Battalapalli Dheerendranath, Chakraborty Purbali, Jain Disha, Obaro Stephen K, Gurkan Umut A, Bonomo Robert A, Draz Mohamed S
Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
Division of Pediatric Infectious Diseases, Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL 35233, USA.
Polymers (Basel). 2023 Aug 5;15(15):3316. doi: 10.3390/polym15153316.
Rapid and reliable point-of-care (POC) diagnostic tests can have a significant impact on global health. One of the most common approaches for developing POC systems is the use of target-specific biomolecules. However, the conjugation of biomolecules can result in decreased activity, which may compromise the analytical performance and accuracy of the developed systems. To overcome this challenge, we present a polymer-based cross-linking protocol for controlled and directed conjugation of biological molecules. Our protocol utilizes a bifunctional thiol-polyethylene glycol (PEG)-hydrazide polymer to enable site-directed conjugation of IgG antibodies to the surface of screen-printed metal electrodes. The metal surface of the electrodes is first modified with thiolated PEG molecules, leaving the hydrazide groups available to react with the aldehyde group in the Fc fragments of the oxidized IgG antibodies. Using anti- carbapenemase-2 (KPC-2) antibody as a model antibody used for antimicrobial resistance (AMR) testing, our results demonstrate a ~10-fold increase in antibody coupling compared with the standard -hydroxysuccinimide (NHS)-based conjugation chemistry and effective capture (>94%) of the target KPC-2 enzyme antigen on the surface of modified electrodes. This straightforward and easy-to-perform strategy of site-directed antibody conjugation can be engineered for coupling other protein- and non-protein-based biological molecules commonly used in POC testing and development, thus enhancing the potential for improved diagnostic accuracy and performance.
快速且可靠的即时检测（POC）诊断测试对全球健康会产生重大影响。开发POC系统最常用的方法之一是使用针对特定靶点的生物分子。然而，生物分子的偶联可能会导致活性降低，这可能会影响所开发系统的分析性能和准确性。为了克服这一挑战，我们提出了一种基于聚合物的交联方案，用于生物分子的可控和定向偶联。我们的方案利用双功能硫醇 - 聚乙二醇（PEG） - 酰肼聚合物，实现将IgG抗体定点偶联到丝网印刷金属电极表面。电极的金属表面首先用硫醇化的PEG分子进行修饰，使酰肼基团可与氧化的IgG抗体Fc片段中的醛基反应。以抗碳青霉烯酶 - 2（KPC - 2）抗体作为用于抗菌药物耐药性（AMR）测试的模型抗体，我们的结果表明，与基于标准N - 羟基琥珀酰亚胺（NHS）的偶联化学方法相比，抗体偶联增加了约10倍，并且在修饰电极表面有效捕获（>94%）了目标KPC - 2酶抗原。这种直接且易于操作的定点抗体偶联策略可设计用于偶联POC测试和开发中常用的其他基于蛋白质和非蛋白质的生物分子，从而提高诊断准确性和性能的潜力。
