Construction Of Several Biosensors Based On Photoelectrochemical Response

In recent years,photoelectrochemical?PEC?technology increasingly becomes a powerful alternative analytical method for bioassays.The photocurrent generation mechanism is based on the photoexcitation of photoactive materials that undergo a process of charge separation,resulting in photo-potential or photocurrent.According to the different measurement parameters,PEC sensors can be divided into two kinds of potential and current type.The quantitative mechamism of the current type PEC sensors are based on the physical and chemical interactions between the analytes and photoactive materials,which caused the changes in photocurrent intensity and the relationship between the difference and the concentration of analyte was obtained.In this thesis,several PEC biosensors based on photocurrent response were constructed by using quantum dots and applied to the detection of analytes.The preparation process and sensing mechanism were investigated emphatically.The specific contents are as follows:The first chapter is the introduction.Firstly,we summarized the concept and characteristics of photoelectrochemistry,the mechanism of photocurrent and its research progress.Secondly,the applications of current type PEC sensors in the determination of biomolecules were reviewed.The future prospects of PEC sensing analysis were discussed.Finally,we proposed our working assumptions.In the second chapter,the water-soluble CdTe quantum dots?QDs?modified with3-mercaptopropionic acid were prepared and used for the construction of PEC sensors.A novel signal-on PEC biosensing platform based on the energy transfer between CdTe QDs and Ag nanoparticles?NPs?,which coupled with the catalytic hydrolysis of protease,was constructed for highly sensitive detecting cancer marker type IV collagenase.Firstly,the CdTe QDs were assembled onto the surface of the indium-tin oxide?ITO?electrode by means of electrostatic attraction between the negatively charged CdTe QDs and positively charged poly?dienylpropylmethyl?.The peptide chain containing a special sequence with positively charges arginines at the one end and cysteine-labeled terminal at the other end,which can be modified with Ag NPs,was designed.The designed peptide was connected to the ITO electrode through electrostatic attraction between positively charged arginines and negatively charged QDs,which have been successfully combined to the electrode surface.The as-obtained modified electrode showed a lower photocurrent under irradiation.The photocurrent intensity was increased towards the addition of type IV collagenase,which can specifically hydrolytic cleave Gly-Pro-Ala,leading to Ag NPs away from the electrode surface.The linear range of 0.5 to 50.0?g/mL and the detection limit as 0.096?g/mL for the type IV collagenase were obtained.The peptide-QDs based PEC detection has excellent specificity against type IV collagenase showing a promising potential for other proteases or kinases detection when we change the substrate of peptide.In the third chapter,it is reported that the amplitude of the resulting photocurrent was determined by the concentration of the donor compounds in a certain range in the PEC sensors.Among kinds of electron donor species,ascorbic acid?AA?was widely used as a nontoxic and efficient electron donor.Here,we wanted to develop a templated synthesis strategy based on a protein cage to prepare AA encapsulation,and then enzymatic hydrolysis the encapsulation,in situ to release AA as electron donors to produce photocurrent.In this work,we exploited apoferritin?APO?as a protein cage.The encapsulation of AA inside the APO cavity?formation of APOAA?is realized in the fact that the assembly-disassembly mechanism of the APO protein cage undergoes pH dependent.The APOAA with different AA contents was successfully synthesized.It was examined that the encapsulation yield increased with the increasing applied AA concentration.When joining the maximum amount of AA,by calculation,a molecular APOAA can hold about 4000 molecules AA.The amount of AA released from APOAA increased with pH from weakly alkaline to strongly acidic.At the physiological pH,APOAA is stable,and the prepared APOAA can be used in subsequent PEC experiments.In the fourth chapter,a signal-on PEC sensor based on the as-obtained APOAA for protease detection is described.Cd Te QDs modified ITO electrodes were used as the working electrodes,and the buffer solution containing APOAA was used as the detection solution.The constructed PEC sensor was used for quantitatively detection of trypsin.The sensing mechanism was based on that the loaded AA could be released from the central cavity of APO into the buffer solution as sacrificial electron donor to capture the photo-generated holes of CdTe QDs when light is turned on.In this system,the sensor relied on monitoring the photocurrent intensity as a result of enzymatic substrate proteolysis in the homogeneous aqueous solution.A low detection limit of 2.7 ng/mL and the linear range from 30 to 450ng/mL for trypsin were achieved.We believed that such a sensing system not only hold the potential ability as a probe for trypsin activity assay,but also could be used for the corresponding inhibitor-screening.The recoveries of the proposed PEC sensor for the detection of trypsin in biological samples were in the range from 89%to 102%.In the fifth chapter,a novel and sensitive signal-on of PEC immunosensor was constructed for detecting tumor marker.Alpha-fetoprotein?AFP?was chosen as a model analyte to investigate the prepared procedure and the analytical performance of the exploited sensor.In order to construct the sensor,CdSe QDs were used as photoactive material,biotin conjugated AFP antibody as detecting probe,streptavidin as signal capturing unit,and biotin functionalized APOAA?Bio-APOAA?as amplification unit,which were assembled onto the electrode.The signal sensing strategy was based on in situ enzymatic hydrolysis of Bio-APOAA to release AA as sacrificial electron donor to produce photocurrent.The photocurrent from the immunosensor was monitored as a result of AFP concentration.The constructed sensing platform displayed high selectivity and good sensitivity for detecting AFP.Under optimal conditions,a wide linear range from 0.001 to1000ng/mL and a low detection limit of 0.31pg/mL were obtained.The developed immunosensor is expected to be used to determine AFP and other tumor markers in human plasma in clinical laboratories either for pre-cancer screening or cancer monitoring.The sixth chapter,a PEC sensor for the detection of metal ion was constructed.The ITO/ZnS was used the working electrode and dopamine?DA?as the detection solution.When different amounts of Cu²⁺were added,PDA-Cu?II?was formed based on the interaction between copper?II?and dopamine?DA?,which destroyed the electron transfer between the dopamine and the photogenerated holes,resulting in a decrease of photocurrent.The detection of Cu²⁺was achieved using the reduced photocurrent.The linear range of 1 to 1000 pM and a detection limit of 0.45 pM were obtained.Thus,a novel,simple,selective,sensitive and environmentally friendly PEC sensor for Cu²⁺detection was constructed.The PEC sensor based on the change of electrode surface state can realize the sensitive detection of Cu²⁺.Finally,all the work in this thesis was summarized and our working assumptions for future were proposed.