Novel Recognition And Detection Approaches Of Glycoproteins Based On Molecular Imprinting Technology

Glycoproteins participate in many key biological processes such as molecular recognition,signal transduction,cell adhesion and immune response.The abnormal changes in structures and expression of glycoproteins are closely associated with the occurrence and development of many diseases and thus a lot of glycoproteins have been employed as disease biomarkers.Moreover,glycoprotein hormones,especially erythropoietin(EPO),have been abused by athletes as doping agents to improve performance in sports,which have been included in prohibited substances by the World Anti-doping Agency(WADA).Because of the limited concentration of glycoprotein biomarkers or hormones in biological samples as well as the coexistence of highabundance interfering species,establishment of specific and sensitive analytic approaches for trace glycoproteins in complex real-world samples is of important scientific significance and practical value.Molecular imprinting has been an efficient technology to create artificial receptors with high selectivity and affinity to proteins.Notably,our group has developed a series of methods to prepare high-quality molecularly imprinted polymers(MIPs)for glycoproteins based on boronate affinity,which have successfully found applications in various fields such as disease diagnosis and cell imaging.On the other hand,Raman spectrometry and mass spectrometry are robust tools for detection and identification of trace amounts of glycoproteins.To solve the existing problems in separation and analysis of glycoproteins,here we present fast,convenient and efficient recognition and detection approaches for glycoproteins by combining molecular imprinting technology with plasmon-enhaced Raman scattering(PERS)and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry(MALDI-TOF-MS)successively.Firstly,we developed an approach called plasmonic immunosandwich assay(PISA)for specific,sensitive and fast determination of trace glycoproteins in complex real-world samples.This approach enjoys the specific recognition ability of the boronate affinity-based MIPs and the high sensitivity of PERS,as well as the superiority of price and stability compared with normal immunoassays that rely on biomolecules like antibodies and enzymes.Taking EPO in human urine as a model glycoprotein,we fabricated EPO-imprinted arrays and high Raman active nanotags,to specifically capture trace EPO in samples and gently labeling,thus forming sandwichlike "immunocomplexes".Surface plasmon wave is generated on the gold thinlayer substrate of the MIP array upon exposure to a laser beam of the Raman spectrometer,which further enhances the SERS signal of silver-based nanotags and thereby enables the fast detection of down to 2.94 × 10-14 M EPO in human urine(S/N=4).We presented a new standard addition method for quantitative analysis via Raman spectrometry and achieved to meassure EPO concentration in human urine,which opens a new avenue for determination of EPO in clinical diagnosis and anti-doping analysis.Secondly,we undertook an initial attempt to on-line hyphenation enrichment based on MIP-modified target plate with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry(MALDI-TOF-MS).We fabricated a MIP-modified MALDI target plate via boronate affinity-based oriented surface imprinting,which revealed potential in on-line recognition and capture of glycoprotein in real-world samples,as well as rapid detection capability via mass spectrometry.