A Study On Application Of New Bioanalytical Technology In Environment Medicine Monitoring

With the progress of society civilization and the development of science innovation, effects have been made on public health and living conditions. However, the effects of environmental pollution on human health have become serious problems. Food safety issues often occur. The number of patients with diabetes and cancer is increasing dramatically than before.All of them are regarded as public health problems, which threat to people health seriously. Effective analytical methods play a central role in assisting food safety and chronic disease. With the development of science and technology, a number of new bioanalytical techniques, such as Enzyme Thermistor biosensor and microarray, have been developed. Vice verse, they not only promote the development of science but also expand their practical application.The thesis intends to make a study on application of Enzyme Thermistor(ET) and Protein Microarray in environment medicine monitoring to provide a powerful technological platform to keep people health.Enzyme Thermistor is a new type of biosensor that employs thermal signal transduction to detect the heat generated from immobilized enzymatic breakdown of analytes. The signal is proportional to substrate concentration. The dual-signal flow injection analysis scheme uses an enzyme and a reference column. The reference column makes it possible to independently monitor and correct for non-specifically generated heat, thereby eliminating the need for sample pre-treatment. ET is able to continuously detect urea, lactate and glucose in milk or blood without pretreatment. Compared to traditional methods, ET has advantages of simple operation, rapid detection and environment friendly, which has attracts more and more interest by biologist and become an important role in bioanalytical analysis.With the development of proteomics, protein microarray technology with advantages of high-throughput and rapid detection has made much progress than before."XNA on GoldTM" microarray is a new type of microarray that combines self-assembling monolayers with the biotin-streptavidin system to provide a robust, versatile immobilization scheme. A hydrophobic film is patterned onto the surface creating an array of tension wells that eliminates evaporation effects thereby keeping protein activity to which biomolecules are exposed to during immobilization. The streptavidin linker layer makes it possible to adapt and/or develop microarray based assays using virtually any class of biomolecules including:carbohydrates, peptides, antibodies, receptors, as well as the more traditional DNA based arrays. The article intends to present that some useful biomarkers, such as urea and lactate in milk, glucose and lactate in blood, were detected by ET, which will improve methodology research of biological sample and practical application,accumulate scientific information,provide technical support and explore practical application in order to make a good foundation for analysis standard system in China. In additional, lung cancer gene biomarker epidermal growth factor receptor(EGFR) was detected using ET based on ELISA signal amplification theory as well as protein biomarker carcinoembryonic antigen (CEA)was detected by "XNA on GoldTM" microarray. It is significance for us to test lung cancer biomarkers at the level between gene and protein and make some useful results. In the future, our aim is to develop a high-throughput technique to test multiple biomarkers and investigate their value in patients group. More important,our work will focus on discovering the relationship among environment factors, early diagnose of lung cancer, disease epidemiology at multiple levels. The whole study is composed of the following four Parts: Part I Determination of urea and lactate in milk using Enzyme ThermistorObjection:A method of determination of urea and lactate in milk using Enzyme Thermal biosensor has been developed. Method:Detection of specific analytes in food stuffs normally requires sample pretreatment regimes in order to remove interfering compounds. Here we report the design and construction of a dual-signal flow injected analysis device which eliminates the need for sample pretreatment. The device employs thermal signal transduction to detect the heat generated from enzymatic breakdown of analytes. The dual-signal flow injection analysis scheme uses an enzyme and a reference column. The reference column makes it possible to independently monitor and correct for non-specifically generated heat. In order to test the device, lactate and urea concentrations were determined in milk samples without any prior treatment. Result:In buffer, the urea assay had a linear range from0.1to50mM (R=0.996), while the lactate assay had a linear range from0.025to5.0mM (R=0.9998). The regression values for urea in milk samples were0,994,0,992and0,990for0.5%,1.5%and3.0%fat milk, respectively. The linear regression value for the milk lactate measurements was0,999in all cases. The assay cycle time was5min. Elimination of sample pretreatment shortens assay times, simplifies testing and reduces training requirements. In addition, the dual-signal design improves measurement reproducibility, sensitivity and stability. Conclusion:The method has advantage of simple operation and rapid detection,which is able to meet requirement of large samples detection and provide a platform for healthcare, agriculture and environmental testing. Part II Continuous and simultaneous determination of blood glucose and lactate without pretreatmentObjection:A method of continuous and simultaneous determination of blood glucose and lactate without pretreatment has been developed. Method:Patients undergoing diabetes therapy, as well as intensive care patients have their glucose levels monitored continuously in order to eliminate this risk factor. Glucose levels are widely used as a measure of health status, but alone the results are often times inadequate to make an accurate determination about the general health status of the individual. Lactate, another energy metabolite, is produced under anaerobic conditions and can be used to monitor the balance between aerobic and anaerobic metabolism. Tested together, these metabolite levels can provide prodiagnostic information that improves patient outcomes. Glucose and lactate were determined simultaneously in whole blood using a split-flow dual channel Enzyme Thermistor biosensor with immobilized glucose oxidase for glucose analysis and lactate oxidase for lactate analysis. Result:No detectable clogging or interference was observed using whole blood samples. The linear detection range for both the glucose and lactate assays was0.5-45mM. Glucose linear equation was that Y=2.11x+8.09(r2=0.999). Lactate linear equation was that Y=1.86+1.60(r2=0.999). The assay cycle time was2.5minutes. Comparative analysis between our device and the HemoCue device showed an excellent correlation. The device was stable for hundreds of injections over a period of45days. The linear ranges and sensitivities fulfill clinical analysis requirements. The ability of the device to analyze whole blood without any pretreatment makes it possible to develop real-time analysis of glucose and lactate in a clinical environment. Conclusion:The method has advantage of simple operation and rapid detection, which is able to meet requirement of continuous and simultaneous determination of blood glucose and lactate without samples pretreatment in clinical patients. Part Ⅲ A preliminary study on thermal signal determination of EGFR using Enzyme Thermistor based on DNA hybridizationObjection:A novel method of detecting DNA hybridization regarding lung cancer biomarker EGFR based on Enzyme Thermistore has been developed. Method:The biotin-DNA was injected and hybridized to the bound DNA after immobilizing the probe DNA on the CPG. After this, AP-strep conjugate was injected and bound to the biotin-DNA.Then, pNPP was injected to detect a thermal signal. Result:Tris buffer was used as mobile phase and substract solution with flow rate0.5mlmin-1. Substract pNPP concentration was10.0mgmL-1. Conclusion:Thermal signal d detection of DNA hybridization was investigated using Enzyme Thermistor at primary level in order to make a foundation to further research in the future. Part IV A preliminary study on determination of CEA using protein microarrayObjective:To establish a method of constructing protein microarray based on sandwich immunoassay to test CEA to provide a powerful platform for early diagnosis of lung cancer. Methods:"XNA on GoldTM" microarray were robotically printed with CEA capture antibodies by BioDot instrument based on sandwich immunoassay to test CEA concentration. Biotinyled-CEA-antibody were immobilized on slides as capture antibody, then dilutions of the CEA antigen were applied to the arrays and the proteins were detected with cy3-labeled detection antibody. A laser confocal scanner was used to obtain the images and the signal intensity was proportional to CEA concentration. Result:Various factors in the production of protein microarrays were analyzed. The signal intensities increased with increasing concentration of capture antibody and detection antibody. Both of them were0.1mgmL-1. Conclusion:A preliminary method for constructing protein microarray based on sandwich immunoassay was established which might lay a foundation for fabrication of protein microarray with multiplexing and quantitative capacity.