Study On The Analytical Methods Of Biomarkers Associated With Behaviors And Diseases And Their Applications

Biomarkers, providing early warning before the body severely damaged, not only play important roles in clinical diagnosis and environmental assessment, but also have aroused wide concern in the social science fields of behavior, psychology and so on. To establish rapid, effective and accurate methods of qualitative and quantitative analysis for biomarkers has important implications for the research and development of related disciplines. Monoamine neurotransmitters (MANTs), histidine (His) and cortisol (Cor) have been proved as biomarkers associated with behaviors and diseases. In this study, the analytical methods of MANTs, His and Cor were developed and applied to the determination of actual samples.This research focused on the five aspects as follow:(1) Developmet of a pretreatment method for the extraction of monoamine neurotransmitters in human plasma samples based on composite nanofibers material and a high performance liquid chromatography for the determination of MANTs; (2) Developmet of a fast pretreatment method based on metal ion modified nanofibers solid phase extracion, and establishment of an accurate method for the determination of histidine in saliva by HPLC-UV; (3) Development of a rapid and sensitive method for the determination of salivary cortisol based on polystyrene nanofibers as the adsorbent of solid phase extraction; (4) Appliction of the new method for determination of salivary cortisol to the research of psychological and behavioral experiments;(5) Exploration of salivary histidine response to stress.The main aspects of this paper were summarized as follows:1. Packed fiber solid-phase extraction of monoamine neurotransmitters in human plasma using composite nanofibers composing of polyvinyl pyrrolidone and polystyrene as sorbentA simple, rapid and high-efficiency sample pretreatment method is developed for the extraction of monoamine neurotransmitters in human plasma using composite nanofibers composing of polyvinyl pyrrolidone and polystyrene as sorbent in the packed fiber solid-phase extraction (PFSPE) column. The plasma samples without protein precipitation and derivatization were loaded on the PFSPE column and the analytes were eluted from the solid-phase with sulfuric acid-methanol mixture (1:9, v/v) before separation and fluorescent detection by HPLC. The absolute extraction recovery of the composite nanofibers is between 60.3%-72.5%.2. Polystyrene and zinc acetate composite nanofiber as absorbing material for solid phase extraction of histidine in human saliva and determination with high performance liquid chromatographyA new polystyrene and zinc acetate composite nanofiber is prepared and used as absorbing material in packed fiber solid-phase extraction column, and a high performance liquid chromatography was established for quantification of histidine in human saliva. The samples were concentrated and purified with PFSPE column after pre-column derivatization with azo coupling, and then the target analytes were eluted with water-methanol mixture (v/v=4/6) containing 3%(v/v) 1.45M sodium carbonate from the PFSPE and monitored by diode array detector at a wavelength of 463nm.Under the optimized conditions, the detection limit is 0.03μg/mL, the quantification limit is 0.1μg/mL, and the linear range is from 0.1μg/mL to 5μg/mL. The extraction rate of histidine is 68.5%. This method was successfully applied to quantify histidine in human saliva due to its good selectivity and high sensitivity.3. The HPLC-MS method for the determination of salivary cortisol based on polystyrene nanofibers solid phase extraction.In this paper, polystyrene nanofibers solid-phase extraction column is used for sample pretreatment, and a rapid method for the determination of salivary cortisol by high performance liquid chromatogramand and electrospray ionization mass spectrometry (HPLC-ESI-MS) is developed. The method has good accuracy and precision, and the detection limit is lOng/L. Besides being simple to operate, the method saves considerable time and sample consumption.4. The application of the proposed method for the determination of salivary cortisol in psychological and behavioral experiments.In order to explore the impact of anxiety state and personality traits on the salivary cortisol response to stress task, the new detection method of salivary cortisol was applied to measure the salivary cortisol levels. The results showed that salivary cortisol levels of the subjects increased significantly (p<0.05) after the speech was completed, and recovered twenty minutes later. The salivary cortisol levels were significantly positively correlated with the score of STAI (r=0.497, p=0.001). Moreover, the rate of cortisol before and after the stress had a significant positive correlation with the level of neuroticism (r=0.395, p=0.016). In conclusion, salivary cortisol response to stress is not only significant, but also corresponds to personality traits.5. Applications of the salivary histidine analytical method in the research of psychological and behavioral.Human response to stressful events and its biomarks, such as cortisol (Cor) and salivary alpha amylase (sAA), have been widely paid attention to in the academia. Histidine, an essential amino acid, is important for the growth of infants and animals, but studies on its response to stress have been limited. In this research, eighty healthy participants were presented with a control condition and two stressful tasks consisting of a cold pressor stress (CPS) and a keynote speech. We intended to verify the feasibility of histidine as a stress marker and explore the impact of anxiety state and personality traits on the histidine response to stress tasks.The results show that histidine level increase in about half of the participants and decline in the other half, which are statistically significant (p<0.05). Analysis of the anxiety state and personality traits parameter indicate histidine response in the keynote speech has a significant positive correlation with the score of STAI (r= 0.49, p= 0.002) and the level of neuroticism(r = 0.37, p= 0.021). In the CPS task, the similar results were obtained, which means that histidine response has a significantly positive correlated to the score of STAI(r= 0.78, p= 0.000) and the level of neuroticism (r= 0.52, p= 0.018). In conclusion, histidine in human saliva can be used as a biomarker for the study of stress, and its response characteristic corresponds to personality traits.