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Performance Improvement For The Wavelength Modulation Surface Plasmon Resonance Sensor And Its Application

Posted on:2007-05-21Degree:DoctorType:Dissertation
Country:ChinaCandidate:X LiuFull Text:PDF
GTID:1118360185455300Subject:Analytical Chemistry
Abstract/Summary:PDF Full Text Request
The first surface plasmon resonance (SPR) sensor was developed in 1983by Liedberg et al. In recent years, along with rapidly advancing in the moleculelevel research, and SPR sensor's features of real time monitoring the associationor dissociation of biomolecules, no label, high selectivity, great progress hasbeen made in the field of SPR for the determination of antibody-antigeninteractions, peptide/protein-protein interactions, DNA hybridization conditions,bio-compatibility of polymers, biomolecule-cell receptor interactions andDNA/receptor-ligand interactions in these years.SPR sensor is a reliable tool for investigating interaction betweenbiomolecular all along. However, along with advancing rapidly of SPRinstrument and internet applications, a great deal investigation work isperformed in small molecules based on SPR sensor and made great progress,especially in drug. In addition, studying new methods of SPR is mostly forenhancing sensitivity of SPR sensor, except for enlarging application range ofSPR. So, actively exploring various analytical methods for enhancing SPRsensitivity is also a mainly topic. The work includes two aspects for enhancingsensitivity of SPR sensor. On the one hand, the research system is improved.Such as, improving SPR sensor surface structure and changing component ofreagent solution. On the other hand, the SPR instrument is improved includingoptimizing wave-guide element, renovating detection system et al. In the firstchapter, the element theory of SPR sensor is simple introduced. Theconfigurations and principles for wavelength modulation SPR sensors areoutlined and some applications of these sensors presented. The latestdevelopments in application of SPR sensor are reviewed on small moleculesand enhancing sensitivity aspects.The wavelength modulation SPR instrument has been developed fordetermining tetanus toxin. Molecular self-assembling on the surface of thesensor is applied to form the sensing membrane on the gold substrate. Thoilterminal of MPA is easily adsorbed to gold surface, the COO-terminal group isan active group that can bind to antibody. In practice, MPA was firstimmobilized onto the gold film, and then the COO-group was acylated. Then,tetanus antibody was injected into flow cell to monitor its assembling on theactivated biosensor surface. The interaction between tetanus antitoxin andtetanus toxin were studied. The tetanus toxin was determined in theconcentration range of 0.028-0.085Lf/ml. In addition, The method of SPRbiosensor regeneration was investigated (including 0.08 mol/L H3PO4, 0.1mol/L H3PO4, 0.1mol/L HAc, 0.01 mol/L NaOH, 0.1 mol/L NaOH, 0.01 mol/LHCl, 0.1 mol/L HCl, 0.03 mol/L HNO3 and 0.3 mol/L citrate solution). Thecitrate buffer (0.3 mol/L) solution is chosen as regeneration solution.Methods of enhancing SPR sensitivity were investigated by determiningcomplement C4 and transferrin. The Complement C4 antibody wasimmobilized on the SPR sensor surface by using amine coupling method. Then,Complement C4 was determined by direct detection mode, sandwich detectionmode, colloidal gold enhanced mode and colloidal gold enhanced sandwichmode. The colloidal gold was used as label by antigen (antibody) conjugating toenhance sensitivity of SPR biosensor. The complement C4 was determined inthe concentration range of 2-20μg/mL , 0.2-20μg/mL , 0.1-20μg/mL ,0.05-5μg/mL, respectively. The rational explanation was given for everyexperiment phenomenon. The method of SPR biosensor regeneration wasinvestigated. The 0.1 mol/L NaOH solution is chosen as regeneration solution.The transferrin was determined using direct assay, double-linker(MEA-SPA) assay and colloidal Au amplified assay. The 2-Mercaptoethylamine(MEA) was immobilized on the biosensor surface with traditional aminecoupling method. The interaction between colloidal Au nanoparticles and MEAwas investigated. The anti-transferrin was immobilized on the biosensor surfaceprepared with staphylococcal protein A (SPA). The interaction of the antibodyand antigen was monitored in real time. The colloidal gold was not as label butas active components to enhance sensitivity of SPR biosensor. The goodresponse was obtained in the concentration range 1-20μg /mL, 0.1-20μg /mLand 0.05-20μg /mL for directly immune assay, double-linker assay and colloidalAu-amplified assay. The rational explanation was given for every experimentphenomenon. The 0.1 mol/L NaOH solution is chosen as regeneration solution.The interaction for seven kinds of the antibiotic drug (penicillin V K,cefoperazone, cefotaximum natricum, oxcillin, amoxicillin, enxacin, andnorfloxacin,) and serum albumin (HSA and BSA) was studied. The serumalbumin was immobilized on the SPR sensor surface by using amine couplingmethod, and then drug solution of different concentration was flowed cross SPRsensor surface in turn. The kinetic constant, thermodynamic constant andbinding percentages were obtained, respectively. These results indicated thatseven kinds of the antibiotic drug show differences in their affinities towardsthe serum albumin. Binding ability of drugs to HSA should be as follows,penicillin V K> cefoperazone > cefotaximum natricum > oxacillin > amoxicillin> enoxacin > norfloxacin. For BSA, this ability follows the pattern: penicillin VK > cefoperazone > cefotaximum natricum > oxacillin > enoxacin > amoxicillin> norfloxacin. The percentage data range of drugs binding to HSA is between90.00% -40.82%. However for BSA, the binding percentage data range isbetween 85.92% -50.37%. The binding percentages determined with SPRsensor are compared with these obtained by other methods. In addition, thereproducibility and stability of the serum albumin immobilized on the sensorsurface were studied under the experiment condition. The method of SPRbiosensor regeneration was investigated. The NaOH (0.01mol/L) solution waschosen as regeneration solution. Under the experiment condition, the sensor hasa good sensitivity, repeatability, and reversibility. Thoil terminal of MPA iseasily adsorbed to gold surface, the COO-terminal group is an active group thatcan bind to protein. The typical process of amine coupling method is that MPAwas first immobilized onto the gold film, and then the COO-group was acylated.Serum albumin is assembled on the surface in correct direction. Now, the SPRsensor is ready to detect interaction between the drug and serum albumin. Theinteraction for the anthraquinone (including emodin, chrysophanol andphyscion) and human serum albumin was studied using the same method. Thekinetic constant, thermodynamic constant, and binding percentages wereobtained, respectively. Binding ability of drugs to HSA should be as follows,emodin > physcion > chrysophanol. The rational analysis is given for boundmechanism of anthraquinone and human serum albumin. The interaction for theginsenosides (including Rb1, Rb2, Rb3, Rc, Rd, Re) and serum albumin (HSAand BSA) was studied with a known concentration of the ginsenosides. Thekinetic constant, thermodynamic constant, and binding percentages wereobtained, respectively. The binding ability of ginsenosides to HSA should be asfollows: Re> Rc > Rb2 > Rb1 > Rd > Rb3. For BSA, this ability follows thepattern: Rb3 > Rb1= Rb2 > Re > Rd > Rc >. The percentage data range ofginsenosides binding to HSA is between 97.10% and 88.33%. However forBSA, the binding percentage data range is between 97.10% and 68.47%. Thesame method was used in order to study the interaction β-CD and serumalbumin (HSA and BSA). The binding ability of HSA, BSA toβ-CD should beas follows, HSA > BSA.The interaction for the indomethacin, ketoprofen and cyclooxygenase werestudied. Cyclooxygenase (drug target) was immobilized on the SPR sensorsurface by using amine coupling method, the drugs were injected acrossimmobilized cyclooxygenase surface. The interaction between theindomethacin, ketoprofen and cyclooxygenase were monitored in real time. Thekinetic constant, thermodynamic constant were obtained. The binding ability oftwo kinds of drugs to cyclooxygenase should be as follows: ketoprofen >indomethacin. Under selected experimental conditions, the sensor has a goodsensitivity, repeatability and reversibility.
Keywords/Search Tags:Performance
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