| Optical biosensors have been widely developed for the convenient detection of environmental pollutions and biological analysis, attributing to their outstanding advantages of high specificity and excellent biocompatibility. In this paper, we focused on the development of new biomaterials, new bio-reaction and new biological sensing strategies to establish novel biosensors to environmental analysis. These biosensors owned satisfied optical response performance, which could be utilized to transform the recognition behavior of specific targets to optical signals and achieve the detection of target objects. The detail contents of this dissertation are listed as follows:1. A sensitive fluorescent biosensor for the detection of copper ion inspired by biological recognition element pyoverdineIn this work, we have developed a fluorescent biosensor based on biological recognition element pyoverdine to selectively detect copper ion. The fluorescence of pyoverdine is quenched obviously after binding with copper ion. A good linearity within the range of 0.2–10 μM(R=0.997) is attained and the detection limit is 50 n M. The biosensor has been successfully utilized for the detection of copper ion in drinking water, seawater and bio-samples and the results agree well with those obtained by the inductively coupled plasma mass spectrometry. Therefore, the established biosensor is a creditable method to detect copper ion with high sensitivity and selectivity, which can be utilized as a powerful tool to monitor copper pollution in the environment.2. Pyoverdine secreted by Pseudomonas aeruginosa as a biological recognition element for the fluorescent detection of furazolidoneIn this work, a method based on the rapid fluorescence quenching effect of furazolidone to pyoverdine for the detection of furazolidone was developed. Pyoverdine secreted by a Pseudomonas aeruginosa strain PA1 was purified through affinity chromatography and its fluorescent property was characterized. The fluorescence of pyoverdine could be specially quenched by furazolidone, and based on this phenomenon a fluorescent method to detect furazolidone was established. Fluorescence of pyoverdine was quenched by furazolidone due to the electrons transfer from pyoverdine to furazolidone. The optimal p H for the detection was 7.2 in 50 m M 3–(N–Morpholino) propanesulfonic acid solution, and the whole detection process could be completed within seconds. The linear range of the detection was 2–160 μM and the limit of detection(LOD) was 0.5 μM. This study was the first time to develop a fluorescent biosensor for furazolidone detection, and the rapid and specific fluorescent method can be potentially applied for furazolidone detection in the environmental aquatic samples.3. Ultrasensitive colorimetric detection of Cu2+ ion based on catalytic oxidation of L-cysteineIn this work, a simple, facile colorimetric sensor for the ultrasensitive determination of Cu2+ ion was developed based on the following principle: L-cysteine and 1-chloro-2,4-dinitrobenzene(CDNB) could be conjugated to form the yellow product 2,4-dinitrophenylcysteine(DNPC), which was measurable at absorbance of 355 nm; however, upon addition of Cu2+ ion, the absorbance of DNPC would be decreased owing to the Cu2+ ion catalytic oxidation of L-cysteine to L-cystine in the presence of O2. Thus, the colorimetric detection of Cu2+ ion could be achieved. The optimal p H, buffer, temperature and incubation time for the colorimetric sensor were obtained of p H 6.8 in 0.1 M HEPES solution, 90 oC and 50 min, respectively. A good linearity within the range of 0.8–10 n M(R=0.996) was attained, with a high detectability up to 0.5 n M. Analyses of Cu2+ ion in drinking water, lake water, seawater and biological samples were carried out and the method performances were found to agree well with that obtained by ICP-MS. The developed simple colorimetric sensor proved applicable for Cu2+ ion determination in real samples with high sensitivity and selectivity.4. Simultaneous bioremediation and biodetection of mercury ion through surface display of carboxylesterase E2 from Pseudomonas aeruginosa PA1In this work, carboxylesterase E2 from mercury-resistant strain Pseudomonas aeruginosa PA1 has been successfully displayed on the outer membrane of E. coli Top10 bacteria to simultaneously adsorb and detect mercury ion. The transmission electron microscopy analysis shows that mercury ion can be absorbed by carboxylesterase E2 and accumulated on the outer membrane of surface-displayed E. coli bacteria. The adsorption of mercury ion is followed by a physicochemical, equilibrated and saturatable mechanism, which well fits the traditional Langmuir adsorption model. And the surface-displayed system can be regenerated by regulating p H values. In addition, the activity of carboxylesterase E2 can be inhibited by mercury ion, which has been successfully utilized to detect mercury ion. Therefore, the developed surface display system is of great potential in the simultaneous bioremediation and biodetection of environmental mercury pollution.5. A near-infrared ratiometric fluorescent probe for cysteine detection over glutathione indicating mitochondrial oxidative stress in vivoIn this work, we establish a near-infrared(NIR) ratiometric fluorescent probe Cy-NB for the selective detection of cysteine(Cys) over glutathione(GSH) and homocysteine(Hcy) in mitochondria to indicate oxidative stress. Heptamethine cyanine dye is chosen as the fluorophore of Cy-NB whose emission locates in NIR region. And p-nitrobenzoyl is employed as the fluorescent modulator due to its capability of selective-Cys response. Once triggered by Cys, the uncaged p-nitrobenzoyl rearranges the polymethine π-electron system of the fluorophore, which leads to a remarkable spectrum shifts in absorption and emission profiles. Taking advantage of these spectroscopic properties, we construct a ratiometric fluorescent signal for the detection of Cys with a detection limit of 0.2 μM within 5 min. Our probe Cy-NB can sensitively detect the mitochondrial Cys pool changes under different oxidative stress status in Hep G2 cells. We also successfully employ Cy-NB to imaging Cys level changes in living mice. It suggests that mitochondrial Cys can be used as an oxidative stress biomarker with simple potential clinical applications. And our probe Cy-NB is of great potential for further utilizing in exploring the physiological function of Cys in biological systems.6. Cyanine-based colorimetric and fluorescent probe for the selective detection of diethylstilbestrol in seawater, shrimp and fish samplesIn this work, we have developed a new colorimetric and fluorescent probe Cy-DES for the detection of DES with high sensitivity and selectivity. As a near-infrared probe, Cy-DES is able to avoid autofluorescence of dissolved organic compounds and maximize signal-to-background contrast. Taking advantage of the strong electrostatic interaction between the probe Cy-DES and DES, the spectroscopic properties of probe Cy-DES can be obviously changed in presence of DES. Under testing conditions, there is an excellent linearity within the range of 1- 8 μM(r = 0.9997) and the detection limit is 0.2 μM. The probe Cy-DES is successfully applied for the detection of DES in spiked seawater, shrimp and fish samples. Additionally, the detection of DES can be directly achieved by naked eyes with the utilizing of probe Cy-DES. The developed method is of great potential for application in the on-site detection of DES. |
PDF Full Text Request