在线再生压电和阻抗免疫传感器，用于在定向抗体金表面检测 C 反应蛋白。

Online regeneration of a piezoelectric and impedimetric immunosensor for the detection of C-reactive protein on the oriented antibody gold surface.

机构信息

Department of Chemistry, Rhodes University, P. O. Box 94, Makhanda, 6140, South Africa.

Institute for Nanotechnology Innovation, Rhodes University, P. O. Box 94, Makhanda, 6140, South Africa.

出版信息

Anal Methods. 2023 Mar 2;15(9):1157-1167. doi: 10.1039/d2ay01245f.

DOI:10.1039/d2ay01245f

PMID:36787150

Abstract

Cardiovascular diseases (CVDs) refer to diseases that affect the heart and blood vessels. CVDs are considered silent killers, which are fatal (death occurs abruptly) particularly when their diagnosis is delayed. Among the biomarkers related to cardiovascular diseases, C-reactive protein (CRP) is expressed or found in high concentrations during a cardiac event. Therefore, CRP is an excellent biomarker to diagnose cardiac events, and therefore quantitative monitoring of CRP is necessary. Herein, we report the fabrication of an immunosensor for the detection and monitoring of CRP. The anti-CRP monoclonal antibody (anti-CRP-mAb), which is specific to the CRP antigen, was immobilized on the surface of gold pre-modified with 4-mercaptophenyl boronic acid (MPBA) to act as a capture antibody. The self-assembled monolayer (SAM) of 4-mercaptophenyl boronic acid (4-MPBA), Au-MPBA, was used for the oriented immobilization of anti-CRP-mAb for piezoelectric (mass-sensitive) and piezoelectric (impedance) measurements. Controlling the orientation of anti-CRP-mAb was crucial to eliminate false positive and negative results during sample analysis. The quartz-crystal microbalance with dissipation (QCM-D) measurements enabled us to follow the covalent immobilization of anti-CRP-mAb on AuCQC-MPBA in real-time. QCM-D was further used to follow the affinity reactions between anti-CRP-mAb and CRP and further with the anti-CRP polyclonal antibody (anti-CRP-pAb). The changes in frequency (Δ, Hz) were related to the changes in the mass (Δ, ng cm) of CRP up to a concentration of 0.10 μg mL, which is equivalent to 0.10 mg L. CRP was detected using the direct affinity immunoassay (anti-CRP-mAb < CRP) and also a sandwich immunoassay using anti-CRP-pAb for signal enhancement. The developed piezoelectric CRP detection protocol was translated to a gold disc electrode for electrochemical impedance spectroscopy (EIS) measurements. The limit of detection (LOD) using both methods was at the μg mL or mg L level. Furthermore, the developed electrode could be regenerated using acidic buffer (0.10 M HCl). The detected signal could be reproduced to within 5% relative standard deviation (% RSD) in buffer and serum samples, showing excellent selectivity and specificity toward CRP.

摘要

心血管疾病（CVDs）是指影响心脏和血管的疾病。心血管疾病被认为是沉默的杀手，当诊断延迟时尤其致命（突然死亡）。在与心血管疾病相关的生物标志物中，C 反应蛋白（CRP）在心脏事件发生时表达或浓度升高。因此，CRP 是诊断心脏事件的优秀生物标志物，因此需要对 CRP 进行定量监测。在这里，我们报告了一种用于检测和监测 CRP 的免疫传感器的制造。抗 CRP 单克隆抗体（抗 CRP-mAb），特异性针对 CRP 抗原，固定在经过 4-巯基苯硼酸（MPBA）预修饰的金表面上，用作捕获抗体。4-巯基苯硼酸（4-MPBA）的自组装单层（SAM），Au-MPBA，用于定向固定抗 CRP-mAb 进行压电（质量敏感）和压电（阻抗）测量。控制抗 CRP-mAb 的取向对于消除样品分析过程中的假阳性和假阴性结果至关重要。石英晶体微天平与耗散（QCM-D）测量使我们能够实时跟踪抗 CRP-mAb 在 AuCQC-MPBA 上的共价固定。QCM-D 进一步用于跟踪抗 CRP-mAb 与 CRP 之间的亲和反应，以及与抗 CRP 多克隆抗体（抗 CRP-pAb）之间的反应。频率的变化（Δ，Hz）与 CRP 质量的变化（Δ，ng cm）有关，直至浓度达到 0.10μg mL，相当于 0.10mg L。CRP 是通过直接亲和免疫测定（抗 CRP-mAb < CRP）和使用抗 CRP-pAb 进行信号增强的夹心免疫测定来检测的。开发的压电 CRP 检测方案已转化为金盘电极，用于电化学阻抗谱（EIS）测量。两种方法的检测限（LOD）均在μg mL 或 mg L 水平。此外，该开发的电极可以使用酸性缓冲液（0.10 M HCl）进行再生。在缓冲液和血清样品中，检测信号的重现性在 5%相对标准偏差（% RSD）以内，对 CRP 表现出优异的选择性和特异性。