| Potentiometry as a main branch of electrochemical analysis shows the advantages of low cost,ease of operation,and rapid response.It has been widely used in the fields of environmental monitoring,medical diagnosis,and food analysis.Potentiometric sensors include solid-state membrane electrodes based on surface charge change and polymeric liquid membrane ion-selective electrodes.The former show drawbacks of low sensitivity and limited analytes,and the latter are mainly used for ions and small molecules,but not for whole cells.As the new recognition molecules,aptamers and peptides can recognize the targets with high affinities,and show the advantages of easy modification,good stability,and capability to recognize a wide range of target molecules.In recent years,aptamers and peptides have been successfully applied in the fields of biosensors,gene chips,and medicine development.Aptamers and peptides can recognize a variety of targets,including organic molecules and cells,and thus may significantly expand the applications of potentiometric sensors.Moreover,the introduction of aptamers and peptides can improve the selectivities of potentiometric sensors due to the high affinities of aptamers and peptides to their targets.In this thesis,a series of potentiometric sensors based on aptamers and peptides have been developed.The contents are shown as follows:1. Solid-state membrane potentiometric aptasensor for bisphenol A based on surface charge changePotentiometric analysis based on surface charge change shows lower sensitivity and limited applications for detection of neutral molecules.In this work,a novel potentiometric aptasensing method for detection of neutral molecules is presented,using bisphenol A?BPA?as a model.The working electrode can be prepared by layer-by-layer assembling of carboxylated multiwall carbon nanotubes,poly?diallyldimethylammoniumchloride??polycation?,and aptamer?polyanion?.The presence of BPA induces the conformational change and thus detachment of the aptamer at the electrode surface,which leads to a variation of the surface charge and therefore a potential change.Thus,the electrode can be used for the detection of the target.Under optimum conditions,the present sensor shows a stable response to BPA in the concentration range from 3.2×10-8 to 1.0×10-6 M with a detection limit of 1.0×10-8 M.The proposed methodology can be used for sensitive potentiometric sensing of other targets involved in aptamer/target binding events.2. Solid-state membrane potentiometric aptasensor for bisphenol A based on coulometric signal readoutNewly developed signal readouts for the potentials of polymeric membrane ion-selective electrodes show the advantages of high sensitivity and selectivity.However,using new signal readouts for determination of neutral molecules has not been explored.In this research,we have fabricated a solid-state membrane electrode based on aptamer for the determination of the neutral small molecules.BPA has been used as a model.The signal readout is based on constant potential coulometry.By electropolymerization and/or physical adsorption,polyaniline and the BPA aptamer can be modified on the glass carbon electrode successively.The aptamer on the electrode surface can interact with the target,thus leading to a conformational change,This change could weaken the interactions between polyaniline and the aptamer and cause the detachment of the aptamer.The charge distribution change of the electrode surface induced by the detachment of the aptamer can induce a potential change.Since the potential between the indicator electrode and the reference electrode is enforced to remain constant,the potential change gives rise to a reducing/oxidizing current that can be measured.The cumulated charge is linearly proportional to the change of the logarithm of the target concentration.Under optimum conditions,BPA can be detected in the concentration range from 3.2×10-9 to 3.2×10-7 M with a detection limit of 1.0×10-9 M.Compared to the traditional solid-state membrane potentiometric sensors,this developed methodology using the coulometric signal readout shows higher sensitivity.The proposed electrode has been applied in the determination of BPA in environmental samples with satisfactory results.3. Polymeric liquid membrane electrode for potentiometric detection of Listeria monocytogenes using a short peptide pair as recognition moleculesPotentiometric sensors based on peptides have only been used for detection of ions,which limits the applications of peptides in the field of potentiometry.In this research,a new potentiometric sandwich assay based on a short peptide pair for bacterial cells is demonstrated,using Listeria monocytogenes?LM?as a model.The original peptide for LM can be shortened into two fragments to serve as the peptide pair for the sandwich assay?capture fragment and detection fragment?.The capture fragment is conjugated to magnetic beads.Horseradish peroxidase is labeled to the detection fragment for catalyzing the oxidation of 3,3?,5,5?-tetramethylbenzidine with H2O2,which induces the potential change on the polymeric membrane electrode.After conjugation of the two short fragments with LM,magnetic separation can be used to eliminate the interferences from the sample matrix.Under optimal conditions,the concentration of LM can be determined potentiometrically in a linear range of 1.0×102 to 1.0×106 CFU m L-1 with a detection limit of 10 CFU m L-1?3??.The proposed sensor has been applied in the determination of LM in seawater,and the results agree well with those obtained by the plate count method.4. Magnetic field-driven potentiometric response of a polymeric liquidmembrane electrode to Staphylococcus aureus based on peptide-modified magnetic beadsIn the previous research,we developed a new potentiometric sandwich sensor based on a short peptide pair for LM.However,this method still needs the introduction of chemical labels and/or additional indicators.In this research,we propose a potentiometric sensor for detection of bacteria using the peptide as both the recognition molecule and indicator.Staphylococcus aureus?S.aureus?has been selected as a model.Peptides as zwitterionic molecules can be positively or negatively charged in the buffer solution with a specific p H and thus induce a potentiometric response on the polymeric membrane electrodes.The peptide is connected with magnetic beads?MBs?via biotin-streptavidin interactions.By applying a magnetic field,the peptide modified MBs can be extracted into the polymeric membrane,and induce a potential change.The modification of peptide on the MBs does not interfere with the recognition of peptide to bacteria.After incubation with the bacterial cells,the surface charge change or charge density change of the peptide can lead to a potential decrease,which can be used for potentiometric sensing.Under optimal conditions,S.aureus can be measured at levels down to 10 CFU m L-1.The developed potentiometric sensing methodology without the need for probe labeling and indicator addition could broaden the applications of the polymeric membrane ISEs.5. Polymeric liquid membrane potentiometric sensor array based on peptide-modified magnetic beads for discriminating bacteriaIn recent years,many biosensors have been developed for the detection of bacteria.However,the simultaneous detection and pattern evaluation of several bacteria are still rare.Previously,we developed a simple potentiometric protocol for the detection of a bacterium strain.In this research,we propose a potentiometric sensor array based on the previous protocol for the pattern evaluation of bacteria.Four peptides have been used as the recognition molecules for the fabrication of the biosensors.With the array,we have investigated the potentiometric response of eight different bacteria.Linear discriminant analysis can be used for the pattern recognition of the bacteria.Eight selected bacteria can be discriminated into eight clusters.Meanwhile,the blind test of seawater reveals that the developed array exhibits high accuracy for the pattern evaluation of bacteria. |
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