Direct detection of protein biomarkers in human fluids using site-specific antibody immobilization strategies

Abstract

Design of an optimal surface biofunctionalization still remains an important challenge for the application of biosensors in clinical practice and therapeutic follow-up. Optical biosensors offer real-time monitoring and highly sensitive label-free analysis, along with great potential to be transferred to portable devices. When applied in direct immunoassays, their analytical features depend strongly on the antibody immobilization strategy. A strategy for correct immobilization of antibodies based on the use of ProLinker™ has been evaluated and optimized in terms of sensitivity, selectivity, stability and reproducibility. Special effort has been focused on avoiding antibody manipulation, preventing nonspecific adsorption and obtaining a robust biosurface with regeneration capabilities. ProLinker™-based approach has demonstrated to fulfill those crucial requirements and, in combination with PEG-derivative compounds, has shown encouraging results for direct detection in biological fluids, such as pure urine or diluted serum. Furthermore, we have implemented the ProLinker™ strategy to a novel nanoplasmonic-based biosensor resulting in promising advantages for its application in clinical and biomedical diagnosis.

Figure 1.

ProLinker™ strategy (a) Immobilization protocol: (i) surface coating with ProLinker™ B; (ii) antibody immobilization and blocking step with bovine serum albumin (BSA); and (iii) specific antigen detection; (b) Proposed mechanism for antibody capturing by ProLinker™ molecule. Main contribution to coupling is attributed to the host-guest interaction between ionized amino groups from the protein and the crown-ether moiety. Hydrophobic interactions between methoxy group of the linker and hydrophobic residues of the protein are also involved. End-on orientation is induced by dipole-dipole interactions. Figure adapted from [14,17].

Figure 2.

(a) Comparison of antibody immobilizations at different antibody concentrations (5, 10, 20, 50, 100 μg/mL) with different strategies. Grey: covalent strategy; Green: Protein G strategy ([Protein G] = 50 μg/mL); Purple: ProLinker™ strategy; (b) Antigen detection performed with hCG/anti-hCG for covalent strategy (black), Protein G strategy (green) and ProLinker™ strategy (purple). Concentration of anti-hCG antibody was 10 μg/mL in all cases. Dashed lines represent adsorption of nonspecific proteins onto antibody functionalized surfaces for covalent strategy (black), Protein G strategy (green) and ProLinker™ strategy (purple). Blue dotted line indicates additional control for the ProLinker™ strategy, based on the detection of hCG onto a nonspecific antibody (10 μg/mL) immobilized over ProLinker™ layer (same experimental conditions as with specific antibody).

Figure 3.

Calibration curves for (a) FAK protein and (b) CRP protein, performed with 10 μg/mL of specific antibody and following both Protein G strategy (green) and ProLinker™ strategy (purple). Dashed red lines represent linear fit of the linear region of the curves.

Figure 4.

(a) Sensogram of biosensing measurement with ProLinker™-coated surface: antibody immobilization (anti-CRP 10 μg/mL), blocking step (BSA 1 mg/mL), antigen detection (CRP 1 μg/mL) and regeneration (HCl 5 mM); (b) Detection cycles performed by consecutive interaction of specific target at 1 μg/mL and regeneration with HCl 5 mM using PBS in flow; (c) Detection cycles performed by consecutive interaction of specific target at 1 μg/mL and regeneration with HCl 5 mM using PBST in flow.

Figure 5.

(a) SPR sensograms for pure urine spiked with different CRP concentrations (b) Calibration curves for CRP detection using ProLinker™ strategy with 10 μg/mL of specific antibody and performed in: PBS (black), PBST 0.5% (purple) and undiluted urine (orange); (c) Serum nonspecific adsorption onto sensor surface blocked with different agents (BSA, amine-dextran, diamine-PEG and PLL-PEG) diluted 1:10 with different buffers (PBS, PBS + 1%BSA, SuperBlock^®, PBST 0.5% and HBB buffer).

Figure 6.

(a) Immobilization protocol for gold nanodisks substrates: (i) ProLinker™ layer formation; (ii) Antibody immobilization and blocking step with PLL-PEG; and (iii) Specific antigen detection; (b) Scanning electron microscopy (SEM) images of gold nanodisks fabricated by hole-mask colloidal lithography (c) CRP detection curves performed with the nanoplasmonic biosensor at different concentrations of antibody immobilized (10, 20, 50 μg/mL) with ProLinker™ strategy; (d) Calibration curves for CRP detection performed on gold film (orange) and gold nanodisks nanoplasmonic (blue). Antibody concentration was 20 μg/mL and PLL-PEG was employed as blocking agent for both substrates. Dashed lines represent linear fit of linear region; (e) Nonspecific adsorption study of serum at different concentrations (10%, 25%, 50%, 100%) using different buffers in flow (PBST 0.5% and HBB) performed for both sensor substrates: SPR gold film (orange) and LSPR gold nanodisks (blue).

Figure 6.

(a) Immobilization protocol for gold nanodisks substrates: (i) ProLinker™ layer formation; (ii) Antibody immobilization and blocking step with PLL-PEG; and (iii) Specific antigen detection; (b) Scanning electron microscopy (SEM) images of gold nanodisks fabricated by hole-mask colloidal lithography (c) CRP detection curves performed with the nanoplasmonic biosensor at different concentrations of antibody immobilized (10, 20, 50 μg/mL) with ProLinker™ strategy; (d) Calibration curves for CRP detection performed on gold film (orange) and gold nanodisks nanoplasmonic (blue). Antibody concentration was 20 μg/mL and PLL-PEG was employed as blocking agent for both substrates. Dashed lines represent linear fit of linear region; (e) Nonspecific adsorption study of serum at different concentrations (10%, 25%, 50%, 100%) using different buffers in flow (PBST 0.5% and HBB) performed for both sensor substrates: SPR gold film (orange) and LSPR gold nanodisks (blue).