Regioselective S_N2-Type Reaction for the Oriented and Irreversible Immobilization of Antibodies to a Glass Surface Assisted by Boronate Formation

Abstract

Antibodies have exquisite specificities for molecular recognition, which have led to their incorporation into array sensors that are crucial for research, diagnostic, and therapeutic applications. Many of these platforms rely heavily on surface-bound reactive groups to covalently tether antibodies to solid substrates; however, this strategy is hindered by a lack of orientation control over antibody immobilization. Here, we report a mild electrophilic phenylsulfonate (tosylate) ester-containing boronic acid affinity ligand for attaching antibodies to glass slides. A high level of antibody coupling located near the Fc region of the boronated antibody complex could be achieved by the proximal nucleophilic amino acid driven substitution reaction at the phenylsulfonate center. This enabled the full-length antibodies to be permanently tethered onto surfaces in an oriented manner. The advantages of this strategy were demonstrated through the individual and multiplex detection of protein and serum biomarkers. This strategy not only confers stability to the immobilized antibodies but also presents a different direction for the irreversible attachment of antibodies to solid supports in an orientation-controlled way.

Surface Modification of Polystyrene with Boronic Acid for Immunoaffinity-Based Cell Enrichment

“…An appreciable enhancement (54%, Figure ) of nitrogen concentration in PS-APBA-Ab versus PS-APBA indicates the presence of nitrogen from IgG, as IgGs are proteins and are enriched with amino (NH 2 ) groups. In addition, the ratio of nitrogen to carbon concentration ([N]/[C]) is increased 74% (from 0.12 to 0.21%) in PS-APBA-Ab surface (Figure ) which supports the presence of protein on the surface after antibody immobilization. , Another indicator of Ab existence on the surface is sulfur which is expected due to the disulfide bridge of Ab structure. , The concentration of sulfur at 0.3% is slightly less than the approximate sulfur content in an antibody (i.e., 0.45%) and just above the detection limit of XPS (about 0.1%) . This suggests a high-density layer of Abs.…”

“…In addition, the ratio of nitrogen to carbon concentration ([N]/[C]) is increased 74% (from 0.12 to 0.21%) in PS-APBA-Ab surface (Figure 7) which supports the presence of protein on the surface after antibody immobilization. 67,68 Another indicator of Ab existence on the surface is sulfur which is expected due to the disulfide bridge of Ab structure. 64,65 The concentration of sulfur at 0.3% is slightly less than the approximate sulfur content in an antibody (i.e., 0.45%) and just above the detection limit of XPS (about 0.1%).…”

“…In XPS characterization, only the top 10 nm of the surface is exposed to the X-ray. 68,70 So, the X-ray will mostly scan the added Ab layer in the PS-APBA-Ab surface, provided the Abs are nearly confluent. The sublayers that are APBA, GPTES, and Dextran have a lower chance to be exposed with X-ray and Additionally, the ratio of N: B elements in a single molecule of APBA is 1:1.…”

Silver-Coated Cu2O Nanoparticle Substrates for Surface Azide–Alkyne Cycloaddition

“…Subsequently, the tosyl group was displaced by a nucleophile on the antibody through an S N 2 substitution. After that, antibodies can be successfully immobilized onto the slide in a covalent orientation . However, due to the cross-reactivity between boronic acid and copper ions, the functionalization of the azido-BA-tosyl probe on the slides emerges as an additional problem to be solved.…”

“…After that, antibodies can be successfully immobilized onto the slide in a covalent orientation. 41 However, due to the crossreactivity between boronic acid and copper ions, 15 the functionalization of the azido-BA-tosyl probe on the slides emerges as an additional problem to be solved. By contrast, alkynylated Ag-coated Cu 2 O NP slides with SAAC activity make the surface CuAAC feasible for the immobilization of In a proof of concept study, RCA 120 , a useful lectin surrogate for the bioterrorism agent ricin toxin, was chosen as a model protein target.…”