| 其他摘要 | Potentiometric sensors based on polymeric membrane ion-selective electrodes (ISEs) have advantages of simple equipment, rapid response, easy miniaturization and are suitable for the on-site detection. These sensors have been widely used in various fields including environmental detection, food inspection and medical diagnosis. However, the conventional polymeric membrane ISEs are mainly used for the detection of metal ions and electrolyte ions. Their ionophores are hydrophobic chemical compounds.In recent years, with the introduction of hydrophilic bioreceptors and new sensing principles, a broad range of different targets can be detected by ISEs. As a new bioreceptor, peptide has unique features such as small molecular, flexible design and good affinity. Notably, peptide with zwitterionic property can be positively or negatively charged, which could induce a potentiometric response on the ISEs. Our groups developed potentiometric sensor array for the detection of bacteria, in which peptide was used for both target recognition and signal transduction. Peptide-functionalized magnetic beads (MBs-peptide) could be extracted into the polymeric membrane under a magnetic field and induce obvious potentiometric responses. By analyzing the potentiometric responses using linear discriminant analysis, the pattern recognition of bacteria could be achieved. However, the proposed potentiometric assay for a specific bacteria detection can not be easily achieved and is still required. Moreover, the potentiometric sensing mechanisms of the magneto-controlled assay need further theoretical clarification. In this study, the potentiometric response mechanisms of the magneto-controlled peptide on polymeric membrane ISEs have been explored. New potentiometric polymeric membrane sensors based on magneto-controlled peptide have been developed for the selective detection of the specific bacteria. The contents are as follows:1. Potentiometric response mechanisms of magneto-controlled peptide on polymeric membrane ISEsPeptide is one kind of polymer formed by natural or synthetic amino acids. Amino acids containing both acidic carboxyl groups and basic amino groups are linked by peptide bonds. Hence, peptide with zwitterionic property can produce a potentiometric response on the polymeric membrane ISEs. However, peptides are hydrophilic molecules that can not be easy extracted into polymeric membrane. MBs-peptide can be extracted into polymeric membrane to induce a potential response under the magnetic filed. In this study, the mechanisms and the theoretical formula of potential response of MBs-peptide on polymeric membrane have been explored. The results show that the peptide on the MBs and its interactions with the ion exchanger (dinonylnaphthalenesulfonate, DNNS) in the membrane could lead to a quasi-steady-state potential response. The magnetic field intensity influences the thickness of the diffusion layer of MBs-peptide in solution. The number of charges or charge density of peptide has an effect on the ion exchange process at the membrane/solution interface. Therefore, the potentiometric responses of peptide can be enhanced with the increase of magnetic field strength and the number of positive charges of peptide.2. Magneto-controlled potentiometric assay for E. coli based on cleavage of peptide by outer-membrane protease TEscherichia coli (E. coli) has been used as an indicator for routine analysis of the water environment. The development of accurate and efficient detection of E. coli is an urgent need. In this work, we describe a magneto-controlled potentiometric assay for specific detection of E. coli J96 by making use of the E. coli outer-membrane protease T (OmpT). OmpT can specifically cleave peptide at the peptide bond in consecutive basic amino acids (such as -R (arginine) -R -, -K (lysine) -K-, -K-R-, and -R-K-). Hence, a peptide with a number of positively charged arginines is designed and immobilized on the magnetic beads via streptavidin-biotin interaction. MBs-peptide can be extracted into the the polymeric membrane electrode to induce a potential response. In the presence of E. coli, OmpT on the surface of E. coli can selectively cleave the peptide, which leads to the amounts of arginine decrease and charge density change on the surface of MBs-peptide. Such changes of surface charge can lead to a decrease of potentiometric response of MBs-peptide. The change of the potentiometric response can be used for the quantitative detection of E. coli J96. A linear relationship between the potential change and logarithmic value of E. coli J96 concentrations is ranging from 5.0 × 103 to 1.0 × 107 CFU mL-1. The detection limit for E. coli is 5.0 × 103 CFU mL-1. This potentiometric sensor can achieve highly selective detection of E. coli J96 and provide a new avenue for rapid and selective bacteria analysis.3. Magneto-controlled potentiometric biosensor for E. coli apoptosis evaluation based on phosphatidylserine-binding peptideIn the above charpter, a magneto-controlled potentiometric sensor for the selective detection of E. coli have been achieved by using peptide as a bioreceptor. But this method could not be used for the detection of apoptotic bacteria. Accurate evaluation of bacteria apoptosis is of great importance in many biological fields such as antibiotic susceptibility test and drug screening. Phosphatidylserine (PS) externalization, translocation from the inner to the outer leaflet of the plasma membrane, is a characterization of bacteria apoptosis. In this study, a potentiometric sensor array for apoptotic bacteria detection based on the specific interaction between peptide and phosphatidylserine on the apoptotic bacteria is designed. The peptide with positively charged arginines and lysines is immobilized on the magnetic beads via biotin-streptavidin interaction. MBs-peptide can be extracted into the polymeric membrane to induce a large potential change under magnetic filed. In the presence of apoptotic bacteria, MBs-peptide can capture apoptotic bacteria via the efficiently interaction between peptide and PS of the apoptotic bacteria. Negatively charged bacteria causes the charge density change of MBs-peptide and prevents the peptide from being extracted into polymeric membrane, leading to a decrease of the potential response of MBs-peptide. Apoptotic bacteria can be detected based on the potential response changes of MBs-peptide. Taking E.coli as an example, the sensor can produce a linear response to 1.0 × 107 CFU mL-1 E.coli with apoptosis rate ranging from 10% to 50%. Based on this potentiometric sensor, the antibacterial activities of five antibiotics and silver nanoparticles with different particle sizes are evaluated. The antibacterial activities against E.coli rank as follows: norfloxacin, mytomycin C, ampicillin, gentamicin, and spectinomycin. With the increase of particle size, the antibacterial activity of silver nanoparticle decreases. The results of antibacterial properties evaluations from this potentiometric method are consistent with those from annexin V-based flow cytometric assay. The potentiometric sensor with merits of easy operation and fast analysis is suitable for direct detection of apoptotic bacteria and rapid evaluation of activities of antibacterial materials. Key Words: Potentiometric Sensor, Polymeric Membrane, Peptide, Escherichia coli, Apoptotic Bacteria |
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