Oriented Antibody Covalent Immobilization for Label-Free Impedimetric Detection of C-Reactive Protein via Direct and Sandwich Immunoassays

Abstract

The detection and monitoring of biological markers as disease indicators in a simple manner is a subject of international interest. In this work, we report two simple and sensitive label-free impedimetric immunoassays for the detection of C-reactive protein (CRP). The gold electrode modified with boronic acid–terminated self-assembled monolayers afforded oriented immobilization of capture glycosylated antibody (antihuman CRP monoclonal antibody, mAb). This antibody-modified surface was able to capture human CRP protein, and the impedance signal showed linear dependence with CRP concentration. We confirmed the immobilization of anti-CRP mAb using surface sensitive X-ray photoelectron spectroscopy (XPS) and electrochemical impedance. The oriented covalent immobilization of mAb was achieved using glycosylated Fc (fragment, crystallizable) region specific to boronic acid. The direct immunoassay exhibited a linear curve for concentration range up to 100 ng ml−1. The limit of detection (LoD) of 2.9 ng ml−1, limit of quantification (LoQ) of 9.66 ng ml−1, and sensitivity of 0.585 kΩ ng−1 ml cm−2 were obtained. The sandwich immunoassay was carried out by capturing polyclonal anti-CRP antibody (pAb) onto the CRP antigen immunoreaction. The impedance signal after pAb capture also showed linear dependence with CRP antigen concentration and acted as a CRP antigen detection signal amplifier. The detection of the CRP antigen using sandwich pAb immunoassay improved LoD to 1.2 ng ml−1, LoQ to 3.97 ng ml−1, and enhanced the sensitivity to 0.885 kΩ ng−1 ml cm−2. The real sample analysis, using newborn calf serum, showed excellent selectivity and % recovery for the human CRP ranging from 91.2 to 96.5%. The method was reproducible to 4.5% for direct immunoassay and 2.3% for sandwich immunoassay.

One-Dimensional HKUST-1-Decorated Glassy Carbon Electrode for the Sensitive Electrochemical Immunosensor of NS1 Dengue Virus Serotype-3

“…Furthermore, the C 1s spectrum was observed in the region of 280–290 eV as seen in Figure c. Carbon sp 2 is represented by the peak at 284.2 and 284.8 eV in bare SPCE and modified SPCE, respectively. , The binding event between S40 and antibody is marked by the peak at binding energies of 286.07 and 288.27 eV, which are attributed to the carboxyl group of −COOH and the amide bond of N–CO. , The addition of BSA, which serves as a blocking protein to minimize nonspecific interaction, slightly reduced the peak, indicating that the sensing surface was covered mainly by the high-density immobilized antibody.…”

“…43,44 The binding event between S40 and antibody is marked by the peak at binding energies of 286.07 and 288.27 eV, which are attributed to the carboxyl group of −COOH and the amide bond of N−C�O. 45,46 The addition of BSA, which serves as a blocking protein to minimize nonspecific interaction, 47 slightly reduced the peak, indicating that the sensing surface was covered mainly by the high-density immobilized antibody. The addition of target NS1 protein notably reduces the peak further, showing a negligible faradaic current.…”

A Review on Bio- and Chemosensors for the Detection of Biogenic Amines in Food Safety Applications: The Status in 2022

“…Chemical binding of antibodies to sensor surfaces can induce an upward orientation of IgGs (binding sites available) together with better resilience against mechanical forces due to washing steps. This was demonstrated recently by Adesina and Mashazi, using glycosylated antibodies against CRP (complement reactive protein) and gold electrodes with a monolayer of boronic acid [ 237 ]. For further reading on random and oriented antibody immobilization strategies, comprehensive reviews are available [ 238 , 239 ].…”

Scalable biomimetic sensing system with membrane receptor dual-monolayer probe and graphene transistor arrays

“…Although both types of probes exhibit high sensitivity and specificity, antibodies require immunogenicity, are expensive to produce, and lack batch-to-batch consistency, while aptamers are sensitive to nuclease degradation and unstable in biofluids such as human serum. Their detection range can also fall out of clinical relevance ( 6 ). An alternative type of bioprobes without these issues is natural receptor proteins, which are cells’ input/output machineries and mostly reside on plasma membranes, including G protein–coupled receptors (GPCRs) that comprise the largest protein family in human proteome ( 7 ).…”