A STUDY OF DETECTION OF ENANTIOMERS AND CHEMICAL ANALOGUES BY MOLECULAR IMPRINTED POLYMER COATED QUARTZ CRYSTAL MICROBALANCE TECHNIQUE BY LIU XIAO NATIONAL UNIVERSITY OF SINGAPORE 2006 A STUDY OF DETECTION OF ENANTIOMERS AND CHEMICAL ANALOGUES BY MOLECULAR IMPRINTED POLYMER COATED QUARTZ CRYSTAL MICROBALANCE TECHNIQUE BY LIU XIAO (B. Sc., Jilin University) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2006 Acknowledgement I would like to express my deepest gratitude to my supervisors, Professor Hardy Chan for his invaluable guidance of this work. His encouragement, support and friendly personalities were helpful and precious to the success of this research work and will always remain in my mind. I deeply appreciate the kind assistance from, Dr. Zhang Weiguang, and Dr. Xiao Changyou; Particularly I will appreciate to the great help from Dr. Liu Feng for his stimulating discussion and useful suggestions. I also profoundly give my sincere thanks to my colleagues and friends who studied and worked in the same research laboratory, Haibing Xia, Xuedong Zhou, Daming Chen, Weihua Tang, Lee Teck Chia, Sheng Zhang, Huijuan Che, particularly Mdm Frances for her generous help and priceless discussion during the research. Last but not least, I also want to thank National University of Singapore for awarding the research scholarship and for providing the facilities to carry out the research work reported herein. Table of Contents Acknowledgment Summary………………………………………………………………v List of Tables………………………………………………………….vi List of Figures……………………………………………………… .vii List of Abbreviations Symbols……………………………………….ix 1. Chapter one: Introduction…………………………………………1 1.1 Principle, function of binding groups, and general application of molecular imprinted polymers (MIPs)…… 1.1.1 Principle of the Molecular Imprinted Polymers……………….1 1.1.2 Function of the Binding groups…………………………………5 1.1.2.1 Noncovalent interactions……………………………….6 1.1.2.2 Covalent interactions………………………………… .7 1.1.3 Applications of Molecular Imprinted Polymers………………10 1.1.3.1 Liquid chromatography……………………………….10 1.1.3.2 Solid phase extraction………………………………….11 1.1.3.3 Binding assays………………………………………….11 1.1.3.4 Sensors………………………………………………….12 1.1.3.5 Catalysis……………………………………………… .13 1.2 Principle and applications of Quartz Crystal Microbalance (QCM)……………………………………………………… 14 1.2.1 Principle………………………………………………………….15 i 1.2.2 Mass Sensitivity………………………………………………… 17 1.2.3 Applications of Quartz Crystal Microbalance…………………18 1.2.3.1 Gas phase detection…………………………………….19 1.2.3.2 Immunosensors…………………………………………19 1.2.3.3 DNA-based sensors…………………………………… 20 1.2.3.4 Detection of Cells……………………………………… 21 1.3 Combination of Molecular Imprinted Polymers and Quartz Crystal Microbalance………………….……………………… 22 1.4 Research projective and scope……………………………… 26 2. Chapter two: Experimental……………………………………… .28 2.1 Materials………………………………………………………29 2.2 Synthesis of functional monomer………………………… .29 2.3 Procedures for Modification and Characterization of the Surface of QCM…………………………………………… .30 2.3.1 Self-assembly of thiol groups onto the gold electrode………………30 2.3.2 File preparation and polymerization……………………………… .31 2.3.2.1 MIPs for L-try……………………………………………….31 2.3.2.2 MIPs for SMZ……………………………………………… 32 2.3.3 Instrumentation……………………………………………………….33 2.3.3.1 QCM systems……………………………………………… .33 2.3.3.2 Atomic force microscopy (AFM)……………………………34 2.3.3.3 Scanning Electron Microscope (SEM)………………… …34 ii 2.3.3.4 UV spectroscopy…………………………………………… 35 2.3.3.5 Electrochemical measurement…………………………… .35 2.3.3.6 Measurement of the thickness of the MIPs films………….36 3. Chapter three: Result and Discussion…………………………… 37 3.1 Effect of enantioselective molecular imprinting polymer coated QCM for the recognition of L- tryptophan…………………38 3.1.1 Characterization of the thiol monolayer and MIP film-modified gold electrode………………………………………………………38 3.1.2 Response time and reproducibility of the QCM sensor………… .41 3.1.3 Sorption characteristics of the enantioselective sensor……………42 3.1.4 Influence of the cross-linking monomer concentration on the sensor performance……………………………………………………… 45 3.1.5 Application of the enantioselective sensor…………………………47 3.2 Study of the detection of molecularly imprinted polymer coated QCM for sulfamethazine (SMZ) and its chemical analogue with novel methodology……………………………………….49 3.2.1 Choice of functional monomers…………………………………… .49 3.2.2 Effect of PVAc on the adsorption and thickness of MIPs………….53 3.2.3 Sorption characterization of SMZ-sensor………………………… .57 3.2.4 Characterization of polymer films with AFM and SEM………… .58 iii 4. Chapter four: Conclusion and further work…………………… .61 4.1 Conclusion…………………………………………………62 4.2 Further work………………………………………………63 References………………………………………………………………65 iv Summary Molecular imprinted polymers (MIPs) and Quartz Crystal Microbalance (QCM) techniques have been widely used in the detection and separation of chemical compounds. In this work, a new quartz-crystal microbalance (QCM) sensor that provides enantioselectivity to tryptophan enantiomers, with a high selectivity and sensitivity, was fabricated by the use of the molecularly imprinted polymers (MIPs) as the artificial biomimetic recognition material. The preparation of the thin permeable film coatings on QCM surface is described as well as the results and discussion on the sensitivity and selectivity of the coatings to tryptophan enatiomers under different conditions. The influence of the cross-linking agent concentration on the sensitivity and selectivity of the fabricated polymer films was investigated and optimized. The combination technique of molecular imprinted polymers and QCM was also applied in discriminating chemical analogues, sulfamethazine (SMZ) and sulfamethoxazole (SMO). Some improvements were made to obtain the better selectivity of the MIPs by incorporating poly (vinyl acetate) (PVAc) and low-volatility solvent diethylene glycol dimethyl ether to facilitate better adsorption by the formation of a more porous and open structure of the polymer. All of the results show that good reproducibility, sensitivity and selectivity can be achieved. And the thickness of the films is controllable. AFM and SEM are used to characterize the morphology of the polymer film coated on QCM. v List of Tables Table 1.1 Covalent interactions during the imprinting process. Table 1.2 Functional monomer and template utilized in the combination technique Table 2.1 Synthesis condition of SMZ MIPs Table 3.1 Sensitivity and enantioselectivity of the QCM Sensors Table 3.2 UV results of interactions between SMZ and functional monomers Table 3.3 Amount of PVAc and water absorbed vi List of Figures Figure 1.1 Schematic representation of interaction in imprinted polymers Figure 1.2 Schematic representation of process of adsorption and desorption in MIP Figure 1.3 Schematic of a typical piezoelectric crystal. Figure 1.4 AT-cut of a quartz crystal Figure 1.5 Schematic representation of a MIP-coated QCM sensor. Figure 3.1 AFM images gold electrode surface Figure 3.2 Typical cyclic voltammogram of QCM electrode Figure 3.3 Frequency change of the MIP-coated QCM sensor Figure 3.4 Sorption characteristics of the enantioselectivity of QCM sensor Figure 3.5 Scatchard plot for adsorption curve Figure 3.6 Effect of the crosslinker on QCM sensor Figure 3.7 Frequency change of QCM sensor for different enantionmeric composition Figure 3.8 Structures of different functional monomers and analytes Figure 3.9 UV spectra for SMZ and functional monomers Figure 3.10 Proposal model for SMZ and AAM interaction Figrue 3.11 The adsorption and selectivity of MIP-coated QCM Figure 3.12 frequency change of the QCM as a function of concentration of PVAc Figure 3.13 Thickness of MIP films Figure 3.14 Sorption characteristics of the QCM sensor to the SMZ and SMO. Figure 3.15 AFM images for SMZ-IMP Figure 3.16 SEM images for SMZ-IMP vii 3.2.4 Characterization of polymer films with AFM and SEM In order to understand the influence of PVAc on MIPs morphology, we use AFM and SEM to study the morphologies of polymer films. Figure 3.15 and 3.16 show the dramatically different images between the films with and without PVAc. In fig. 3.15a and 3.16a, there is hardly any porosity and cavity on polymer film, which is very crucial in the following detection and adsorption testing. However, in fig. 3.15b and 3.16b, excellent and obvious cavities can been seen, which is caused by the adding of PVAc. PVAc gives a brilliant facility to the SMZ-MIPs to obtain good adsorption and discrimination. The crack in fig. 3.16a is caused by the shrinking of the imprinted polymers, after the solvent involved evaporated. However we not observed them in fig. 3.15b with PVAc. It shows that the addition of PVAc probably is able to improve the mechanical properties. a b Figure 3.15 AFM images for SMZ-IMP a) SMZ-IMP without PVAc b) SMZ-IMP with PVAc 59 a) b) Figure 3.16 SEM images for SMZ-IMP a) SMZ-IMP without PVAc b) SMZ-IMP with PVAc 60 Chapter Conclusion and further work 61 4.1 Conclusion In this work we have described a quartz crystal biomimetic sensor for the detection of enantiomeric composition using artificial recognition films prepared via molecular imprinting technology. The influence of the cross-linking agent concentration on the sensitivity and selectivity of the fabricated polymer films was investigated and optimized. The enantioselectivity of the MIPs coating has also been investigated for the l- and d-tryptophan enantiomers giving an enantiomeric selectivity coefficient of 6.4. Four different bio-mimetic polymers for l-tryptophan have been synthesized by molecular imprinting. Through comparison, the MIP2 sensor using TRIM/AM (molar ratio) value of 2.21:1 as the cross-linking monomer concentration shows the highest sensitivity and enantioselectivity for the analyte. The composition of l- and d-tryptophan enantiomer mixtures can be determined by measuring the frequency shifts of the sample with reference to the calibration plot. The detection limit of the analyte is 8.8µM. In another system, the effect of porogen generator PVAc and low-volatility solvent DEGDM on the improvement to the reproducibility and controllability was studied using SMZ as the template molecule. On this work we can see that the thickness of the polymer films coated onto the QCM surface could be controlled accurately through adjusting the content of PVAc. It was also found that the adsorption of SMZ-MIPs is proportional to the percentage of the PVAc added each time. Good 62 reproducibility as well as discrimination was obtained. Meanwhile, we also studied the influence of the morphology of polymer films containing PVAc and DEGDM. The AFM and SEM images showed the morphologies of films with and without PVAc are dramatically different. The porosities generated by PVAc was clearly observable. 4.2 Further work The most important and promising applications of this kind of MIP-QCM sensors lie in their ability to discriminate one of the enantiomors from their racemates and to study interactions in biological systems such as interactions between antibody and antigen, DNA and its complementary oligonucleotide. First of all, we can select a certain chiral compound found in drugs to make the template in MIPs. It is expected that this PVAc/DEGDM/MIPs system should be more suitable for chiral discrimination, because of their good reproducibility, controllability and high porosities. Secondly, DNA or antibody could be introduced into imprinted polymer films as a functional component to detect their complementary oligonucleotide and antigen. However, some necessary modification should be made in order to connect these DNA and antibodies to the polymer matrix. Carbon double bond can be introduced into DNA and antibody, so that by using the radical polymerization functional components will be connected firmly in the polymer matrix. Finally, we also can study the biological systems with the method reported by Cooper in 2003 63 [138]. The essence of their idea is to keep the resonant frequency constant and change the voltage applied to adjust the amplitude of the oscillation of the crystal. With the increase of oscillation, the interaction between analytes and substrate will be decreased. At last the bond will be broken and the signal is obtained. By analyzing these signals, the relative bond strengths and intensity of the bond could be studied. 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Liao HP, Zhang ZH, Li H, Nie LH, Yao SZ, Electrochimica Acta 49 (2004) 4101-4107 74 [...]... viscoelastic behavior of the mass deposited upon the metal electrode surface of the quartz crystal 1.2.1 Principle of QCM A piezoelectric quartz crystal resonator is a precisely cut slab from a natural or synthetic crystal of quartz Quartz crystal in its perfect natural form can be seen in Fig.1.3 a) A quartz crystal microbalance (QCM) consists of a thin quartz disk with electrodes plated on it as can be seen... non-biological alternatives to antibodies in a competitive radiolabelled molecularly imprinted sorbent assay, MIA [53] The assay 11 is analogous to that of a competitive immunoassay, or limited reagent assay Sample, containing analyte, and a fixed concentration of marker, a labelled derivative of the analyte, are incubated with a limited number of antibody binding sites or imprints Analyte and marker compete for... thickness of the device is a multiple of a half wavelength of the acoustic wave The quartz crystal may provide a large variety of different resonator types depending on the cut angle with respect to the crystal lattice AT-cut crystals, which are predominately used for QCM devices, operate in the TSM and are Figure 1.4 AT-cut of a quartz crystal prepared by slicing a quartz wafer with an angle of 35¼°... chromatographic materials for analytical purposes, noncovalent interactions are usually preferred, as the materials are more readily obtainable and an excess of binding groups apparently does not have a detrimental effect on the separations For the constructions of catalysts, the orientations of the binding groups and catalytically active groups in the cavity are of greater significance, so that covalent... b) a) b) Figure 1.3 a) The assignment of axes of quartz crystal b) Schematic of a typical piezoelectric crystal 15 The application of an external electrical potential to a piezoelectric material produces internal mechanical stress As the QCM is piezoelectric, an oscillating electric field applied across the device induces an acoustic wave that propagates through the crystal and meets minimum impedance... amplitude of the shear vibration depends on energy dissipation and therefore on the kind of load on the quartz The radial distribution of the shear amplitude can be described empirically by a Gaussian function 1.2.3 Applications of Quartz Crystal Microbalance QCMs have traditionally been used in vacuum deposition systems and have found a plethora of other applications: thin film deposition control; estimation... environmental pollutants [89, 90] and chromatography detectors [91] The first gas phase immunosensor was described by Guilbault and Ngeh-Ngwainbi [92], using parathion antibodies coated on a PZ crystal surface 1.2.3.2 Immunosensors The high specificity of antigen-antibody reactions and the ability to generate antibodies against a variety of biological and nonbiological substances opened up a means to develop... MIPs, a cavity has to first be made with a defined shape corresponding to the shape of the substrate or, even better, to the shape of the transition state of the reaction At the same time, functional groups are incorporated that act as binding sites, coenzyme analogs, or catalytic sites within the cavity and in a defined stereochemical manner [67] These artificial polymeric catalysts are more durable and. .. 1999, the preparation and characterization of electrosynthesized poly (o-phenylenediamine) (PPD) imprinted by glucose is reported as the first case of an electrosynthesized polymer molecularly imprinted by a neutral template by Cosimino et al [111] In this work, good adsorption was obtained and the approach offers an easy way to the preparation (and, in persperctive, to the miniaturization) of biomeimetic... to separate the enantiomers Typical values for the enantioseparation factor a are between 1.5 and 5, although in some cases much higher values have been obtained If the molecule of interest contains more than two chiral centers, as is the case with carbohydrates, these properties of molecularly imprinted materials become even more relevant; in a study in which polymers were imprinted against a glucose . A STUDY OF DETECTION OF ENANTIOMERS AND CHEMICAL ANALOGUES BY MOLECULAR IMPRINTED POLYMER COATED QUARTZ CRYSTAL MICROBALANCE TECHNIQUE BY LIU XIAO NATIONAL UNIVERSITY. UNIVERSITY OF SINGAPORE 2006 A STUDY OF DETECTION OF ENANTIOMERS AND CHEMICAL ANALOGUES BY MOLECULAR IMPRINTED POLYMER COATED QUARTZ CRYSTAL MICROBALANCE TECHNIQUE BY LIU XIAO (B Summary Molecular imprinted polymers (MIPs) and Quartz Crystal Microbalance (QCM) techniques have been widely used in the detection and separation of chemical compounds. In this work, a new