Engineering peptide linkers for scFv immunosensors

Abstract

Using A10B single-chain fragment variable (scFv) as a model system, we demonstrated that the flexibility of scFv linker engineering can be combined with the inherent quick and adaptable characters of surface coupling chemistry (e.g., electrostatic, hydrogen bonding, or covalent attachment) to attach scFv to preformed functionalized self-assembled monolayers (SAMs). Six arginines, which were separated by glycine or serine as spacer, were incorporated in the peptide linker to form a 15-mer peptide linker (RGRGRGRGRSRGGGS). The polycationic arginine peptide was engineered into the A10B scFv-RG3 to favor its adsorption at anionic charged template surface (11-mercaptoundecanoic acid (MUA) and poly(sodium 4-styrenesulfonate (PSS))). This new approach was compared with the other engineered scFv constructs. Our results demonstrated that the anionic charged SAM template facilitated the oriented immobilization of scFvs on the SAM template surface as well as reduced the possibility of protein denaturation when directly immobilized on the solid surface. A 42-fold improvement of detection limits using MUA/A10B scFv-RG3 (less than 0.2 nM experimentally determined) was achieved compared to A10B Fab antibody and a 5-fold improvement was observed compared to A10B scFv that was engineered with a cysteine in the linker sequence. Using protein A-coated gold nanoparticles, a picomolar experimental detection limit was achieved. With 20 amino acids to choose from, engineered recombinant scFv in combination with SAM technology and nanoparticle mass amplification provide an emerging strategy for the development of highly sensitive and specific scFv immunosensors.

Figure 1. ELISA results for different scFvs on rabbit IgG, human IgG, rat IgG, goat IgG, bovine IgG, and BSA

(A) scFv-RG3, (B) scFv-Cys, (C) scFv-His, (D) scFv-ZnS4, (E) scFv-CDS6, (F) scFv-RS, and negative controls: (G) I-20 scFv-RS specific for P450 CYP1B1, (H) D-11 scFv-Cys specific for isoketal protein adduct. Insertion depicts ELISA results for varying concentrations of rabbit IgG (1.25 to 0.019 µg/mL) binding with scFv-RG3.

Figure 2

(a) CVs of 1 mM K₄Fe(CN)₆/K₃Fe(CN)₆ in 0.1 M NaClO₄ on bare gold electrode (black), MUA (red), MUA/scFv-RG3 (green), and MUA/scFv-RG3 binding with rabbit IgG (blue) modified electrode. Scan rate, 100 mV/s. (b) EIS Nyquist plots. Frequency range is 0.1 Hz–100 kHz. Bias potential equals to open circuit potential. ac amplitude is 10 mV.

Figure 3. Frequency change vs time curves

When (A) MUA/scFv-RG3, (B) PSS/scFv-RG3, (C) 1-dodecanethiol /scFv-RG3, and (D) cysteamine/scFv-RG3 QCM sensors were exposed to the 132 nM rabbit IgG.

Figure 4

(a) MUA/scFv-RG3 modified Au QCM electrodes exposed to various concentrations of rabbit IgG (0.22–198 nM). (b) Calibration curve, frequency change vs rabbit IgG concentration.

Figure 5

Negative controls (132 nM mouse IgG, 66 nM PTEN mouse IgG1, 10 µg/mL HSA, 20 µg/mL yeast, 66 nM anti-human IgG, 122 nM goat anti rabbit IgG Fab) and 22 µg/mL FBS.

Figure 6

Plot of [rabbit-IgG]₀/ΔM vs [rabbit-IgG]₀ for MUA/scFv-RG3-modified QCM sensor.

Figure 7. Comparison of various engineered scFv-based QCM sensors’ sensitivity

All above modified scFv QCM sensors were exposed to 132 nM rabbit IgG.

Figure 8. Comparison of sensor selectivity and sensitivity for MUA/scFv-RG3 (black), scFv-Cys (red), scFv-His (green), scFv-ZnS4 (megenta), and scFv-CdS6 (blue)

For each sensor, 20 µL of FBS was added three times to 1 mL of PBS buffer. This was followed by the addition of 20 µL of rabbit IgG to 1 mL of PBS buffer evaluating antigen detection in a complex matrix. The final concentration of FBS is 22 µg/mL; rabbit IgG is 20 µg/mL (132 nM).

Figure 9. Detection of rabbit IgG by MUA/scFv-RG3-modified Au QCM

Binding signal amplified using protein A-coated Au nanoparticle. Insertion (zero dose blank): the addition of gold nanoparticle (ANP) solution and protein A-coated gold nanoparticle (ProA-ANP) solution.

Chart 1. Schematic Illustrationa

^a Amino acid abbreviation: A, alanine; C, cysteine; F, phenylalanine; G, glycine; H, histidines, I, isoleucine; L, leucine; N, asparagine; P, proline; R, arginine; S, serine; V, valine; and W, tryptophan)

Chart 2. Schematic Illustration of Direct Detection of Rabbit IgG (I) and Gold Nanoparticle Amplification (II)