Construction And Applications Of Potentiometric Sensors Based On Chemiluminescence Readout

As a kind of electrochemical sensor,a potentiometric sensor detects the analyte concentrations by measuring the potential differences between the indicator and reference electrodes.It shows the advantages of simple instrument,rapid response,low cost and good selectivity,and has been widely applied in the fields of environmental monitoring,food analysis,and medical diagnosis.Potentiometric sensors can be classified into polymeric liquid membrane electrodes and solid-state membrane electrodes.The potentiometric response of a polymeric liquid membrane electrode to the activity of the ion in the sample solution obeys the Nernst equation,which is not sensitive to a small activity change,and the measurements are limited to the detection of simple ions.The potentiometric response of a solid-state membrane electrode is based on the change in surface charge,which suffers problems of low sensitivity and poor reproducibility.In recent years,the transduction modes of potentiometric sensors have been developed from the traditional zero-current potential detection to a variety of new sensing strategies such as chronopotentiometry,coulometry,amperometry and optical methodologies.The introduction of these new signal readouts improves the analytical performances of potentiometric sensors and broadens their applications.As an ideal transduction mode for potentiometric sensors,chemiluminescence analysis has the advantages of high sensitivity,wide linear range,simple equipment and easy automate.In this thesis,a series of new readout technologies based on chemiluminescence analysis for potentiometric sensors are described.An aptasensor has been developed for the detection of Escherichia coli based on the immobilization of electrochemiluminescent reagents and a moving-part-free fluid flowing system.A flow-injection chemiluminescence system for the determination of calcium ion has been developed based on the controlled release of a chemiluminescence reagent driven by the membrane potential.A rapid screening platform for plastic degrading microorganisms based on calcium ion sensing has been developed via using the designed and synthesized Fe₃O₄-Ca₃（PO₄）₂@polystyrene（PS）magnetic microspheres.Based on the surface blocking effect of sensitive membrane,the detection of Staphylococcus aureus has been realized by using a pulsed constant current to regulate the chemiluminescence ion fluxes.The detailed contents of this thesis are as follows:1.Solid-state membrane potentiometric aptasensor based on electrogenerated chemiluminescence signal readout for detection of Escherichia coliThe solid-state membrane potentiometric sensors are based on the surface charge change of electrode,which have low sensitivity and poor reproducibility.In this work,we constructed a highly sensitive aptasensor for amplifying the potentiometric response of Escherichia coli based on the electrogenerated chemiluminescence readout technique,in which the electrode immobilized with electrochemiluminescence reagent was used as the signal-converting element,and the solid-state membrane electrode modified with recognition molecule acted as the recognition component.The immobilization of electrochemiluminescence reagent greatly improved the luminescence efficiency and reduced the consumption of luminescent reagents,and the molecular modified electrode ensured the high selectivity of target detection through the specific recognition of target-aptamer.Furthermore,a motor-driven reagent flow technology was also introduced to accelerate the electron transfer and transmit between the luminescent reagent-electrode and the co-reactant solution in the detection compartment,enhancing the strength and stability of the electrogenerated chemiluminescence signals.Applying a constant potential between the components of signal conversion and recognition,the potentiometric responses generated by Escherichia coli with different concentrations were directly converted into the electrogenerated chemiluminescence signals under the zero-current potential detection.The electrogenerated chemiluminescence readout strategy showed a good linear relationship with the logarithm of Escherichia coli in the concentration range of 5-1000 CFU·m L^-1,and the detection limit was up to 2 CFU·m L^-1（3σ）.Compared with the zero-current potential detection strategy,the developed new readout strategy had higher detection accuracy(the relative standard deviation of three repeated measurements was 1.8%for 50 CFU·m L^-1 Escherichia coli).Furthermore,this transduction mode had been successfully applied to the detection of Escherichia coli in spiked seawater.2.Polymeric membrane potentiometric sensor based on chemiluminescence signal readout for determination of calcium ion in seawaterThe potentiometric response of the traditional polymeric membrane ion-selective electrode is limited by the thermodynamic Nernst equation,leading to the low slope sensitivity for detection of targets with small activity changes.The chemiluminescence detection of ions in water can be realized by utilizing the ion-exchange process between the chemiluminescent reagents in polymeric sensitive membrane and target ions in sample solution.However,the static chemiluminescent detection mode was employed in the previously reported technique,which had low luminescence efficiency and poor reproducibility,and the detection system was susceptible to the interference of the complex matrix of the sample.In this work,calcium ion was chosen as the target,and a flow-injection chemiluminescence method was developed by using the releasing technology of chemiluminescent reagent driven by sensitive membrane potential,which effectively resolved the problems of low luminescence efficiency,poor reproducibility and complex sample matrix interference caused by the static chemiluminescence mode.A calcium ion-selective electrode(Ca²⁺-ISE)was used for the recognition of target,and a lucigenin releasing electrode acted as the signal-converting element.The two electrodes were placed separately and connected by a salt bridge,and a constant potential was applied between them.When the Ca²⁺-ISE was in contact with different activities of calcium ion in the sample solution,which would influence the potential of the working electrode,and subsequently regulate the release of lucigenin indicator ion from the inner filling solution to the membrane surface.Under alkaline conditions,the released lucigenin reagent was oxidized by H₂O₂ and then generated chemiluminescence signals,which can realize the chemiluminescent detection of calcium ion in the activity range of 2.0×10^-3-2.9×10^-3 M.Furthermore,the chemiluminescent platform was applied to determination of calcium ion in seawater obtained from different depths,and the obtained results were basically consistent with the inductively coupled-plasma mass spectrometry method.Compared with the traditional zero-current potential detection,the accuracy of calcium ion detection was significantly reduced.3.Polymeric membrane potentiometric sensor for rapidly screening plastic degrading microorganismsMicrobial degradation of plastics shows the advantages of little pollution and no secondary pollution,so it has a great potential in solving the problem of plastic pollution.Several methods evaluated the ability of bacteria to degrade plastics usually require a degradation period of several days or even months,which is not suitable for the rapid screening of the plastic degrading microorganisms.In this work,we combined the indicator ion fluxes regulation method with polymeric membrane ion-selective electrode,and proposed a novel polymeric membrane electrode sensing method which can quickly detect the microbial degradation efficiency of plastics.The Fe₃O₄-Ca₃（PO₄）₂@PS magnetic material was synthesized,in which PS coating was used as a recognition element to identify the specific microorganisms.Ca₃（PO₄）₂ coating layer was used to release the indicator ions after medium conversion.Fe₃O₄ magnetic particles were used as the substrate of the composites,by which the medium can be changed easily by the magnetic separation method.The mathematical relationship among the electrode signals,the indicator ion fluxes and the microbial degradation efficiency was established by regulating the indicator ion release fluxes via the microbial degradation of the PS layer.The bacteria obtained from the deep sea that can degrade PS plastics was used as experimental bacteria.Traditional methods,such as scanning electron microscope（SEM）observation,were used as the qualitative method for comparison.The analytical performance of the constructed rapid screening platform for plastic degrading microorganisms based on polymer membrane potentiometric sensor technology was evaluated.In terms of qualitative analysis,the analysis results of the constructed detection platform for the three bacterial colonies were consistent with those results obtained from SEM analysis methods.In terms of quantitative analysis,the detection platform based on electrochemical sensing only taken 6 hours for the measurement of the microbial degradation efficiency of plastics,but the weight loss rate analysis method always required at least 30 days for degradation.The above results indicate that the screening method based on electrochemical sensing technology can greatly improve the screening efficiency of the plastic degrading microorganisms.4.Polymeric membrane potentiometric sensor based on chemiluminescence signal readout for rapidly screening of polystyrene plastic degrading bacteria in coastal environmentThe coastal environment always suffers from the serious plastic pollution problems caused by anthropogenic perturbations.As one of the common coastal plastic pollution,the degradation process of polystyrene（PS）plastic in the natural environment is very slow.In order to solve the problem of PS plastic pollution in coastal areas,the PS plastic degrading bacteria was selected as the screening object.The accumulation of the potential bacteria that could degrade PS plastics was carried out by placing the PS plastic film into the coastal sea of Yantai.Polymer membrane ion-selective electrode detection method was used to quickly screen the collected microorganisms to obtain the microorganisms with high efficiency for degrading PS plastics.In order to improve the efficiency of screening,the PS coating thickness of the Fe₃O₄-Ca₃（PO₄）₂@PS microspheres was reduced to shorten the time required for the microbial degradation experiment.In order to improve the accuracy of the polymer membrane sensor,the potentiometric signals of the polymer membrane ion-selective electrode were converted into the sensitive chemiluminescence signals.Based on the above key technologies,the polymeric membrane sensor based on the chemiluminescence signal readout taken only3 h for the quantitative analysis of the bacterial degradation efficiency of PS plastics.The obtained results showed that the strains of YTZ-2 and YTZ-3 exhibited the potential for degrading PS plastic.5.“Blocking”polymeric membrane potentiometric sensor based on chemiluminescence signal readout for detection of Staphylococcus aureusCurrently,polymeric membrane potentiometric sensors based on the surface blocking effect of sensitive membranes can realize the detection of Staphylococcus aureus and other pathogenic bacteria.However,these sensors generally suffer from low detection sensitivity and long detection time.The previously developed method of solid-state membrane potentiometric aptasensor based on electrogenerated chemiluminescence signal readout can also apply to the sensitive detection of Staphylococcus aureus by replacing the corresponding aptamer,unfortunately,the system involves complicated readout device.In this work,we developed a highly sensitive,rapid and simple chemiluminescence readout system for the detection of Staphylococcus aureus via the combination of highly sensitive chemiluminescence readout strategy and“blocking”polymeric membrane potentiometric sensor.The sensing principle was as follows:the chemiluminescent reagent（lucigenin ion）of the inner filling solution of the polymeric liquid membrane electrode modified the aptamer of Staphylococcus aureus can be released quickly and controllable by applying the current,and then,the lucigenin ion released into the solution can directly generate chemiluminescence signal to realize the signal readout,which effectively avoided the complicated chemiluminescence readout device.The release of lucigenin ion would be hindered by the presence of target in the sample,realizing the detection of Staphylococcus aureus.Under the optimal conditions,we realized the highly sensitive,rapid and accurate detection of Staphylococcus aureus through the chemiluminescence readout system.The proposed chemiluminescence readout strategy showed a good linear relationship with the logarithm of Staphylococcus aureus in the concentration range of 50-5000 CFU·m L^-1 with a detection limit of 10 CFU·m L^-1（3σ）,and the relative standard deviation for the detection of 3000 CFU·m L^-1 Staphylococcus aureus was 1.8%（n=3）.Furthermore,we applied this new readout system to accurately detect Staphylococcus aureus in seawater.