Study And Application Of Novel Fluorescence Sensor For Determination Of Lactic Acid Enantiomer

Lactic acid is the final product of anaerobic decomposition of glucose in human tissues.There are two isomers,L-lactic acid and D-lactic acid.L-lactic acid is the main form of lactic acid in healthy people.If its content is too high,it will lead to acidosis.Its isomer D-lactic acid is only 1% of L-lactic acid in the body,while the increase of Dlactic acid level in blood and urine is usually the result of excessive secretion of intestinal bacteria,infection,ischemia or traumatic shock,which has become a clinical marker for the diagnosis of related diseases.In daily life,lactic acid often exists in the form of enantiomer.Therefore,it is of great significance to establish a rapid,sensitive and effective method for the identification of lactic acid enantiomers in food quality control and medical diagnosis.Currently,the commonly used methods for the determination of lactic acid enantiomers often require strict sample pretreatment and complex derivatization process.The detection is time-and labor-consuming,and requires expensive equipment and professional personnel.Point of care testing(POCT)biosensor has the advantages of good selectivity,high sensitivity and fast analysis speed.It is considered to be a low-cost and high-performance detection tool,and has developed rapidly in recent years.At present,the electrochemical biosensor based on immobilized enzyme electrochemical analysis is mainly used to detect L-lactic acid in biological fluid,but there are few reports on the simultaneous analysis of lactic acid enantiomers.In this thesis,based on the specific catalytic reaction of lactate dehydrogenase to lactic acid enantiomers,the following studies were carried out on the rapid and sensitive detection of lactic acid enantiomers.The main results are as follows:1.A paper-based sensor based on sodium alginate hydrogel system for simultaneous detection of lactic acid enantiomer was developed.Lactate dehydrogenase-glutamic pyruvic transaminase(LDH-GPT)dual enzyme was embedded on cellulose paper with sodium alginate.The dual enzyme cooperated with lactic acid enantiomer to produce NADH,the fluorescence signal was collected by smart phone and processed.The analytical performance of the sensor was systematically investigated and studied,and the experimental results verified the feasibility and reliability of the method.The LOD of L-lactic acid and D-lactic acid was 3.3 ?M,and the linear detection ranges were 0.01-3.0 mM and 0.01-0.5 mM,respectively.It has been successfully applied to the simultaneous determination of Llactic acid and D-lactic acid in human serum.The enantiomeric lactic acid paper-based sensor has the advantages of simple preparation,portability,high sensitivity and good accuracy,and can be used for on-site test,and has a good application prospect in other biological samples and food samples.2.A multi-channel microstructure fiber-based fluorescence sensor for highperformance detection of lactic acid enantiomer was developed.Dual enzymes(LDHGPT)were immobilized on the inner wall of multi-channel microstructure fiber by chemical bonding,and the dual enzyme synergistic catalytic reaction was used to promote the conversion of lactic acid enantiomers to fluorescent product NADH.Multichannel microstructure fiber greatly reduced the amount of samples,and its threedimensional structure with large specific surface area increased the signal strength,which was beneficial to effective fluorescence collection.In addition,the diffusion distance of the microchannel is short,which resulted in the high mass transfer efficiency and short signal response time.The results show that the sensor is rapid and sensitive,and has been successfully applied to the determination of lactic acid enantiomers in human serum.The recovery of serum samples was between 96.1% and 105.5%.There was a good correlation between the concentration of lactic acid enantiomers and the results of clinical kit with the correlation coefficients of 0.9969 and 0.9973 respectively.The proposed sensor is expected to play a role in disease diagnosis and clinical monitoring.