Study On The Fabrication Method Of Dehydrogenase Based Electrochemical Biosensor

Dehydrogenase based electrochemical biosensors （DBEB） are thedevices that detect target molecules based on the dehydrogenase, whichmodified on the electrode with the presence of cofactor NAD+. Thedehydrogenase reactions have a great significance in maintaining thenormal human’s function and cell metabolism. Electrochemical biosensorspossess the distinguished features of rapid detection, portable device andlow cost. Dehydrogenases have a high specificity toward target molecules.It has an important value to combine the excellent property ofelectrochemical biosensor and dehydrogenase for meeting the clinician’sdemands of rapid detection. This thesis is carried out focused on the keypoints and challenges of the DBEB research. The two works can be brieflysummarized as follows.（1） Constructed a new MDB/GMC/CS nanocomposite for one-stepfabrication of NADH electrochemical biosensor. MDB was adsorbed on theGMC, and the CS was utilized to disperse and immobilize GMC with its specialty of polycation and excellent film formation. Characters of thebiosensor toward NADH with the presence of physiological electroactivemolecules （ascorbic acid, uric acid, dopamine, et al） and metal ions (K+,Na⁺, Ca²⁺, Mg²⁺) were investigated. Under the optimized operatingpotential, the NADH biosensor showed the high sensitivity （10.36nA/μM）,fast response （5s）, very low limit of detection （0.186μM） and long termstability.（2） Constructed a novel MDB/GMC/ADH/NAD+/CS nanobiocomposite forone-step fabrication of integrated ethanol electrochemical biosensor. thenanobiocomposite was prepared by simply mixing the ADH and NAD+with MDB/GMC/CS suspension. The disposable ethanol biosensor wasprepared with simple and rapid drop-casting procedure. The ADH wassteadily immobilized on the electrode by CS with its excellent filmformation and favourable biocompatibility. The performance of theproposed ethanol biosensor was investigated after optimizing the enzymeloading （ADH and NAD+） and detecting condition （temperature and pHvalue）. Under the optimized conditions, ethanol biosensor showed a goodsensitivity （67.28nA/mM）, wide linear range （0.5-15mM）, low LOD （80μM）, good stability （40days） and outstanding reproducibility （RSD<5%）.The stability of the ethanol and NADH biosensor was estimated bymeasuring UV-vis spectrums of MDB under the different pH value. Theproposed ethanol biosensors were successfully used to detect real blood samples and the recovery of97.2%-106%was obtained.In summary, we used ADH as a research model, a novel method forDBEB fabrication via simple and rapid drop casting was developed basedon rational design and fabrication of the nanobiocomposite. The variancefrom batch to batch caused by multiple fabricating steps could be decreasedvia one step formation, and the precision and the stability were obviouslyimproved. Moreover, a series of DBEB could be made by changing thetype of dehydrogenase and other carbon nano-materials. The proposedDBEB could provide a rapid and portable testing tool to clinical diagnosis.