triggered together the in situ formation of AuNPs-based network architecture on the electrode surface. Nevertheless, the formation of peptide-kinase complex on the electrode surface made the peptide-triggered AuNPs assembly difficult. Electrochemical impedance spectroscopy was used to measure the change in surface property in the binding events. When a ferrocene-labeled peptide (Fc-peptide) was used in this design, the network of AuNPs/Fc-peptide produced a good voltammetric signal. The competitive assay allowed for the detection of protein kinase A with a detection limit of 20 mU/mL. This work should be valuable for designing novel optical or electronic biosensors and likely lead to many detection applications.

Keywords: aptameric peptide; colorimetric assay; electrochemical biosensor; gold nanoparticle; protein kinase A; signal amplification.

Figure 1

(A) UV–Vis absorption spectra of AuNPs in various systems: curve a and tube 1, AuNPs; curve b and tube 2, AuNPs + IP₂₀; curve c and tube 3, AuNPs + IP₂₀/PKA. (B) TEM images of AuNPs in the presence of IP₂₀ (top) and IP₂₀/PKA (bottom). Notes: The final concentrations of AuNPs, IP₂₀, and PKA were 2.4 nM, 5 µM and 500 U/mL, respectively. Abbreviations: Abs, absorbance; AuNPs, gold nanoparticles; PKA, protein kinase A; TEM, transmission electron microscope.

Figure 2

Dependence of A₆₇₀/A₅₂₀ on the concentration of IP₂₀. Notes: The concentration of AuNPs was 2.4 nM. The error bars in the data points show the absolute standards. Abbreviation: AuNPs, gold nanoparticles.

Figure 3

(A) UV–Vis absorption spectra of 2.4 nM AuNPs in the presence of 2 µM IP₂₀ and various concentrations of PKA. The inset shows the corresponding photos. (B) Dependence of A₆₇₀/A₅₂₀ on PKA concentration. Note: The error bars in the data points show the absolute standards. Abbreviations: Abs, absorbance; AuNPs, gold nanoparticles; PKA, protein kinase A; UV–Vis, ultraviolet–visible.

Figure 4

SPR sensorgrams after injecting three concentrations of PKA into the IP₂₀-functionalized chips surface. Abbreviation: SPR, surface plasmon resonance.

Figure 5

(A) EIS Nyquist diagrams of IP₂₀-functionalized electrode after incubation with different solutions: curve a, PBS; curve b, peptide; curve c, AuNPs; curve d, AuNPs/peptide; curve e, PKA; curve f, PKA + AuNPs/peptide. (B) DPV of IP₂₀-functionalized electrode after incubation with various solutions (black curve, Fc-peptide; red curve, AuNPs; blue curve, AuNPs/Fc-peptide; green curve, PKA + AuNPs/Fc-peptide. Notes: The final concentrations of AuNPs, peptide, Fc-peptide, and PKA were 2.4 nM, 4 µM, 4 µM, and 5 U/mL, respectively. Abbreviations: AuNPs, gold nanoparticles; DPV, differential pulse voltammetry; EIS, electrochemical impedance spectroscopy; Fc, ferrocene; PBS, phosphate-buffered saline; PKA, protein kinase A.

Figure 6

Dependence of i_pa on the Fc-peptide/AuNPs ratio (A) and the final concentration of AuNPs (B). In panel A, the concentration of AuNPs was 2.4 nM. In panel B, the Fc-peptide/AuNPs ratio was kept at 104:1. Note: The error bars in the data points show the absolute standards. Abbreviations: AuNPs, gold nanoparticles; Fc, ferrocene; i_pa, peak current.

Figure 7

(A) DPV of IP₂₀-functionalized electrode after incubation with different concentrations of PKA, followed by incubation with the mixture of AuNPs/Fc-peptide. (B) Dependence of i_pa on PKA concentration. The inset shows the linear part of the fitting curve. Notes: The final concentrations of AuNPs and Fc-peptide were 1.2 nM and 125 µM, respectively. The error bars in the data points show the absolute standards. Abbreviations: AuNPs, gold nanoparticles; DPV, differential pulse voltammetry; Fc, ferrocene; i_pa, peak current; PKA, protein kinase A.

Figure 8

Selectivity of the proposed electrochemical method to PKA (1 U/mL), BSA (5 µM), lysozyme (5 U/mL), thrombin (1 µM), and Src (5 U/mL). Note: The final concentrations of AuNPs and Fc-peptide were 1.2 nM and 125 µM, respectively. Abbreviations: AuNPs, gold nanoparticles; BSA, bovine serum albumin; Fc, ferrocene; i_pa, peak current; PKA, protein kinase A.

Scheme 1

Illustration of the AuNPs-based colorimetric detection of PKA. Abbreviations: AuNPs, gold nanoparticles; PKA, protein kinase A.

Scheme 2

Illustration of the AuNPs-based electrochemical detection of PKA. Abbreviations: AuNPs, gold nanoparticles; PKA, protein kinase A.