Reading School of Pharmacy, University of Reading, Whiteknights, Reading RG6 6AD, UK.
Capillary Film Technology Ltd, 2 Daux Road, Billingshurst, RH14 9SJ, UK.
Analyst. 2017 Mar 13;142(6):959-968. doi: 10.1039/c6an02622b.
This study reports for the first time the sensitive colorimetric and fluorescence detection of clinically relevant protein biomarkers by sandwich immunoassays using the covalent immobilisation of antibodies onto the fluoropolymer surface inside Teflon®-FEP microfluidic devices. Teflon®-FEP has outstanding optical transparency ideal for high-sensitivity colorimetric and fluorescence bioassays, however this thermoplastic is regarded as chemically inert and very hydrophobic. Covalent immobilisation can offer benefits over passive adsorption to plastic surfaces by allowing better control over antibody density, orientation and analyte binding capacity, and so we tested a range of different and novel covalent immobilisation strategies. We first functionalised the inner surface of a 10-bore, 200 μm internal diameter FEP microcapillary film with high-molecular weight polyvinyl alcohol (PVOH) without changing the outstanding optical transparency of the device delivered by the matched refractive index of FEP and water. Glutaraldehyde immobilisation was compared with the use of photoactivated linkers and NHS-ester crosslinkers for covalently immobilising capture antibodies onto PVOH. Three clinically relevant sandwich ELISAs were tested against the cytokine IL-1β, the myocardial infarct marker cardiac troponin I (cTnI), and the chronic heart failure marker brain natriuretic peptide (BNP). Overall, glutaraldehyde immobilisation was effective for BNP assays, but yielded unacceptable background for IL-1β and cTnI assays caused by direct binding of the biotinylated detection antibody to the modified PVOH surface. We found NHS-ester groups reacted with APTES-treated PVOH coated fluoropolymers. This facilitated a novel method for capture antibody immobilisation onto fluoropolymer devices using a bifunctional NHS-maleimide crosslinker. The density of covalently immobilised capture antibodies achieved using PVOH/APTES/NHS/maleimide approached levels seen with passive adsorption, and sensitive and quantitative assay performance was achieved using this method. Overall, the PVOH coating provided an excellent surface for controlled covalent antibody immobilisation onto Teflon®-FEP for performing high-sensitivity immunoassays.
这项研究首次报道了使用共价固定化抗体到聚四氟乙烯-氟聚合物（Teflon®-FEP）微流控设备内部氟聚合物表面，通过三明治免疫测定法对临床相关蛋白质生物标志物进行灵敏的比色和荧光检测。Teflon®-FEP 具有出色的光学透明度，非常适合高灵敏度比色和荧光生物测定，但这种热塑性塑料被认为是化学惰性的，非常疏水。与塑料表面的被动吸附相比，共价固定化可以通过更好地控制抗体密度、取向和分析物结合能力来提供优势，因此我们测试了一系列不同的新型共价固定化策略。我们首先用高分子量聚乙烯醇（PVOH）对 10 孔、200μm 内径 FEP 微毛细管膜的内表面进行功能化，而不会改变 FEP 和水之间匹配的折射率所提供的装置的出色光学透明度。戊二醛固定化与使用光活化连接子和 NHS-酯交联剂将捕获抗体共价固定到 PVOH 上进行了比较。针对细胞因子 IL-1β、心肌梗死标志物心肌肌钙蛋白 I（cTnI）和慢性心力衰竭标志物脑钠肽（BNP），我们测试了三种临床相关的三明治 ELISA。总体而言，戊二醛固定化对 BNP 测定有效，但由于生物素化检测抗体直接与改性的 PVOH 表面结合，导致 IL-1β 和 cTnI 测定产生不可接受的背景。我们发现 NHS-酯基团与 APTES 处理的 PVOH 涂层氟聚合物反应。这为使用双功能 NHS-马来酰亚胺交联剂将捕获抗体固定到氟聚合物装置上提供了一种新方法。使用 PVOH/APTES/NHS/马来酰亚胺实现的共价固定化捕获抗体的密度接近被动吸附的水平，并且使用这种方法实现了灵敏和定量的测定性能。总体而言，PVOH 涂层为 Teflon®-FEP 上控制共价抗体固定化提供了出色的表面，用于进行高灵敏度免疫测定。
