State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University , Nanjing 210023, China.
ACS Appl Mater Interfaces. 2017 Apr 5;9(13):12082-12091. doi: 10.1021/acsami.7b00628. Epub 2017 Mar 21.
Assays of glycoproteins hold significant biological importance and clinical values, for which immunoassay has been the workhorse tool. As immunoassays are associated with disadvantages such as poor availability of high-specificity antibodies, limited stability of biological reagents, and tedious procedure, innovative alternatives that can overcome these drawbacks are highly desirable. Plasmonic immunosandwich assay (PISA) has emerged as an appealing alternative to immunoassay for fast and sensitive determination of trace glycoproteins in biosamples. Plasmonic substrates play key roles in PISA, not only in determining the specificity but also in greatly influencing the detection sensitivity. Herein, we report a new type of molecularly imprinted plasmonic substrates for rapid and ultrasensitive PISA assay of trace glycoproteins in complex real samples. The substrates were fabricated from glass slides, first coated with self-assembled monolayer (SAM) of gold nanoparticles (AuNPs) and then molecularly imprinted with organo-siloxane polymer in the presence of template glycoproteins. The prepared molecularly imprinted substrates exhibited not only a significant plasmonic effect but also excellent binding properties, ensuring the sensitivity as well as the specificity of the assay. Alkaline phosphatase (ALP) and α-fetoprotein (AFP), glycoproteins that are routinely used as disease markers in clinical diagnosis, were used as representative targets. The limit of detection (LOD) was 3.1 × 10 M for ALP and 1.5 × 10 M for AFP, which is the best among the PISA approaches reported. The sample volume required was only 5 μL, and the total time required was within 30 min for each assay. Specific and ultrasensitive determination of ALP and AFP in human serum was demonstrated. Because many disease biomarkers are glycoproteins, the developed PISA approach holds great promise in disease diagnostics.
糖蛋白分析具有重要的生物学意义和临床价值，免疫测定一直是其主要工具。然而，免疫测定存在一些缺点，如高特异性抗体的可用性有限、生物试剂的稳定性有限以及操作繁琐等，因此非常需要能够克服这些缺点的创新替代方法。等离子体免疫夹心测定（PISA）作为一种替代免疫测定的方法，因其能够快速、灵敏地测定生物样品中的痕量糖蛋白而备受关注。等离子体基底在 PISA 中起着关键作用，不仅决定了测定的特异性，而且极大地影响了检测的灵敏度。在此，我们报道了一种新型的分子印迹等离子体基底，用于快速、灵敏地测定复杂实际样品中的痕量糖蛋白。该基底由载玻片制备而成，首先通过自组装单层（SAM）涂覆金纳米粒子（AuNPs），然后在模板糖蛋白存在的情况下，通过有机硅氧烷聚合物进行分子印迹。制备的分子印迹基底不仅表现出显著的等离子体效应，而且具有优异的结合性能，确保了测定的灵敏度和特异性。碱性磷酸酶（ALP）和甲胎蛋白（AFP）作为临床诊断中常用的疾病标志物，被用作代表性靶标。ALP 的检测限（LOD）为 3.1×10^-9 M，AFP 的检测限为 1.5×10^-9 M，这是报道的 PISA 方法中最好的。每个测定所需的样品体积仅为 5 μL，总时间在 30 min 内。在人血清中对 ALP 和 AFP 进行了特异性和超灵敏的测定。由于许多疾病标志物是糖蛋白，因此开发的 PISA 方法在疾病诊断中具有广阔的应用前景。
