Study Of Optical Chemical Sensor With Eggshell Membrane

Optical chemical sensor is a device based on spectrochemistry, optical waveguide and measurement techniques to express the chemical massages and optical properties of analyte. It uses the change of light propagation characteristic to determination, which cause by the change of physicochemical property with the reaction between the analyte and sensitive membrane. The most important characteristics of optical chemical sensor are its high resolution, good security, capability in remote or real time monitoring and strong anti-interference ability.Developing solid-phase optical chemical sensor with homogeneous sensor principle is an effective approach to the practical application of optical chemical sensor. Compared with homogeneous sensor, solid-phase optical chemical sensor can be repeatedly used, easy to be a device and do not pollute the system. Nowdays, it is very active to study on membrane optical chemical sensor, thus reseach closely related to the design of membrane optical chemical sensor have became a focal point of research, such as selection of sensing element, novel immobilization method and matrix.Eggshell membrane is a kind of cheap protein film with high specific surface area, which is composed of biological molecules and highly cross-linked protein fibers, and possesses excellent gas and water permeability. It is reported that eggshell membrane is semipermeable, through which the small molecules can be selectively permeated and the lager ones can not. Eggshell membrane can be used as an ideal bio-platform for antibody immobilization, which is helpful for antibody to keep activity. In this study, some novel optical chemical sensors were explored based on the eggshell membrane as a solid support, which combine with several common analytical methods. The main contents and some conclusions of the thesis are as follows:Several physical and chemical properties of eggshell membrane were investigated. The microstructure of the eggshell membrane was observed with scanning electron microscope, it can be seen that there are many highly cross-linked protein fibers and cavities, which make the membrane possess excellent gas-and water-permeability. The amino acid contents of eggshell membrane were detected by high performance liquid chromatography with acid hydrolysis and the chemical compositions were also analyzed using XPS technique, it was found that the main compositions are N, O, C and S, among which the contents of N. O, C are more, but the one of S is less. The infrared spectrum of eggshell membrane was investigated, it was shown that the main absorption peaks are at 3000-3400,1650,1540,1400cm-1, which caused by N-H stretching vibration of amide's A bond, C-H stretching vibration of amide's B bond, C=O stretching vibration of polypeptide skeleton and vibrations ofⅠ,Ⅱ,Ⅲbond of amide, respectively. The intrinsic fluorescence excitation and emission spectra were studied and resulted that the optimal excitation and emission peaks were at 280 nm and 350 nm. The effects of pH value and temperature to the fluorescence spectrum were also investigated. It was observed that the fluorescence intensity of eggshell membrane hardly changed with pH value from 3.4 to 10.5, or at different temperature from 4 to 60℃. However, the eggshell membrane became denaturation over 80℃. This result was corresponded with the data measured by thermal analysis. In addition, the adsorption between eggshell membrane and Eosin Y was studied, including effects of Eosin Y concentration and adsorption time, interaction between human serum albumin and Eosin Y adsorbed on eggshell membrane.A novel fluorescence immunosensor using eggshell membrane was developed. The immunosensor was fabricated by immobilizing antibody on the eggshell membrane with glutaraldehyde. due to the natural amino groups of eggshell membrane. Based on the immunoreactions of primary antibody, target antigen and secondary antibody, the sandwich complex were formed on the eggshell membrane and FITC labeling secondary antibody could be employed to detect the target antigen. It was observed by scanning electron microscope and fluorescence spectra whether the antibody was immobilized onto eggshell membrane. The effects of experimental parameters, including choice of the blocking reagents, concentration of glutaraldehyde and dilution ratio of primary and secondary antibody, on the fluorescence intensity of system were investigated. Under the optimal conditions, the fluorescence intensity was in proportion to the concentration of HIgG and HIgM, the linear range of two systems were 20-100 ng mL-1 and 5-60 ng mL-1, the detection limits were 15 ng mL-1 and 4.3 ng mL-1. The proposed sensors were successfully applied to the determination of HIgG and HIgM in the standard blood serum, and the results were satisfactory compared with that obtained by general immunonephelometric method. Stability of the proposed sensors was investigated. It was shown that the intrinsic fluorescence intensity of different fresh eggshell membranes were basically same and the fluorescence intensity did not change obviously in the storage period of 30 days. The fluorescence intensity of eggshell membrane with sandwich immunoassay was nearly retained. It was demonstrated that the proposed sensors have remarkable storage stability, permeability and highly biocompatibility.R-phycoerythrin (R-PE) possess several unique characteristics that make them attractive for use as fluorescence labels, a fluorescence sensor was fabricated using R-PE immobilized on eggshell membrane with glutaraldehyde. It was found that salbutamol (SAL) can significantly quench the fluorescence of R-PE. which could be reversible after a running PBS buffer. Therefore, a new and rapid analysis method of SAL content in urine was established. Scanning electron microscopy, fluorescence microscopy and fluorescence spectra were used to investigate whether R-PE was successfully immobilized on eggshell membrane. The detailed study was made on the effect of blocking reagent, glutaraldehyde content, pH value and reaction time. The results indicated that there was a good linear relationship between the change of system fluorescence intensity and the concentration of SAL in the range 5.00-100 ng mL-1, the detection limit was 3.50 ng mL-1. The relative standard deviation (n=4) was 3.22% at 100 ng mL-1 SAL solution. Under the optimal conditions, it was found that most of the ions and amino acids examined did not interfere with determination, whereas some could produce slight interference on the determination. The proposed sensor was applied to the determination of SAL in urine samples. Recovery experiment was done and the recoveries were in the range from 84% to 101%. The reproducibility and stability of proposed sensor was investigated and the results demonstrated that the sensor had a highly reproducibility. In addition, it was found that the membrane sensor could be stored without any remarkable change in its fluorescent intensity for at least 50 days.A label-free immunoassay system using eggshell membrane as matrix was combined with light scattering technique, which could centralize high selectivity of immunoassay and high sensitivity of light scattering. Poly(diallyldimethylammonium chloride) (PDDA) is a cationic polyelectrolyte with positive charges. It can be concluded that PDDA reacts with Ag-Ab with a certain pH value, which results in the formation of a new three-component complex, the LS intensity of the complex was higher than those of Ag, Ab and PDDA. A series of experimental conditions was optimized, such as pH value, reaction time, PDDA concentration and Ab concentration. The linear range of the system was 5-500 ng mL-1 and the detection limits was 2.31 ng mL-1. In order to evaluate the proposed method, HIgG concentration of human serum samples were analyzed. The results obtained by the proposed method are consistent with those obtained by immunonephelometric method.