Molecularly Imprinted Plasmonic Substrates for Specific and Ultrasensitive Immunoassay of Trace Glycoproteins in Biological Samples.

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 × 10-12 M for ALP and 1.5 × 10-14 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.

Molecularly Imprinted Polymer-Based Plasmonic Immunosandwich Assay for Fast and Ultrasensitive Determination of Trace Glycoproteins in Complex Samples.

Glycoproteins play significant roles in many biological processes. Assays of glycoproteins have significant biological importance and clinical values, for which immunoassay has been the workhorse tool. However, immunoassay suffers from some disadvantages, such as poor availability of high-specificity antibodies and limited stability of biological reagents. Herein, we present an antibody-free and enzyme-free approach, called molecularly imprinted polymer (MIP)-based plasmonic immunosandwich assay (PISA), for fast and ultrasensitive detection of trace glycoproteins in complex samples. A gold-based boronate affinity MIP array was used to specifically extract the target glycoprotein from complex samples. After washing away unwanted species, the captured glycoprotein was labeled with boronate affinity silver-based Raman nanotags. Thus, sandwich-like complexes were formed on the array. Upon being shined with a laser beam, the gold-based array generated a surface plasmon wave, which significantly enhanced the surface-enhanced Raman scattering (SERS) signal of the silver-based Raman nanotags. The MIP ensured the specificity of the assay, while the plasmonic detection provided ultrahigh sensitivity. Erythropoietin (EPO), a glycoprotein hormone that controls erythropoiesis or red blood cell production, was employed as a test glycoprotein in this study. Specific detection of EPO in solution down to 2.9 × 10-14 M was achieved. Using a novel strategy to accommodate the method of standard addition to a logarithmic dose-response relationship, EPO in human urine was quantitatively determined by this approach. The analysis time required only 30 min in total. This approach holds promising application prospects in many areas, such as biochemical research, clinical diagnosis, and antidoping analysis.