Research On New Chromatography/Spectrum Analytical Methods For Detecting Monoamine Neurotransmitters And Uric Acid

Monoamine neurotransmitters including catecholamines(norepinephrine(NE), epinephrine(EP) and dopamine(DA), etc.) and indoleamines(5-hydroxytryptamine(5-HT), etc.) play a vital function in evaluating the activity of the central nervous systems, explaining the disease mechanism and supporting the diagnosis of many diseases. Abnormal levels of monoamine neurotransmitters in brain could bring about many diseases. For example, the deficiency of DA concentrations in brain may lead to Parkinson’s disease and schizophrenia. 5-HT is an indolamine that makes a significant contribution to brain functions such as sleep, thermoregulation, and food intake, as well as in psychopathological states such as depression and anxiety. Hence, it is significant to develop a simple, rapid, and sensitive method to determine monoamine neurotransmitters due to their important roles in clinical diagnosis and medical treatment. Unfortunately, because of the extremely low concentrations in biological specimens and easy oxidations of the catechol groups of monoamine neurotransmitters, the sensitive detection of these compounds remains a significant challenge.Uric acid is the excreted end product of purine catabolism with the normal level of 0.13-0.46 m M in serum. Hyperuricemia is related to the variations of uric acid concentration in serum and urine and causes several diseases like arthritis, cardiovascular and neurological diseases. Therefore, the frequent monitoring and tight control of uric acid in serum and urine are of great importance for preventing from hyperuricemia and reducing the associated complications. Analytical methods including chemiluminescence, fluorescence, electrochemical, high performance liquid chromatography and capillary electrophoresis have been reported for the detection of uric acid. Among them, some methods lacked analytical selectivity, while others have the disadvantages of time-consuming. For instance, the coexisted ascorbic acid in biological sample could affect the specific determination of uric acid because there had an overlapping oxidation potential on the solid electrodes. Hence, the development of a selective and sensitive method for the analysis of uric acid is very important in clinic diagnosis and therapy of hyperuricemia.Chemiluminescence as a analysis method is developed rapidly in recent years, and the analysis method has high sensitivity and simple equipment. Especially the metal nanoparticle-involved chemiluminescence analysis developed quickly in recent years, which has improve sensitivity in a great extent, and it also become applied to detection the trace substances in organism as a kind of important tool. In the present work, remarkable enhancement of the CL intensity of luminol-[Ag(HIO6)2]5- CL reaction by NE, EP, DA and 5-HT was observed, and a highly sensitive and selective method was established firstly for the simultaneous determination of NE, EP, DA and 5-HT in rat brain by coupling HPLC with the luminol-Ag(III) complex CL detection. This HPLC-CL system can be used for the simultaneous detection of catecholamines and indoleamines with the merits of excellent stability. The experimental conditions for the HPLC separation and maximal CL intensities were systematically optimized. A possible mechanism of CL was suggested via the UV-visible absorption spectra. In addition, colorimetric biosensing exhibit excellent properties such as low-cost equipment, no need purification and real-time detection reaction has become one of the research hot points. A colorimetric biosensing method for the detection of uric acid was established based on the morphology transition of Ag nanoprisms by H2O2 etching. The Ag nanoprism–uricase sensor offered the advantages in theoretical simplicity, and low technical and instrumental demands. Compared with the existing(noble metal nanomaterials)-uricase sensing systems, the strategy designed in this work could make it more easy-to-use and more readily adopted. More importantly, the proposed biosensor was successfully applied to the selective and sensitive detection of uric acid in human serum. The present work was illustrated as follows:Part I. Determination of monoamine neurotransmitters by coupling HPLC with [Ag(HIO6)2]5--luminol chemiluminescence detectionHPLC method with chemiluminescence detection was explored for the simultaneous quantification of monoamine neurotransmitters in rat brain. Based on the enhancing effect of norepinephrine(NE), epinephrine(EP), dopamine(DA) and 5-hydroxytrypatamine(5-HT) on the chemiluminescence reaction between [Ag(HIO6)2]5- and luminol in alkaline solution, the [Ag(HIO6)2]5--luminol chemiluminescence system was coupled with HPLC for the first time. The effects of [Ag(HIO6)2]5- concentration, luminol concentration, p H value and components of HPLC mobile phase were investigated. The separation of NE, EP, DA, and 5-HT was achieved with isocratic elution using a mixture of 0.2% aqueous phosphoric acid and methanol(5:95, v/v) within 11.0 min. The limits of detection for NE, EP, DA, and 5-HT were 4.8, 0.9, 1.9, and 2.3 ng/m L, respectively. The recoveries of monoamine neurotransmitters in rat brain samples were more than 93.4% with the precisions expressed by RSD less than 6.0%. The validated method was applied for the determination of monoamine neurotransmitters in rat brain successfully.Part II Uricase-stimulated etching of silver nanoprisms for highly selective and sensitive colorimetric detection of uric acid in human serumDirect selective and sensitive sensing of uric acid in human serum is of great importance because of its crucial role in physiological and pathological processes. Herein, a simple but effective strategy has been implemented using the morphology transition of Ag nanoprisms for the colorimetric sensing of uric acid. In the presence of uricase, uric acid was oxidized to produce H2O2. Subsequently, Ag nanoprisms were etched from triangle to round by H2O2, leading to a more than 120 nm blue shift of the surface plasmon resonance(SPR) spectral band. The color variation relevant to uric acid concentration can be judged by naked eyes from pale blue to purple. A good linear relationship between SPR peak shift and uric acid concentration ranging from 1.0 to 40 μM was observed. The detection limit of uric acid was 0.8 μM. Some potential interferents in human serum showed no interference. Plasma samples after centrifugation could be directly added into Ag nanoprism-uricase sensing system. The Ag nanoprism-uricase sensor was successfully applied for detecting uric acid in serum samples with the satisfying recoveries over 95%. The obtained results are in agreement with those measured by biochemistry analyzer with the average relative error of 5.1%. The colorimetric sensing can be completed within one hour and do not need expensive instruments, thereby avoiding the disadvantages of current methods such as consuming time and sophisticated equipment. The proposed biosensor offers great advantages including high selectivity and sensitivity, simple sample pretreatment, low-cost instruments, and rapid assay. Thus, the present uric acid sensor has great potential in the applications for biochemical analysis and clinical diagnosis.