Silicon Nanowire Field-Effect Transistor as Biosensing Platforms for Post-Translational Modification

Abstract

Protein tyrosine sulfation (PTS), a vital post-translational modification, facilitates protein-protein interactions and regulates many physiological and pathological responses. Monitoring PTS has been difficult owing to the instability of sulfated proteins and the lack of a suitable method for detecting the protein sulfate ester. In this study, we combined an in situ PTS system with a high-sensitivity polysilicon nanowire field-effect transistor (pSNWFET)-based sensor to directly monitor PTS formation. A peptide containing the tyrosine sulfation site of P-selectin glycoprotein ligand (PSGL)-1 was immobilized onto the surface of the pSNWFET by using 3-aminopropyltriethoxysilane and glutaraldehyde as linker molecules. A coupled enzyme sulfation system consisting of tyrosylprotein sulfotransferase and phenol sulfotransferase was used to catalyze PTS of the immobilized PSGL-1 peptide. Enzyme-catalyzed sulfation of the immobilized peptide was readily observed through the shift of the drain current-gate voltage curves of the pSNWFET before and after PTS. We expect that this approach can be developed as a next generation biochip for biomedical research and industries.

Keywords: polycrystalline silicon nanowire field-effect transistor (pSNWFET); post-translational modifications (PTMs); protein tyrosine sulfation (PTS); protein–protein interaction.

Scheme 1

Schematic diagram of the structure and function of the polysilicon nanowire field-effect transistor (pSNWFET) for biosensing application: the molecular interactions appear on the surface of the nanowire device, left, and their electronic responses, right.

Scheme 2

Schematic illustration of immobilization of P-selectin glycoprotein ligand (PSGL)-1 peptide on the NW surface, and in situ peptide sulfation.

Figure 1

SEM images of the poly-Si nanowire (NW) following surface modification. Surface modification of the pSNWFET device was observed through SEM following the addition of gold nanoparticle-conjugated IgG, as described in the Methods section. (A) Surface treated with 3-aminopropyltriethoxysilane (APTES), glutaraldehyde, and PSGL-1 peptide. Following protein tyrosine sulfation (PTS) on the tyrosine residue of PSGL-1 peptide with phenol sulfotransferase– tyrosylprotein sulfotransferase (PST–TPST) coupled enzyme assay, anti-sulfotyrosine antibody and gold nanoparticle-conjugated IgG were subsequently treated. (B) Surface treated with APTES, glutaraldehyde, PSGL-1 peptide, followed by coupled enzyme system treatment without TPST. Anti-sulfotyrosine antibody and gold nanoparticle-conjugated IgG were subsequently treated.

Figure 2

XPS analysis upon PSGL-1 immobilization on the poly-Si surface (A) Oxygen XPS spectra; (B) Carbon XPS spectra; and (C) Nitrogen XPS spectra at each immobilization step.

Figure 3

In situ determination of PTS by ELISA (A) PSGL-1 peptide was coated on the surface of the 96-well plate and subsequently blocked with milk. The immobilized PSGL-1 was treated with PST–TPST assay to produce protein sulfation. Control experiment was performed in the absence of the critical enzyme TPST, and the PTS reaction through PST–TPST enzyme without the substrate peptide, PSGL-1. Anti-sulfotyrosine antibody and horseradish peroxidase (HRP)-conjugated secondary antibody were used to determine PTS reaction on PSGL-1, as expected. The average value was obtained from three independent measurements. (B) ELISA absorbance value plotted vs. varying concentrations of antibody incubation buffer, bis-tris propane (BTP).

Figure 4

Electrical responses of the functionalized pSNWFET following each modification process. I_D-V_G curve obtained from the unmodified surface following the modification of the pSNWFET with APTES, glutaraldehyde, and PSGL-1 peptide. In set figure represent the changes of voltage at 10⁻⁸ A following each surface modification step.

Figure 5

Electrical responses of the functionalized pSNWFET to synthesized sulfated PSGL-1 (A) I_D-V_G curve obtained from the synthesized sulfated PSGL-1 peptide and interaction with anti-sulfotyrosine, and anti-glutathione-S-transferase antibodies (anti-GST) as the controls. The measurement was conducted at a fixed drain voltage (V_DS = 0.5 V), whereas the gate voltage sweeping rate was kept at 1–2 V to obtain the electrical property as the baseline (M1). The anti-GST antibody was used as a negative control (M2). Anti-sulfotyrosine antibody treatment (M3). The concentration of both antibodies was 250 ng/mL. (B) Average ΔV_G value of Figure 5A; the error bar indicates standard deviation. The average electrical responses were calculated from four devices for each condition, and Figure S3 illustrates other data of electrical response.

Figure 6

Electrical responses of the functionalized pSNWFET to PSGL-1 sulfation (A) Nonsulfated PSGL-1 peptide on the pSNWFET surface as the baseline (M1). I_D-V_G curve obtained through PSGL-1 sulfation (M2) and interaction with anti-sulfotyrosine (M4), and anti-GST antibodies as the controls (M3). (B) Average ΔV_G value of Figure 6A.

Figure 7

Electrical responses of functionalized pSNWFET to PSGL-1 sulfation without TPST (A) I_D-V_G curve obtained from nonsulfated PSGL-1 following coupled enzyme treatment without the critical enzyme TPST and the interaction with anti-sulfotyrosine, and anti-GST antibodies as the controls. (B) Average ΔV_G value of Figure 7A.