Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA.
Analyst. 2019 Sep 7;144(17):5108-5116. doi: 10.1039/c9an01093a. Epub 2019 Aug 2.
We report here the influence of antibody immobilization strategy for protein immunosensors on screen printed carbon electrode arrays in terms of antibody binding activity, analytical sensitivity, limit of detection, and stability. Horseradish peroxidase (HRP) was the model analyte with anti-HRP immobilized on the sensors, and HRP activity was used for detection. Covalently immobilized anti-HRP antibodies on electrodes coated with chitosan, electrochemically reduced graphene oxide (rGO), and dense gold nanoparticle (AuNP) films had only 20-30% of the total immobilized antibodies active for binding. Active antibodies increased to 60% with passively adsorbed antibodies on bare electrodes, to 85% with oriented antibodies using protein A covalently immobilized on AuNP-coated carbon electrode, and to 98% when attached to protein A passively adsorbed onto bare electrodes. Passively adsorbed antibodies on bare electrodes lost activity in 1-2 days, but antibodies immobilized using other strategies remained relatively stable after 5 days. Covalent immobilization gave limits of detection (LOD) of 40 fg mL, while passively adsorbed antibodies or protein A on carbon electrodes had LODs 4-8 fg mL, but were unstable. Sensitivity was highest for antibodies covalently attached to AuNP electrodes (2.40 nA per log pg per mL) that also had highest antibody coverage, and decreased slightly when protein A on AuNP was used to orient antibodies. Passively adsorbed antibodies and oriented antibodies on protein A gave slightly lower sensitivities. Immobilization strategy or antibody orientation did not have a significant effect on LOD, but dynamic range increased as the number of active antibodies on sensor surfaces increased.
我们在此报告了抗体固定策略对基于丝网印刷碳电极阵列的蛋白质免疫传感器的影响，主要涉及抗体结合活性、分析灵敏度、检测限和稳定性。辣根过氧化物酶（HRP）作为模型分析物，将抗-HRP 固定在传感器上，并用 HRP 活性进行检测。壳聚糖、电化学还原氧化石墨烯（rGO）和致密金纳米粒子（AuNP）膜上共价固定的抗-HRP 抗体只有 20-30%的总固定抗体具有结合活性。通过在裸电极上被动吸附抗体，活性抗体增加到 60%，通过在 AuNP 涂层碳电极上共价固定的蛋白 A 定向抗体，活性抗体增加到 85%，当抗体附着在通过蛋白 A 被动吸附到裸电极上时，活性抗体增加到 98%。在 1-2 天内，裸电极上被动吸附的抗体失去活性，但使用其他策略固定的抗体在 5 天后仍保持相对稳定。共价固定的抗体检测限（LOD）为 40 fg mL，而通过在碳电极上被动吸附抗体或蛋白 A 的 LOD 为 4-8 fg mL，但不稳定。对 AuNP 电极共价连接的抗体的灵敏度最高（每对数 pg 每毫升 2.40 nA），且抗体覆盖率最高，当 AuNP 上的蛋白 A 用于定向抗体时，灵敏度略有下降。通过蛋白 A 被动吸附的抗体和定向抗体的灵敏度略低。固定策略或抗体定向对 LOD 没有显著影响，但随着传感器表面上的活性抗体数量增加，动态范围增加。
