Construction And Characterization Of Biosensors Based On Enzyme Specific Binding Ability

In the field of biosensing,a variety of enzyme electrode sensors have been developed using the catalytic properties of enzymes,while the important property of enzyme-substrate specific binding is rarely addressed.In fact,the enzyme-specific binding ability can provide excellent substrate or ligand recognition performance in affinity-based sensors with more rapid and stable signals.Affinity-based sensors that rely on antibodies to provide specific recognition suffer from high preparation costs and harsh storage conditions,which limit their wide application.Therefore,the development of specific recognition strategies with low cost,stable performance and good operability is one of the research priorities in the field of biosensing.The detection principle of fiber optic Surface Plasmon Resonance（SPR）sensors is different from traditional sensors that rely on enzyme-catalyzed electron transfer technology,which can obtain detection signals through the specific binding of the test object and the enzyme immobilized on the fiber optic surface.The assay has several advantages of its own,such as label-free,high sensitivity,rapidity,low cost,small size and real-time monitoring.In this study,based on the specific binding ability of enzymes,several high-performance enzyme-modified fiber optic biosensors were developed and the sensing elements and construction strategies were optimized,with the following main research contents and results:Based on the properties of gold film on the surface of fiber-optic SPR,a protein immobilization technique based on gold-binding peptide（GBP）was developed to achieve direct binding between enzymes and sensors.It was confirmed that the gold-binding peptide has the ability to assist in the targeted binding of proteins on the gold surface,as well as the ability to improve the protein binding coverage and binding efficiency on the gold surface.On the other hand,graphene oxide-Au nanorods（GO-Au NRs）nanocomposites were prepared and characterized,and the modification of the fiber surface using GO-Au NRs achieved nearly 3-fold signal amplification in the same detection concentration range.A Glucose dehydrogenase（GDH）fiber optic biosensor was constructed for the commonly used glucose electrochemical sensor.The GDH used is Af GDH from Aspergillus flavus,which has high substrate specificity and does not use oxygen for catalysis.Specific low concentration detection of glucose was achieved using this sensor with a linear range of 1×10^-5 m M to 10 m M.Based on this,a chiral amino acid fiber optic biosensor was constructed using the stereospecific recognition ability of D-amino acid oxidase（DAAO）for chiral isomers of amino acids.The DAAO used was Re DAAO from the thermophilic fungus Rasamsonia emersonii stain YA,which has high thermal stability and catalytic activity.The sensor can effectively recognize D-type amino acids with a linear range of 5×10^-4 m M to 30 m M,while exhibiting good resistance to chiral isomeric interference.The specific binding ability of the enzyme depends on the specific binding site region.Therefore,expressing the binding site region alone and using it as a recognition element may be a more efficient strategy.To test this hypothesis,degree candidate designed a coronavirus biosensor based on the specific interaction of angiotensin converting enzyme 2（ACE2）with SARS-Co V-2 spike protein.Using the artificially designed spike protein binding miniprotein LCB1 as the recognition element,the low concentration of spike RBD protein was effectively detected on the fiber optic platform with a detection limit of 1.13pg/m L and good specificity.It validated the possibility of the artificially designed miniprotein as the recognition element for biomarker-specific identification.In this study,the degree candidate abandoned the traditional strategy of relying on the catalytic ability of enzymes to construct sensors,and developed a number of novel label-free,miniaturized,low-cost and rapid detection biosensors using enzyme-specific binding ability and fiber-optic SPR technology.They achieved efficient and specific recognition of glucose,chiral amino acids and spike protein,respectively,with detection limits comparable to or lower than those reported in previous studies.The detection limits were comparable to or lower than those reported in previous studies.The biosensor construction strategy developed in this study integrates the specific binding ability of enzyme,the targeted immobilization means and the signal amplification property of nanomaterials,which provides new ideas and directions for the development and application of biosensors.