Raman-based Sensing Method For Glycoprotein Detection And Exploration Of The Interaction Between Nanomedicine And Digestive Protease

Glycoprotein is crucial to human body.It plays key roles in many physiological processes.The abnormal expression of glycoprotein is often related to the develop-ment of diseases.Therefore,many kinds of glycoproteins are used as disease bi-omarkers for clinical diagnosis.For example,a-fetoprotein(AFP)is an oncogenic glycoprotein and an established biomarker for early diagnosis of the most common forms of primary liver cancer.The development of a sensitive and convenient method for glycoprotein detection is important to reveal the relationship between cancer and glycoprotein expression.Currently,immunoassay is mainly used to detect glycopro-tein,however,the preparation of antibodies is complicated and time-consuming,and the storage stability of antibodies is poor.Therefore,it is important to develop a sensi-tive antibody-free assay for the detection of glycoproteins.Fullerenol is considered to be a promising anti-cancer nanodrug because of its strong antioxidant activity and ability to scavenge free radicals.However,the effec-tive chemotherapy of fullerenol for cancer treatment was mainly tested via the injec-tion route through which high risk could be brought by nanodrugs.Oral administra-tion is more acceptable to most patients and has been considered to be the most popu-lar route for clinical treatments.Nevertheless,challenges still exist for oral delivery,especially for the case that the proteolytic enzymes presented throughout the gastro-intestinal tract tends to interact with the drugs and consequently decreases their effi-cacy.Therefore,investigating the interaction between fullerenol and proteolytic en-zymes in gastrointestinal tract is extremely crucial to evaluate the drug activity during oral delivery.SERS-based frequency-shift sensing method possesses the ability of monitoring the dynamic process between probes and targets with high sensitivity,and thus a desirable tool to investigating the interaction between fullerenol and proteins.The results are as follows:1.Preparation of glycoprotein detection sensor.We chose 4-MPBA as probe molecule for glycoprotein detection sensor due to its large cross section and excellent carbohydrate affinity.The reversible change for the peak of 4-MPBA when 4-MPBA/AgNP substrate recognizing the target allows us to simultaneously capture and detect glycoproteins in a single-step reaction,and our system offers sensitive detection(limit of detection(LOD)of 10-12 M glycoprotein).Finally we explored the selectivity of the sensor,and found it possessed high selectivity of glycoproteins over nonglycoproteins in PBS buffer solution.2.The interaction between fullerenol and pepsin/trypsin was demonstrated by using Raman-based frequency shift sensing method.The specific binding domain of fullerenol to pepsin and trypsin respectively was evidenced by introducing their cor-responding inhibitor for the block of active domains on enzymes.Different from the case of pepsin,fullerenol tended to preferentially adsorb in the active domain of tryp-sin.In addition,the specific binding configurations and modes of fullerenol to pepsin and trypsin without or with inhibitors were figured out at the sub-molecule level by employing the molecular docking method,and it was further confirmed the results.Fullerenol molecules tend to bind to the abundant charged/polarized residues and little hydrophobic residues of digestive proteases without or with inhibitors according to the hydrophilic/hydrophobic distributions of present fullerenol surface.By regulating the amount and distribution pattern of hydroxyl groups in fullerenol,we could pur-posefully design the hydrophilic/hydrophobic characteristics of fullerenol based oral medicine to realize its high penetration through the digestion shield.Finally,we in-vestigated the cytotoxicity and permeability of fullerol through Caco-2 cells/monolayer.