Boronate Affinity Sandwich Assay Based On Molecularly Imprinted Polymers And Surface-enhanced Raman Scattering

Glycoproteins play vital roles in many biological processes, such as molecular recognition and immune response. As the abnormal expression of glycoproteins is associated with the occurrence of diverse diseases, a large variety of glycoproteins have been employed as disease biomarkers for clinical diagnosis. Because of the limited concentration of glycoprotein biomarkers in biological samples as well as the presence of high-abundance interfering species in sample matrix, detections of glycoproteins in real-world sample require high specifically affinity way and high sensitivity detection tool.Immunoassay has been an essential analytical tool for the analysis of proteins in many areas such as clinical diagnostics. However, antibodies with high specificity toward their targets are difficult to prepare and thereby costly. Antibodies also suffer from poor storage stability and biological activity lose upon external treatment. To detect trace target molecular, immunoassays employ a variety of high sensitivity detection schemes and corresponding labels that are usually chemically conjugated to desired antibody or antigen. Widely used labels include enzymes, radioactive isotopes, DNA reporters, fluorescent and chemiluminescent probes. However, these reagents are associated with obvious drawbacks. Enzymes suffer from limited stability, radioactive isotopes are harmful to health, while fluorescent and chemiluminescent probes are susceptible to sample environment.To overcome the disadvantage of immunoassay, we present a boronate affinity sandwich assay for the specific and sensitive determination of trace glycoproteins in complex samples based on surface-enhanced Raman scattering. Firstly, we prepare boronate affinity-functionalized SERS probe to label cis-diol through boronate affinity. Secondly, we use boronate affinity molecularly imprinted polymers (MIP) array as SERS substrate to capture target. Finally, boronate affinity sandwich assay is established by the combination of a boronate affinity MIP with boronate affinity-functionalized SERS probes. A target glycoprotein is first specifically captured by a boronate affinity MIP array from a sample under test. After unwanted species are washed away, the captured glycoprotein is labeled with boronate affinity SERS probe. The formed MIP-target-probe sandwich is subjected to SERS detection. The procedure is straightforward, taking only 30 min totally. The feasibility for real-world applications is demonstrated with the assay of trace a-fetoprotein (AFP) in human serum.The boronate affinity sandwich assay based on molecularly imprinted polymers and surface-enhanced Raman scattering overcomes the drawbacks of conventional immunoassays and has many advantages including ultrahigh sensitivity, less susceptible to sample environment, rapid readout speed and possibility for on-site or field detection.