Investigations And Applications Of Optical Chemo/Biosensing Technologies Based On Continuous And Renewable Drops

Optical chemical biosensing is a technique applied to selectively express real-time information of chemical and biological components with optical signals in detection system. As a product of crossing and penetrating subjects, the technology is becoming one of pioneering researches in analytical chemistry, with the rapid development of the modern analytical chemistry, organic synthetic, biospecific molecular, polymer chemistry and wave-guide apparatus. It is used more and more widely in many important application fields such as life sciences, environment monitoring, illness diagnosis, medication screening, food industry and material sciences.Meanwhile, the wide application of optical chemical sensor requires the improvement of its methods and theories. In view of the principles of chemosensing, the molecular recognition reaction is expected to be reversible, in order to obtain continuous response signals when the sample concentrations increase or decrease, and most of sensing reactions, available up to know, fulfill this requirement. However, still other reactions with high sensitivity and specificity to the analyze have been limited in applying to optical chemical sensor owing to the irreversibility. Furthermore, most of optical sensors are based on the technique of polymer membranes by immobilizing sensing materials in the membrane physically, the leaching of components from membrane phase to solution phase remains a series problem. The sensing technique based on renewable drop provides a most efficient solution to this challenge, which has the function of continuously producing fresh reagents and reacting with a fresh sample. The infections of optical signal, which caused by the absorption and scattering of cell surface and some contaminations of regents and samples to cell in the conventional photometric analysis, can be avoided. The unique features of a liquid drop can be characterized in its reproducibility, renewability, defined volume, and lack of containment walls. The features, individually or in combination, may result in high sensitivity and accuracy for analytes.On the basis of the research work of dynamical droplet optical chemical sensing performed previously in our laboratory, some techniques and parameters involved in selectivity and sensitivity of droplet optical chemical biosensor, such as growth mechanism of liquid drop, transmission and coupling efficiency of light wave, receiving and reproducibility of information, have been investigated by means of fluorescence analysis and modernanalytical technique presented in the paper. In addition to the technique of surface renewability of droplet and its characteristics as a windowless and sampler in reactions has also been studied. Some new ideas and methods have been provided for the application of the irreversible reactions in the domains of optical chemical biological sensing. A novel method of drop-based analysis, which regarding F-2500 and LS55 luminescence spectrometers as its experiment window, has been developed by improving dynamical drop-based fiber optical chemical sensor, which was constructed for principle proven. Many new fluorescence chemical biosensors combined with flow-injection analysis and the technique of dynamical drop-based sensing are developed. A model with automatization, short test time, low reagent cost and real-time assay is established. For the first time, dynamical drop-based sensor with our owner knowledge property right and two functions of absorbance assay and fluorescence detection is manufactured, which provides the possibility for optical chemical biosensors to be developed from mono-signal to multi-signal, multi-dimension, and even intelligence due to the realization of simultaneous determination with multi-channel, multi-component and multi -signal.In detail, the thesis includes the following two parts:Part I : Investigation of the principles and instruments about dynamical drop-based optical chemical biosensing techniques.The mechanism and principle of the dynamic drop-based optical chemical biosensing is systematical described. On the base of dynamical droplet sensor for principle proven based on fiber transmission, a liquid drop sensor was manufactured in combination with F-2500 and LS55 luminescence spectrometer. The apparatus was ameliorated in some components such as light source, monochromator, detector, pump and droplet forming device. Its performance has been improved dramatically. The third era dynamical drop-base instrument integrated with both fluorescence and absorbance detection was developed.Part II : The applications of dynamical drop-based optical chemical biosensing techniques in practical system.1. Applications in the determinations of inorganic ions.A fluorometry based on dynamical liquid drop has been developed for the determination of nitrite and iodate. The method is based on the fluorescence quenching of fluorescein fluoresceiniso-thiocyanate by iodine produced in the oxidation-reduction reaction. The sensors show linear responses in the range from 25 to lOOug/1 nitrite and 200 to 800ug/l iodate. The regression equations are JF=202.04+0.7435C and JF=-28.799+0.386C, respectively. The method is used to determine trace nitrite in water samples and iodate in table salt with satisfactory results.2. Applications in determinations of organic molecular combined with the techniques of immobilized enzyme.(D A drop-based flow-injection fluorometry based on the immobilization of enzyme on gel for determination of trace amount of L-lysine in solutions was proposed. Being illuminated or heated up, gel is formed by cross-linking when propylene amide is used as polymer, N, N'-methene-dipropylene amide as cross-linked agent and ammonium persulfate as attractant. Horseradish peroxidase (HRP) and L-amino acid oxidase are prepared to be enzyme column on the condition of uniformity dispersal of enzyme. L-lysine in solutions is oxidated to be hydrogen peroxide (H2O2) by L-amino acid oxidase. The fluorescence of 3,3',5,5'-tetramethyl-benzidine-dihydrochloride (TMB-d) is quenched by H2O2 in the catalysis of L-amino acid oxidase. The fluorescence change of TMB-d is related to the concentration of L-lysine. Trace amount of L-lysine in solutions is determined based on these mechanisms. Under the optimum conditions, the sensor shows linear response in the range from 2.0xlO"7mol/l tol.lxlO"5mol/l. The regression equation is F(//7=0.9776+2.783xl05C.(2) A method for preparation of enzyme columns based on nanoparticles by sol-gel was developed. Glucose oxidase (GOD) and horseradish peroxidase (HRP) mixed with SiC>2 nanoparticles and polyvinglbuty (PVB) medium were immobilized on the surface of capillary tube, respectively. It is proven that the catalysis effect of separate columns is higher than that of mixed columns. The enzyme columns are featured by high activity, more active centers and effective catalysis. An effective and fast method for assay of micro-amount of glucose was established. In the range from 2.0~400ng/ml, the quench value (F...