A Direct Immunoassay Based on Surface-Enhanced Spectroscopy Using AuNP/PS-b-P2VP Nanocomposites

Abstract

A biosensor was developed for directly detecting human immunoglobulin G (IgG) and adenosine triphosphate (ATP) based on stable and reproducible gold nanoparticles/polystyrene-b-poly(2-vinylpyridine) (AuNP/PS-b-P2VP) nanocomposites. The substrates were functionalized with carboxylic acid groups for the covalent binding of anti-IgG and anti-ATP and the detection of IgG and ATP (1 to 150 μg/mL). SEM images of the nanocomposite show 17 ± 2 nm AuNP clusters adsorbed over a continuous porous PS-b-P2VP thin film. UV-VIS and SERS were used to characterize each step of the substrate functionalization and the specific interaction between anti-IgG and the targeted IgG analyte. The UV-VIS results show a redshift of the LSPR band as the AuNP surface was functionalized and SERS measurements showed consistent changes in the spectral features. Principal component analysis (PCA) was used to discriminate between samples before and after the affinity tests. Moreover, the designed biosensor proved to be sensitive to different concentrations of IgG with a limit-of-detection (LOD) down to 1 μg/mL. Moreover, the selectivity to IgG was confirmed using standard solutions of IgM as a control. Finally, ATP direct immunoassay (LOD = 1 μg/mL) has demonstrated that this nanocomposite platform can be used to detect different types of biomolecules after proper functionalization.

Keywords: LSPR sensor; SERS sensing; direct immunoassay; gold nanoparticles; polystyrene-b-poly(2-vinylpyridine).

Figure 1

Comparison between the Raman spectra of the solid (a) PS, (b) P2VP, and (c) PS-b-P2VP powders and (d) AuNP/PS₁₂₅₀-b-P2VP₁₂₈₅ thin film (SERS spectrum in this case).

Figure 2

SEM images of the AuNP/PS₁₂₅₀-b-P2VP₁₂₈₅.

Figure 3

UV–VIS spectra of each step of the AuNPs/PS₁₂₅₀-b-P2VP₁₂₈₅ substrate functionalization: (a) AuNP/PS-b-P2VP; (b) AuNP/PS-b-P2VP functionalized with MUA/MPA; (c) AuNP/PS-b-P2VP/MUA/MPA further functionalized with EDC/NHS; (d) AuNP/PS-b-P2VP/MUA/MPA/EDC/NHS functionalized with anti-IgG; and (e) full sensor (indicated in (d)) exposed to 150 μg/mL IgG.

Figure 4

Average SERS spectra (n = 100 spectra) of each step of the AuNPs/PS₁₂₅₀-b-P2VP₁₂₈₅ substrate functionalization and detection of the interaction between anti-IgG/IgG: (a) AuNP/PS-b-P2VP; (b) AuNP/PS-b-P2VP functionalized with MUA/MPA; (c) AuNP/PS-b-P2VP/MUA/MPA further functionalized with EDC/NHS; (d) AuNP/PS-b-P2VP/MUA/MPA/EDC/NHS functionalized with anti-IgG; and (e) full sensor (indicated in (d)) exposed to 150 μg/mL IgG.

Figure 5

Binary scatterplot of the PC1 × PC2 scores of functionalization steps and IgG detection (150 μg/mL).

Figure 6

Binary scatterplot of the PC2 × PC3 scores for better discrimination between the sensor before (anti-IgG) and after exposure to 150 μg/mL of the IgG analyte.

Figure 7

Binary scatterplot of the PC1 × PC2 scores of the SERS spectra (n = 2500 spectra) of anti-IgG and IgG samples (concentration range: 1 to 90 μg/mL).

Figure 8

(A) Comparison between the mean SERS spectra of the sensor before (anti-IgG) and after exposure to 100 μg/mL of the IgG and IgM analytes; (B) binary scatterplot of the PC1 × PC2 scores of the SERS spectra of anti-IgG and IgG and IgM samples.

Figure 9

(A) Comparison between the mean SERS spectra (n = 1350 spectra) of anti-ATP and ATP samples (concentration range: 1 to 100 μg/mL); (B) binary scatterplot of the PC1 × PC3 scores of the SERS spectra of anti-ATP and ATP samples.