Nanopore-Based Single-Entity Electrochemistry for the Label-Free Monitoring of Single-Molecule Glycoprotein–Boronate Affinity Interaction and Its Sensing Application

Abstract

Nanopipettes provide a promising confined space that enables advances in single-molecule analysis, and their unique conical tubular structure is also suitable for single-cell analysis. In this work, functionalized-nanopore-based single-entity electrochemistry (SEE) analysis tools were developed for the label-free monitoring of single-molecule glycoprotein−boronate affinity interaction for the first time, and immunoglobulin G (IgG, one of the important biomarkers for many diseases such as COVID-19 and cancers) was employed as the model glycoprotein. The principle of this method is based on a single glycoprotein molecule passing through 4-mercaptophenylboronic acid (4-MPBA)-modified nanopipettes under a bias voltage and in the meantime interacting with the boronate group from modified 4-MPBA. This translocation and affinity interaction process can generate distinguishable current blockade signals. Based on the statistical analysis of these signals, the equilibrium association constant (κ a ) of single-molecule glycoprotein−boronate affinity interaction was obtained. The results show that the κ a of IgG in the confined nanopore at the single-molecule level is much larger than that measured in the open system at the ensemble level, which is possibly due to the enhanced multivalent synergistic binding in the restricted space. Moreover, the functionalized-nanopore-based SEE analysis tools were further applied for the label-free detection of IgG, and the results indicate that our method has potential application value for the detection of glycoproteins in real samples, which also paves way for the single-cell analysis of glycoproteins.

A Nanopore‐Based Saccharide Sensor

“…However, due to extremely similar structures of the monosaccharide components, [18] the need for a nanopore which can fully discriminate between monosaccharides becomes urgent. Though sensing of monosaccharides, [19] polysaccharide [20] and glycoproteins [21] was previously attempted using chemically modified solid state nanopores. Full discrimination between individual monosaccharides using solid state nanopores however has never been reported.…”

A Nanopore‐Based Saccharide Sensor

“…However, due to extremely similar structures of the monosaccharide components, [18] the need for a nanopore which can fully discriminate between monosaccharides becomes urgent. Though sensing of monosaccharides, [19] polysaccharide [20] and glycoproteins [21] was previously attempted using chemically modified solid state nanopores. Full discrimination between individual monosaccharides using solid state nanopores however has never been reported.…”

Boronate-Affinity Cross-Linking-Based Ratiometric Electrochemical Detection of Glycoconjugates

“…Boronate-affinity cross-linking (BAC) has been intensively explored in the separation, enrichment, and sensing of saccharides and glycoconjugates. , For the BAC-based sensing of saccharides (e.g., glucose, fructose, and xylose) and glycoconjugates [e.g., glycated proteins, glycoproteins, , and lipopolysaccharides (LPSs)], the boronic acid (BA)-containing materials are typically used as the recognition elements as the BA group can react with cis -diols to form the cyclic boronate esters . To date, a wide variety of BAC-based fluorescent, , surface-enhanced Raman scattering (SERS), chemiluminescent (CL), photoelectrochemical (PEC), and electrochemical methods have been illustrated for the detection of glycoconjugates, , in view of their great value in disease screening and biomedical research. For example, glycoproteins [e.g., α-fetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 1 (MUC1), and prostate-specific antigen (PSA)] have been well characterized as serum biomarkers in the clinical screening of malignant tumors, while LPSs (also known as endotoxins, the most abundant antigens on the cell wall of most Gram-negative bacteria) are closely associated with the inflammatory reactions and many pathological processes .…”