Preparation And Properties Of Highly Effective Salivary Glucose-sensitive Material

Diabetes is a non-communicable disease that seriously threatens human life and health,and its harm mainly lies in the occurrence of complications:such as renal failure,heart failure,and decreased vision.Therefore,the research and development of fast,convenient,non-invasive and real-time blood glucose monitoring methods is of great significance for the prevention and control of diabetes and the health management of diabetic patients.Saliva has advantages including safety,convenience,and real-time collection,and the glucose level in saliva is highly correlated with the glucose level in blood.Therefore,the glucose in saliva is an ideal indicator for non-invasive blood glucose monitoring.However,the glucose level in saliva is only1/10-1/100 of the blood glucose level,and such a low level requires a highly sensitive glucose biosensor.In addition,saliva contains a lot of proteins,such as mucin,albumin,globulin and enzymes,etc.These proteins will non-specifically adsorb to the surface of the biosensor,resulting in a decrease in its sensitivity,stability and accuracy.In view of this,this paper aims to use the quartz crystal microbalance（QCM）sensing technology and the colorimetric method based on paper strips to achieve sensitive and accurate detection of the glucose level in saliva,so as to realize the non-invasive blood glucose monitoring.The main research contents and results of this paper include the following aspects:（1）Preparation and properties of highly sensitive hybrid hydrogel filmThe phenylboronic acid-functionalized hydrogel film,the sensing material for QCM biosensors,has poor viscoelasticity,which results in instability and low sensitivity of QCM sensor,and limits its application in salivary glucose detection.By introducing boric acid and double bonds into graphene oxide,and the hybrid hydrogel film is prepared by further copolymerizing phenylboronic acid-functionalized graphene oxide with a glucose-sensitive monomer（i.e.,3-acrylamidophenylboronic acid（3-APBA））.It was confirmed by FT-IR,Raman,and SEM that graphene was successfully modified into the hydrogel matrix.Furthermore,it was confirmed by DMA and water contact angle that the introduction of graphene was beneficial to enhance the viscoelasticity and hydrophobicity of the phenylboronic acid-functionalized functionalized hydrogel.The study found that the hybrid hydrogel film has a detection limit of 1 mg/L,and the frequency fluctuation of QCM within120 minutes is only 0.5 parts per million compared with the fundamental frequency,showing excellent stability.This is mainly attributed to the fact that phenylboronic acid-containing graphene can not only provide more reaction sites,but also improve the viscoelasticity of the hydrogel and stabilize the water molecules in the hydrogel.（2）Preparation and properties of interpenetrating polymer network antifouling hydrogel filmAiming at the major challenge of the accuracy and sensitivity reduction of biosensors caused by non-specific adsorption of proteins and other impurities,first,the zwitterionic polymer brush（sulfobetaine polymer:poly SBMA）was grown by the atom transfer radical polymerization（ATRP）method.Secondly,the glucose-sensitive monomer is further penetrated into the polymer brush matrix,and the anti-fouling,glucose-sensitive bifunctional interpenetrating polymer network（IPN）hydrogel film is prepared by the ultraviolet curing method.The IPN hydrogel has excellent antifouling properties and glucose sensitivity since the zwitterionic polymer brush and the glucose-sensitive hydrogel have excellent intertwining effects that can provide a stable hydration layer in the hydrogel film matrix,which solves the current scientific problem of low sensitivity of glucose biosensors caused by steric hindrance of traditional anti-fouling coatings.The study found that the IPN hydrogel film possessed excellent resistance to protein fouling by mucin with reductions in adsorption of nearly 88%and could also enhance the glucose sensitivity by nearly 2folds,compared to the PBA-functionalized hydrogel film.In addition,the IPN hydrogel membrane can realize the dynamic monitoring of low concentration of salivary glucose in the diluted saliva.（3）Preparation and properties of antifouling hydrogel film with a sandwich structure However,complex,time-consuming and high-cost salivary preprocessing（such as solid-phase extraction,heating at 100℃for 30 min,PVDF membrane filtration,ion exchange resin treatment）is needed to remove protein contaminants before salivary glucose detection using IPN hydrogel,which limits its practical application in low salivary glucose level monitoring.The zwitterionic polymer brushes were grown at the surface of the IPN hydrogel membrane by the ATRP method to prepare an antifouling hydrogel film（HSA）with a sandwich structure.Due to the synergistic effect originating from zwitterionic polymer brushes on the surface and substrate of the HSA,which provides a suitable physical barrier（～28 nm）and robust hydration layer for HSA that can enhance its sensitivity and antifouling.The study found that the HSA enhanced the glucose sensitivity by 130%and reduce the adsorption of nonspecific protein in 10%saliva by nearly 90%,compared to IPN hydrogel film.In addition,without the need for complex preprocessing of saliva,HSA can meet the requirements of typical saliva glucose levels（0-50 mg/L）monitoring.（4）In-situ preparation and properties of GOx&HRP@Cu₃（PO4）₂hybrid nanoflowers on the paper stripThe free enzyme loaded by the paper strip has the disadvantages of low activity and low loading,which cannot meet the requirements of the low concentration of saliva glucose detection.A whatman 3#paper strip was immersed into the PBS buffer solution containing inorganic copper sulfate,glucose oxidase（GOx）and horseradish peroxidase（HRP）for the in-situ growth of organic-inorganic nanoflower catalyst（HNF）on the paper matrix,and further use gelatin to fix the chromogenic substrate（TMB）,antifouling agent（BSA,PEG）on the test strip.Due to the close interaction between the smaller crystal nucleus and the wetted paper strip,it is beneficial to increase the load of HNF on the paper strip.The study found that the sensing time（60s）of HNF on the paper strip is 8-fold faster than that（480 s）of free enzyme on the paper strip,and the visible color change by naked eyes exhibits limit of detection for glucose is 10 mg/L.The density function theory（DFT）calculations reveal that the metal sites of Cu₃（PO₄）₂ enhance the binding of both H₂O₂ and enzyme,thus enhance the activity and stability of the HNF.In addition,through the detection of 5 clinical samples of diabetes,the detection results of this method are in good agreement with the detection results of commercial ion chromatography（correlation coefficient R²=0.986）.（5）ConclusionThe work of the paper is mainly divided into two parts:the first part is the modification of phenylboronic acid-funcitionalized hydrogel,the highly sensitive hybrid hydrogel film,IPN antifouling hydrogel film and antifouling hydrogel film with a sandwich structure were fabricated,respectively.In the second part is the mainly about the modification of enzyme,and the organic-inorganic hybrid nanoflowers were fabricated in situ on the paper strip.The main objectives of the two parts of work are to enrich the reaction sites of glucose-sensitive materials,stabilize the hydration layer,and improve the loading of enzyme,so as to achieve sensitive and accurate detection of analytes in complex physiological environment.The above prepared biosensors based on QCM sensor and paper strip sensor have achieved sensitive and accurate detection of glucose molecules in real human saliva.