Dadfar Seyed Mohammad Mahdi, Sekula-Neuner Sylwia, Trouillet Vanessa, Liu Hui-Yu, Kumar Ravi, Powell Annie K, Hirtz Michael
Institute of Nanotechnology (INT), Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein Leopoldshafen, Germany.
Karlsruhe Nano Micro Facility (KNMF), Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein Leopoldshafen, Germany.
Beilstein J Nanotechnol. 2019 Dec 16;10:2505-2515. doi: 10.3762/bjnano.10.241. eCollection 2019.
The level of cancer biomarkers in cells, tissues or body fluids can be used for the prediction of the presence of cancer or can even indicate the stage of the disease. Alpha-fetoprotein (AFP) is the most commonly used biomarker for early screening and diagnosis of hepatocellular carcinoma (HCC). Here, a combination of three techniques (click chemistry, the biotin-streptavidin-biotin sandwich strategy and the use of antigen-antibody interactions) were combined to implement a sensitive fluorescent immunosensor for AFP detection. Three types of functionalized glasses (dibenzocyclooctyne- (DBCO-), thiol- and epoxy-terminated surfaces) were biotinylated by employing the respective adequate click chemistry counterparts (biotin-thiol or biotin-azide for the first class, biotin-maleimide or biotin-DBCO for the second class and biotin-amine or biotin-thiol for the third class). The anti-AFP antibody was immobilized on the surfaces via a biotin-streptavidin-biotin sandwich technique. To evaluate the sensing performance of the differently prepared surfaces, fluorescently labeled AFP was spotted onto them via microchannel cantilever spotting (µCS). Based on the fluorescence measurements, the optimal microarray design was found and its sensitivity was determined.
细胞、组织或体液中癌症生物标志物的水平可用于预测癌症的存在，甚至可以指示疾病的阶段。甲胎蛋白（AFP）是用于肝细胞癌（HCC）早期筛查和诊断的最常用生物标志物。在此，将三种技术（点击化学、生物素-链霉亲和素-生物素夹心策略以及抗原-抗体相互作用的应用）结合起来，实现了一种用于检测AFP的灵敏荧光免疫传感器。通过采用各自合适的点击化学对应物（第一类用生物素-硫醇或生物素-叠氮化物，第二类用生物素-马来酰亚胺或生物素-二苯并环辛炔，第三类用生物素-胺或生物素-硫醇）对三种类型的功能化玻璃（二苯并环辛炔（DBCO）-、硫醇-和环氧基封端的表面）进行生物素化。通过生物素-链霉亲和素-生物素夹心技术将抗AFP抗体固定在这些表面上。为了评估不同制备表面的传感性能，通过微通道悬臂点样（µCS）将荧光标记的AFP点样到这些表面上。基于荧光测量，找到了最佳的微阵列设计并确定了其灵敏度。
