Research On Phenylalanine Ammonia-Lyase Sensor And Detecting System

With the development of science in modern instruments and clinic medicine, some new requirements for detecting the human biochemical parameter will be presented. The trend of the development of medicine will go the way that the therapeutic medicine will be replaced by preventive medicine in the future. Therefore, the early diagnosis of some diseases becomes very important, and in the research field of medicine in recent years, great attention has been given to quickly and effectively detecting the biochemial parameters of some genetic diseases. The biosensor has developed greatly in recent years. It has the following advantages: It is an instrument with simple structure, low cost and better identification. It can easily and quickly identify substances to be detected at molecule level. It also has great application prospects in such areas as monitoring in chemical industry, clinical diagnosing in medicine, drug screening in biomedicine, and poison detecting in various fields and so on. Applying biosensors to detecting amino acid is of great importance in the area of medicine study. This desertation proposes a constructive approach to detecting phenylalanine (Phe), in which a new phenylalanine ammonia lyase (PAL) sensor and the detecting system have been developed to quantitate the level of Phe in a newborn baby in order to treat phenylketonuria (PKU) timely. Funded by the Harbin city government, as part of the study in the project "the Development of a Phenylketonuria Analysis Instrument", one of the Harbin major technology programs, this study focuses on several crucial points in this project. The crucial points in development and making of emzyme sensors are solved, in which PAL sensor, signal adjustment circuit, and controlling and displaying units have been provided, thus developing the idea of incorporating all the above units into one detecting system. The advantage of applications of various technologies in one instrument is obvious and can accelerate research and commercialization. Summary of the work done in the progress of writing this desertation: 1. The design plan and the detecting priciple of the PAL sensor have been expounded. The relationship between the dynamic characteristics of an enzyme reaction and Michaelis-Menten Equation has been discussed. The study provides a theoretical basis for the thickness of the enzyme membrane and enzyme concentration. Based on the characteristics of Phe and methods used in analysing the enzymatic reaction at both stable and transient stages, a mathematic model and the structure of the PAL sensor have been established. 2. Based on the characteristics of PAL, in the research of the way to purify the immobilization enzyme, a new and practical method has been developed in order to achieve the purpose, which is different from the other published purification methods in developing the enzyme sensor. This study solves the problem of further purifications for the commercial enzyme, which is one of the breakthroughs in the development of a PAL sensor. 3. A covalent-cross-link method has been chosen to link PAL onto a sensor after carefully comparing the different methods of the enzyme immobilization methods and comparing the techniques in developing the enzyme sensor. The problems, such as PAL activity lost and the unsatisfied binding ability between PAL and permeate membrane, are well solved in this study, and the newly developed PAL sensor can meet the requirement for its application. 4. Different types of commercial permeate membranes and basic electrodes, the main parts of the PAL sensor, have been selected and tested. We find that the polytetraflouroe thylene (PTFE) membrane and the Model 95-12 ammonia electrode are more suitable to be used for the further development. 5. This study uses computer simulation technology to analyze the relationship between the input and output signals in different PAL sensor internal resistences. The design based on this meets the needs of practical application. The calibration methods of enzyme sensor have been discussed, which helps to find an optimized linearized calibration method and calculations method. 6. This study establishes the control and calculation system on computers, in which suitable hardware and software are designed and developed. The design of a LCD touch screen provides a user-friendly interface. These features increase the reliability of the system, facilitating the communication between theinstrument and its operators, and paving a good way for further study. In this system, the lower limit of detection is 1×10-5 mol/L, the linear range is 1×10-5 ~ 5×10-3mol/L, and the detection time is less than ten minutes. The cost is about 1/10 the fluorescent colorimeter method. Using this detecting system in clinical detection, the disease PKU can be diagnosed earlier, which is beneficial for timely treatment. The PAL sensor and detecting system can be adapted to be used for other amino acid analysis and medical detection. So, it has great value in the application of biochemical detection and academy studies.