| Summary: | 碩士 === 國立成功大學 === 化學工程學系 === 107 === SUMMARY
Creatinine, the final product of the metabolism of creatine in mammals that removed from the body by kidneys. It is a reliable indicator for kidney’s function in human’s blood and urine. Biosensors are already well established in modern analytical chemistry. They have become important tools for clinical diagnostics, environmental analysis, production monitoring, drug detection or screening. Synthetic biomimetic receptors like molecularly imprinted polymers (MIPs) have shown to be a potential alternative to biomolecules as recognition element for biosensing.
In this work, sensors based on poly(N-vinylpyrrolidone-co-1-vinylimidazole) for the selective determination of creatinine are fabricated by heated polymerization, We not only discuss the conclusion of creatinine in MIP by FTIR characterization and morphology, but also optimize creatinine calibration and detection by electrochemical impedance spectroscopy (EIS). Finally, the fabricated creatinine sensor was applied to the detection of urine specimens for feasibility test on clinical application.
Keywords: creatinine, molecularly imprinted polymers, electrochemical sensor, electrochemical impedance spectroscopy, urine specimens
Introduction
Creatinine, is the dehydrogenated form of creatine in ATP metabolism. The normal range of serum creatinine is 0.5~1.5 mg/dL; while in urine, the normal range is 39~259 mg/dL for males and 28~217 mg/dL for females, respectively. The value varies with human’s age, lifestyle and muscle mass. The concentration of creatinine is less affected by the variation of dietary, therefore creatinine in blood and urine is a fairly reliable indicator for clinical evaluation of renal function.
Clinical analysis of creatinine is routinely carried out using colorimetric Jaffé reaction and enzymatical reaction. However, Jaffé reaction suffers from the poor selectivity toward numerous metabolites containing carbonyl group found, such as proteins, glucose, bilirubin, and ascorbic acid. Although the introduction of enzymes may improve specificity, enzymes always suffer from instability, high cost and complex procedures for the immobilization.
In recent years, molecularly imprinted polymers (MIPs) have been used to develop the new generation of biological/chemical sensors due to their superiorities such as high adsorption capacity, high specific recognition of selected analyte, low cost and excellent reusability.
In this work, N-vinylpyrrolidone and 1-vinylimidazole as functional monomers were copolymerized in the presence of creatinine by heat polymerization. Based on the formation of hydrogen bonds of these functional monomers and creatinine, the fabricated electrode can be introduced to improve the specific binding affinity of creatinine. By the linearity between impedance change and creatinine concentration, we can apply in urine specimen detection.
MATERIALS AND METHODS
The gold working electrode was prepared by a sputter coater (sputter coater 108 auto, Cressington). Calculation of the amount of creatinine removed is measured by HPLC (SPD-10A; SIL-9A; C-R6A). Incorporation of creatinine in MIP matrix before and after extraction was compared by FTIR. Morphology of the modified electrodes were observed by SEM images. All electrochemical experiments were performed by using (Electrochemical analyzer) AUT85707 and Zive SP1 controlled and acquired by electrochemical signals of the sample solution. Clinical urine specimens were collected from NCKU hospital for detection test by the MIP modified electrodes.
RESULTS AND DISCUSSION
The proportion of creatinine removed from the MIP film was measured by an HPLC and calculated as 80.2%, which showed good imprinting as well as removal efficiency. FTIR analysis for the MIP films before and after template removal, the peaks at 3,250 cm1 which is amide group (NH stretching) disappeared. Meanwhile, the CN group at 1,108 cm1 decreased, which showed creatinine was removed from the polymer.
From the EIS (electrochemical impedance spectroscopy) simulation of modified electrode, the mechanism of creatinine detection can be confirmed. Because of formation of cavities inside the polymeric film, fascilitating the electron transfer between background electrolyte and working electrode, then with the injection of creatinine solution, creatinine can be rebinded to recognition sites in the cavities, blocked the electron transfer and resulted in the increase of impedance of whole electrode.
The as-prepared MIP electrode was examined by electrochemical detection. The gold electrode was modified by allyl mercaptan and imprinted poly(N-vinylpyrrolidone-co-1-vinylimidazole) was synthesized onto the electrode with the optimal ratio (NVP:NVI= 4:1), The sensing background was optimized at pH 7.4 to get the best calibration curve. Linear calibration curve with a slope of 0.024 ·mg1dL was thus obtained with R2= 0.9971. By comparison between MIP and NIP (Non-molecularly imprinted polymer), the imprinting factor was 12, the modified electrode showed the specific binding towards creatinine molecules. Real urine sample test was accomplished by the modified electrode and HPLC, respectively. Comparison to these methods, the result is good enough for clinical detection and it preliminary feasibility in point of care (POC) detection.
The sensing performance of the MIP electrode in this work such as reusability and storage stability was also investigated, discussing the reusability and storage stability of this MIP electrode. After reused for 50 times, the MIP electrode still remains remarkable stability compared to the same electrode prepared in the very beginning.
Conclusion
The extraction process to remove creatinine from the polymer matrix is confirmed from HPLC measurement and FTIR analysis. Both the preparation of MIP electrode and detection of creatinine were optimized. The as-prepared MIP electrode can detect urine creatinine within the range of 10~120 mg/dL versus the electrochemical impedance change with a sensitivity of 0.024 ·mg1dL and a LOD of 5.68 mg/dL. Additionally, the MIP electrode thus prepared can be applied to the determination of creatinine concentration in urine specimens.
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