Investigation On Molecular Imprinting-Chemiluminescence Analysis

Chemiluminescence (CL) analysis has been widely used in many fields due to its attracting features including low detection limit, wide linear dynamic range and inexpensive instruments. However, the relatively poor selectivity of the CL method itself limits its direct application to the analysis of analyte in complicated sample. Great efforts were made to overcome this shortcoming and the most common used way is that the CL method was combined with some separation techniques such as high performance liquid chromatography, gas chromatography and capillary electrophoresis, but these methods required relatively complicated and expensive instruments. So, it is one of the most urgent and significant works to improve selectivity of the CL method.Molecular imprinting is a technique used for preparing polymers with synthetic recognition sites having a predetermined selectivity for the target molecule. The excellent recognition ability of molecular imprinted polymer (MIP) to the target molecule is comparable to enzyme-substrate, antibody-antigen and receptor-hormone natural molecular recognition system. If a mini-column made of MIP was connected into the CL flow system, utilizing its fine recognition and capture ability to target molecule and separate the target molecule from other coexistent substances, the selectivity of CL method would be improved greatly. Many components in complicated samples can be determined directly by this way. It is the purpose that we establish the molecular imprinting-chemiluminescence analysis.In the first part of this thesis, the basic principle of molecular imprinting technique,the preparation and characterization of MIP and the applications and development tendency of molecular imprinting technique were summarized. It was focus on reviewing the CL mechanisms of the system including manganese with different valences, and the analytical applications of the system.In the second part of this thesis, the research work focused on the application of molecular imprinting technique to the CL systems using strong oxidants (such as MnCV, Ce(IV)) as CL reagent in CL analysis. However, molecular imprinted polymer contained many unsaturated bonds, these strong oxidants could destroy the structure of MIP, and the recognition and capture ability of MIP to target molecule will decrease, even disappear. In our research, this problem has been solved by using some reductive substances as protective reagent of MIP. Using MIP as molecular recognition material and MnO₄^- or Ce(IV) CL system as detection system, some new molecular imprinting-chemiluminesence methods were established. The application of MIP to CL analysis could improve the selectivity of CL method greatly, and make it possible for CL method to become a selective method for the determination of analyte in complicated samples. The adsorption selectivity of the MIP to the target molecule in the aqueous solution was evaluated. The results show that the MIP column exhibits good separation effect for the target molecule and coexisted substances and the MIP exhibits good selective adsorption to the target molecule.In the third part of this thesis, it was found that soluble manganese(IV) could react with more than 30 organic and inorganic compounds to produce strong CL signal in the presence of formaldehyde. The reaction conditions were optimized and a new flow injection CL method for the determination of eight drugs was established. Meanwhile, the CL reaction mechanism was also discussed briefly. Using MIP as molecular recognition material and soluble Mn(IV)-formaldehyde CL system as detection system, some new molecular imprinting-chemiluminesence methods were established. In CL reaction, utilizing highly selectivity and capture ability of MIP to target molecule, the selectivity of the methods was improved greatly. The methods were applied to the determination of these species in biological samples directly.In the fourth part of this thesis, a new CL reaction was observed when codeine, Ni2+, Mg2+, Cd2+ or Zn2+ was injected into the reaction mixture after the finish of CL reaction of alkaline luminol and potassium ferricyanide. A new CL reaction was observed when Ca2+, Sr2+, Ba2+ or organic compound was injected into the reaction mixture after the finish of CL reaction of alkaline potassium ferricyanide and calcein. The experimental conditions of the CL reactions were optimized and the feasibility using the reactions to analyze these species was evaluated.The major contents in this thesis are described as follows:1. Molecular imprinting-chemiluminescence determination of norfloxacin based on Ce(IV)-Na2SC>3-norfloxacin reactionA selective molecular imprinting-chemiluminescence method was developed for the determination of norfloxacin, using norfloxacin-imprinted polymer as recognition material and Ce(IV)-sodium sulfite-norfloxacin chemiluminescence system. The chemiluminescence intensity was responded linearly to the concentration of norfloxacin within 1.0xl0⁷¹.0xl0⁵ mol/L with a detection limit of 3*10⁸ mol/L. The relative standard deviation for 5.0x10'7 mol/L norfloxacin was 2.4 % (n=7). This method was applied to the determination of norfloxacin in urine samples and satisfactory results were obtained.2. Determination of morphine by molecular imprinting-chemiluminescence method based on potassium permanganate chemiluminescence systemThe morphine-imprinted polymer was prepared using methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker in the presence of template molecule of morphine. Using the imprinted polymer as the recognition material, sodium sulfite as the protective agent and potassium permanganate -morphine chemiluminescence system as the detection system, a molecular imprinting-chemiluminescence method for determination of morphine was established. The linear response range of this method was 5.0xl0"9¹.0xl0"6g/rnL(r=0.9981) and the detection limit was 2xlO⁹g/mL. The relative standard deviation for 1.0xl0⁷g/mL morphine solution was 2.8% (n=9). This method was applied to the determination of morphine inthe urine of the druggers with satisfactory results.3. Molecular imprinting-chemiluminescence determination of phenacetin based on potassium permanganate-formaldehyde-phenacetin reactionA molecular imprinting-chemiluminescence method for the determination of phenacetin was established by using phenacetin-imprinted polymer as recognition material and KMnCVphenacetin-formaldehyde reaction as detection system. It was found that a weak chemiluminescence was produced from the reaction of KMnC>4 with phenacetin. The chemiluminescence signal was greatly increased by formaldehyde. Phenacetin-imprinted polymer was synthesized using methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker. The chemiluminescence intensity was responded linearly to the concentration of phenacetin within 5.0xl0"7⁵.0xl0⁵ g/mL with a detection limit of 2xlO"7 g/mL. The relative standard deviation for l.OxlO"6 g/mL of phenacetin was 2.8% (n=ll). The method has been applied to the determination of phenacetin in compound aminopyrine tablets and in urine samples, the results were in good agreement with those obtained by pharmacopoeia method and the recovery results were satisfactory. Meanwhile, the chemiluminescence reaction mechanism was also discussed briefly. 4. Molecular imprinting-chemiluminescence sensor for the determination ofbrucineThe brucine-imprinted polymer was synthesized using methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker in the presence of template molecule of brucine and the binding characteristic of the imprinted polymer to brucine was evaluated by equilibrium binding experiments. Using the imprinted polymer as recognition material, a new chemiluminescence sensor for the determination of brucine was developed based on the chemiluminescence reaction of brucine with potassium permanganate in acidic medium. The sensor displayed high selectivity and high sensitivity. The linear response range of the sensor was from 5.0*10"9to l.OxlO"6 g/mL(r=0.9981) and the detection limit was 2xlO"9 g/mL. The relative standard deviation for the determination of l.OxlO'7 g/mL brucine solution was2.6% (n=9). The sensor was applied to the determination of brucine in urine samples with satisfactory results.5. Molecular imprinting-chemiluminescence determination of trimethoprim based on potassium permanganate-sodium hyposulfite-trimethoprim chemilummescence systemThe trimethoprim-imprinted polymer was synthesized and its adsorption selectivity to trimethoprim in aqueous solution was evaluated. A new molecular imprinting-chemiluminescence method for the determination of trimethoprim was developed. The method was based on the chemiluminescence reaction of potassium permanganate-sodium hyposulfite-trimethoprim using trimethoprim-imprinted polymer as the recognition material. The chemiluminescence intensity responded linearly to the concentration of trimethoprim within 5.0xl0"8⁵.0xl0"6 g/mL range (r=0.9983) with a detection limit of 2x10"8 g/mL. The relative standard deviation for the determination of l.OxlO"7 g/mL trimethoprim solution was 4.8% (n=9). The method was applied to the determination of trimethoprim in pharmaceutical preparations and body fluids and satisfactory results were obtained.6. Chemiluminescence reaction with soluble manganese(IV) as the oxidantIt was found that soluble manganese(IV) could react with more than 30 organic and inorganic compounds to produce strong chemiluminescence signal in the presence of formaldehyde. The reaction conditions were optimized and a new flow injection chemiluminescence method for the determination of four cephalosporin drugs, namely ceftriaxone sodium, cefazolin sodium, cefradine and cefoperazone sodium, was established. The method was applied to the determination of cephalosporins in pharmaceutical preparations. Meanwhile, the chemiluminescence reaction mechanism was also discussed briefly.7. Chemiluminescence determination of trace ascorbic acid with soluble manganese(IV)-formaldehyde systemA novel chemiluminescence method to determine ascorbic acid with flow injection technique, based on formaldehyde enhancing the chemiluminescence from theoxidation of ascorbic acid by soluble Mn(IV) has been developed. Under optimum conditions, the linear dynamic range for the determination of ascorbic acid was from 6.0x10"8 to 2.0x10"5mol/L, and the detection limit was 2x10"8 mol/L. The relative standard deviation for the determination of l.OxlO"6 mol/L ascorbic acid was 2.3% (n=l l).The method has been applied to the determination of ascorbic acid in tablets and injections and the results were in good agreement with those obtained by pharmacopoeia method. Meanwhile, the chemiluminescence reaction mechanism was also discussed briefly. 8. Flow injection-chemilumnescence determination of sulfa drugsBased on formaldehyde enhancing the chemiluminescence from the oxidation of sulfa drugs by soluble manganese(IV), a new chemiluminescence method to determine sulfa drugs such as sulfamethoxazole, sulfadiazine and sulfaguannidine with flow injection technique was developed. Under optimum conditions, the linear ranges for the determination of sulfamethoxazole, sulfadiazine and sulfaguannidine were 6.0x10"8 1.0xl0"5g/mL, 1.0xl0"7⁸.0xl0"6g/mL and 4.0x10"8⁸.0xl0"6g/mL with the limits of detection of 2xlO'8 g/mL, 3xl0"8 g/mL and 2xlO*8 g/mL, respectively. The relative standard deviations for the determination of 6.0x10"6 g/mL sulfamethoxazole, 4.0x10*6 g/mL sulfadiazine and 4.0xl0"6g/mL sulfaguannidine were 1.1%, 1.2% and 2.2% (n=ll), respectively. The method has been applied to the determination of sulfamethoazole in compound sulfamethoxazole tablets and the results were in good agreement with those obtained by pharmacopoeia method. The preliminary mechanism of the reactions was also investigated.9. Molecular iniprinting-chemiluminescence determination of analgin with soluble manganese(IV)-formaldehyde systemChemiluminescence reaction of manganese(IV)-formaldehyde-analgin was proposed. Molecular imprinted polymer of analgin was prepared using methacrylic acid as functional monomer and ethylene glycol dimethacrylate as cross-linker. The binding characteristic of the analgin-imprinted polymer was evaluated by Scatchard analysis. A new chemiluminescence method for the determination of analgin was proposed by thecombination of analgin-imprinted polymer and manganese(IV)-formaldehyde-analgin chemiluminescence system. The linear range is 1.0><10"7¹.0xl0"5mol/L, the detection limit is 4xlO'8mol/L, the relative standard deviation is 2.4% (1.0xlO'6mol/L analgin, n=7).The method was applied to the determination of analgin in human urine samples directly.10. Determination of indomethacin by molecular imprinting-chemiluminescence method based on manganese(IV)-formaldehyde chemiluminescence systemA soluble Mn(IV)-formaldehyde-indomethacin chemiluminescence system was found. Molecular imprinted polymer of indomethacin was prepared and the binding characteristic of the indomethacin-imprinted polymer was evaluated by Scatchard analysis. Using a synthesized indomethacin-imprinted polymer as recognition material and soluble Mn(IV)-formaldehyde-indomethacin as detection system, a new molecular imprinting-chemiluminesence method for the determination of indomethacin was established. The response range of this method was between l.OxlO'7 and 1.0xl0"5g/mL with a linear correlation coefficient of 0.9940. The detection limit was 4xlO'8g/mL. The relative standard deviation for 5.0x10"7g/mL of indomethacin solution was 3.1% (n=7). The method was applied to the determination of indomethacin in human urine with satisfactory results.11. Molecular imprinting-chemiluminescence determination of tamoxifen based on manganese(IV)-formaIdehyde-tamoxifen chemiluminescence systemIt was found the reaction between soluble Mn(IV) and tamoxifen can produce chemiluminescence and formaldehyde can enhance this chemiluminescence reaction. A tamoxifen-imprinted polymer was synthesized and its adsorption selectivity to tamoxifen in aqueous solution was evaluated. Using a synthesized tamoxifen-imprinted polymer as recognition material and soluble Mn(IV)-formaldehyde-tamoxifen chemiluminesence system as detection system, a new molecular imprinting-chemiluminesence method for the determination of tamoxifen was established. The response range of this method was between l.OxlO"7 and 6.0x10"6g/mL with a linear correlation coefficient of 0.9970. The detection limit was 4x10" g/mL. The relativestandard deviation for 5.0xl0"7g/mL of tamoxifen solution was 4.1% (n=9). The method was applied to the determination of tamoxifen in mammary cancer sufferer's urine samples directly.12. Investigation on the post chemiluminescence behavior of codeine in luminol-potassium ferricyanide reactionA new chemiluminescence reaction was initiated when codeine was injected into the reaction mixture of alkaline luminol with potassium ferricyanide. The experimental conditions of the chemiluminescence reactions were optimized. A new chemiluminescence method for the determination of codeine with flow injection technique was developed. The detection limit is 3><10" g/mL, the relative standard deviation is 1.9% (l.OxlO"6 g/mL codeine, n=ll), the liner range is 8.0xl0"8¹.0xl0'5 g/mL. The method has been successfully applied to the determination of codeine in codeine tablets and the results were in good agreement with those obtained by pharmacopoeia method.13. Post-chemiluminescence behaviors of Ni2+, Mg2+, Cd2+ and Zn2+ in potassium ferricyanide-luminol reactionA new chemiluminescence (CL) reaction was observed when Ni2+, Mg2+, Cd2+ or Zn2+ was injected into the reaction mixture after the finish of CL reaction of alkaline luminol and potassium ferricyanide. This reaction is described as a post-chemiluminescence (PCL) reaction. The possible mechanism for PCL reaction was proposed through the studies of the CL kinetic characteristics, the CL spectra and other experiments. The experimental conditions of the CL reactions were optimized and the feasibility using the reaction to analyze these metal ions was evaluated.14. Post-chemiluminescence behaviors of Ca2+, Sr2+, Ba2+, amidopyrine, vitamin Bi, isoniazid, promethazine hydrochloride, thioridazine hydrochloride, phenol and phloroglucinol in calcein-potassium ferricyanide reactionA new chemiluminescence (CL) reaction was observed when Ca2+, Sr2+, Ba2+, amidopyrine, vitamin Bi, isoniazid, promethazine hydrochloride, thioridazine hydrochloride, phenol and phloroglucinol was injected into the reaction mixture after...