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类型 应用研究 预答辩日期 2018-03-09
开始(开题)日期 2014-01-17 论文结束日期 2018-01-18
地点 四牌楼校区李文正楼北312 论文选题来源 国家自然科学基金项目     论文字数 5.7 (万字)
题目 基于流动注射的无创样品中生物标志物检测方法研究
主题词 流动注射,纳米纤维固相萃取,5-羟基色胺,唾液α-淀粉酶
摘要 学习科学的研究旨在探索与人类(特别是儿童)学习能力相关的生物学、心理学和社会学机制,研究影响人类学习能力的生物学、心理学和社会学因素及其客观的评价方法和技术。这些方法和技术包括心理学方法、生理的方法以及生化的方法。生化指标因为客观性较强及易于采集等,越来越受到重视。生物标志物是从生物学介质中可以检测到的细胞、生物化学或分子改变。这里的生物学介质包括各种体液(如血液、尿液)、粪便、组织、头发、呼吸气体等。采血液是目前临床和科学研究中常用的一种方式,但这种方式本身是一种创伤,会对被采样者造成痛苦,导致不配合采样;同时,其本身也是一种急性应激源,可能造成人体应激反应,导致生物标志物瞬时变化,影响研究结果。近年来唾液等无创样品的测定在精神神经内分泌研究中显得日益重要,这种无创性的采样方式简便、易被接受,尤其适合儿童研究。但是无创样品粘稠,成分复杂, 干扰物质多, 且待测物浓度一般都很低, 因此, 往往需要昂贵的大型检测设备进行检测。但是学习科学研究中需要的生化指标,往往需要大样本量的人群数据,用大型设备进行检查费时费力且成本较高。对便携式的检测仪器和方法的需求日益凸显。本研究组用电纺纳米纤维作为固相吸附剂,开展分析样品前处理研究,由于电纺纳米纤维具有极高的比表面积,与目标分子相互作用的位点明显增加,吸附和脱附速度快,与现有萃取填料相比,提取富集效率显著提高;有机溶剂用量大大减少,最大限度的降低对人体的危害;萃取操作简便易行,易于自动化。而固相萃取往往包括活化,上样,淋洗,洗脱等过程,需要的试剂种类较多,此外,唾液淀粉酶等靠催化反应来进行的生物标志物检测,对反应时间的要求非常高,而流动注射技术是溶液自动在线处理及测定的现代分析技术,最多可以将十多种试剂同时或有序进入并进行反应,具有分析速度快、准确度和精密度高、设备和操作简单、通用性强、试样和试剂用量少等一系列优点。因此将流动注射技术用于无创样品中的生物标志物检测,可以提高检测效率的同时,确保检测结果的准确度,恰好可以满足学习科学对于生化指标的检测要求。 在此基础上,本文主要进行了以下几个方面的研究:(1) 基于纳米纤维的分离富集作用,建立液相色谱-流动注射化学发光在线联用检测样品中的5-羟色胺的方法。(2)建立基于流动注射的纳米纤维固相萃取与光度法在线联用的样品检测方法。(3)建立纳米纤维在线固相萃取-电化学检测样品中的铅的方法。(4)建立流动注射-分光光度法检测α唾液淀粉酶活性的方法。(5)将流动注射法测α唾液淀粉酶活性应用于学龄前儿童肥胖相关研究。(6)将流动注射法测α唾液淀粉酶活性应用于应激相关研究。 论文主要工作如下: 一、基于纳米纤维的分离富集作用,建立液相色谱-流动注射化学发光联用检测样品中的5-羟色胺的方法。 5-羟色胺是一种重要的单胺类中枢神经递质,广泛存在于脊椎动物脑内,参与多种生理进程,包括平滑肌收缩、血压调控、中枢和周围神经系统神经传递。与类癌症候群,抑郁症以及神经分裂等病症密切相关。因此, 5-羟色胺的检测有其重要意义。本研究基于纳米纤维的分离富集作用,设计了离线纳米纤维固相萃取,液相色谱-流动注射化学发光在线联用检测样品中的5-羟色胺的方法。并对固相萃取条件,化学发光条件等进行优化。结果表明:该方法在20-1000 ng/mL的范围内线性良好。标准曲线方程为y=-0.0035+0.00178x(R=0.996)。检出限为5 ng/mL。日内和日间精密度分别为7.3%和6.4%,均小于10%。 二、建立基于流动注射的纳米纤维固相萃取与光度法联用的样品检测方法 分光光度法是一种常见的目标物检测方法,本研究以流动注射为基础,将在线纳米纤维固相萃取技术与光度法检测联用,以一种荧光染料罗丹明B为目标物,验证该方法的可行性。与传统的固相萃取技术不同,纳米纤维固相萃取技术的填料用量少,吸附效率高,因此没有现成的在线固相萃取装置可以借鉴。本研究专门设计了在线纳米纤维固相萃取装置,可以实现24个固相萃取柱的自动切换;优化了在线纳米纤维固相萃取的流路和步骤;比较了不同构造的可见光检测器。最终建立了基于流动注射的纳米纤维固相萃取与光度法联用的样品检测方法。该方法在25-1000 ng/mL的范围内线性良好,线性方程为:y=0.0012x+0.0442 (R2=0.9951)。检出限为7.5 ng/mL。日内和日间精密度分别为7.4%和8.1%,均低于10%。样品的加标回收率在94.21%和105.07%之间。 三、建立纳米纤维在线固相萃取-电化学检测样品中铅的方法 铅、镉、铜等重金属会引起严重的环境污染,并且会威胁人类健康。尤其是儿童正处在生长发育阶段,铅超标会影响儿童的智力,包括语言能力、记忆力和注意力等。因此,对铅的检测就显得极为重要。而电化学检测方法是检测样品中重金属的比较简便的方法。本研究以流动注射为基础,设计了在线纳米纤维固相萃取与电化学检测器联用的方法。并对在线预浓缩操作、电化学系统的富集电压、富集时间等参数进行了优化。结果表明,该方法在5-100 μg/L的范围内线性良好,标准曲线方程为y=-0.0035+0.00178x (R=0.998)。检出限为0.38 μg/L。日内和日间精密度分别为4.7%和5.4%,均小于10%。将该方法应用于5名儿童孤独症儿童尿液中的铅含量测定,测定结果为4.97±0.52 μg/L,高于文献报道的三岁儿童中位数2.64 μg/L。 四、建立流动注射-分光光度法检测α唾液淀粉酶活性的方法 唾液蛋白的分泌是交感神经系统和副交感神经系统协调作用的结果。当交感神经兴奋时,唾液α淀粉酶分泌增加,而且反应速度比去甲肾上腺素、皮质醇等激素还要快。因此,随着 α唾液淀粉酶(salivary alpha-amylase,sAA)活性检测的有效性和可靠性日益被认可,它已成为一种反映人体自主神经系统在压力及压力相关刺激下发生变化最为敏感的生物标记物。目前已有比色法,试纸法等α唾液淀粉酶的检测方法,但均存在操作繁琐,成本较高等缺点,还没有标准的方法。我们课题组基于碘淀粉显色反应的机理,提出了用流动注射检测α唾液淀粉酶活性的方法。本研究在此基础上进行改进,提出了一次记录,多次进样的检测模式,并提出了在线校正α唾液淀粉酶活性的设计。研制的仪器无需对样品进行复杂前处理即可检测,操作快速、简便,且具有良好的回收率和准确性。 五、流动注射法测α唾液淀粉酶活性在学龄前儿童肥胖相关研究中的应用 肥胖症是由于机体脂肪过多或脂肪组织与其他软组织比例过高而引起的,是一种营养过剩造成的营养不良性疾病。肥胖会对儿童的身心健康造成影响,出现自卑、缺乏自信、抑郁、焦虑等异常心理和行为,成人后易患上高血压、高血脂、高胰岛素血症、肝功能异常等疾病。因此对儿童肥胖进行评估,并探讨与儿童肥胖相关的生物标志物就有其重要意义。本研究将流动注射法测α唾液淀粉酶活性应用于学龄前儿童肥胖相关研究,检测了肥胖组和对照组共各30例被试,还收集了家长相关信息进行分析,发现儿童体质指数(BMI)和母亲BMI在肥胖组和对照组间的差异有统计学意义(P<0.05),将被试按照母亲BMI分值高低进行分组,发现儿童α唾液淀粉酶的活度值的差异有统计学意义,进一步做相关分析发现被试唾液中α唾液淀粉酶活性与母亲BMI显著相关。国内尚未发现α唾液淀粉酶活性与肥胖相关的研究结果,国外的研究中虽有报道,但研究对象年龄较大(成人,青少年及10岁左右儿童),且不同年龄段α唾液淀粉酶活性与肥胖相关性不同,因此本研究可以填补学龄前儿童这一年龄段的空白。 六、流动注射法测α唾液淀粉酶活性在应激相关研究中的应用 应激是一种当机体内环境稳定受到威胁时,机体对应激源产生的特异性和(或)非特异性反应。心血管疾病、神经精神病、胃肠疾病和内分泌疾病等高发病率的疾病与应激密切相关,也对人的学习和认知过程产生影响。随着现代社会和经济的发展,人的精神压力越来越大,而这些压力,往往会导致疾病的产生。许多研究表明,唾液中的α唾液淀粉酶活性会随着人精神压力的增大而急剧升高。因此,通过检测唾液中的α唾液淀粉酶活性来了解人精神压力状况便成为可能。本研究招募40名大学生作为被试,将5分钟演讲作为应激源,并要求被试填写一般情况问卷和艾森克人格问卷,收集被试演讲开始前,演讲结束时、结束后15分钟和20分钟的唾液样品,并采用已建立的流动注射的方法检测样品中的α唾液淀粉酶活性。按照艾森克人格问卷的内外向性(E维度)进行分组,并用单因素重复测量方差分析的方法进行统计,结果显示高分值组和低分值组α唾液淀粉活性变化间的差异有统计学意义。进一步对E分组的高分值组和低分值组分别进行重复测量的方差分析,发现低值组各个时间点间α唾液淀粉活性的变化无统计学差异(P=0.383>0.05),而高值组各个时间点间α唾液淀粉活性的变化则有统计学差异(P=0.014<0.05)。更进一步对高分值组不同时间点间的α唾液淀粉活性进行两两配对t检验。结果表明在高分值中,演讲前与演讲结束时;演讲结束后15分钟与演讲结束后20分钟的α唾液淀粉活性的差异分别有统计学意义。已有国外研究发现自我怜悯量表Self-Compassion Scale(SCS; Neff, 2003b)中自我怜悯对α唾液淀粉活性应激反应有负性影响;也有研究表明大五人格量表中的神经质以及状态-特质焦虑量表State-trait-anxiety inventory(STAI)中的特质焦虑与被试实验日的α唾液淀粉活性呈正相关。而本研究使用的艾森克人格问卷具有较高的信度和效度,其所测得的结果可同时得到多种实验心理学研究的印证,本研究结果可为用客观测量而非量表的方法来评价人格特质提供依据。
英文题目 Study on the detection of the noninvasive biological markers based on the flow injection assay system
英文主题词 Flow injection assay system, Packed fiber SPE(PFSPE), 5-hydroxytryptamine (5-HT or serotonin), Salivary alpha-amylase,
英文摘要 The purpose of learning science is to explore biological, psychological and sociological mechanisms related to human learning, especially children’s learning ability, and to study biological, psychological and sociological factors that influence human learning ability and the objective evaluation methods and techniques. These methods and techniques include psychological methods, physiological methods, and biochemical methods. The biochemical indexes have been paid more and more attention because of the strong objectivity and easy to collect for them,. A biomarker, or biological marker, generally refers to a measurable indicator of some biological state or condition. The term is also occasionally used to refer to a substance whose detection indicates the presence of a living organism. Biomarkers are often measured and evaluated to examine normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Biomarkers are used in many scientific fields. Various biological fluids have been utilized as the samples, such as plasma, serum, urine, amnioticfluid, and saliva. Comparing with sampling of blood which is invasive and may itself induce stress, salivary measures have become increasingly important in psychoneuroendocrinological research beacause it is noninvasive and more convenient to obtain. A wide array of salivary biomarkers has been proposed over the years. While salivary sample was always sticky, complex composition, interference with more substances and the content of biomarker was low. Sample preprocessing was always the most complex, cumbersome but the most critical step. By comparison of conventional extraction techniques of liquid–liquid extraction (LLE) and SPE etc., a more effective extraction method, packed fiber SPE (PFSPE), was developed for cleaning up and concentration of biomakers before assay. Compared to the conventional SPE technique, PFSPE uses electrospun nanofiber as the sorbent, which possesses large surface area facilitating attachment of target molecules, a little volume of solid phase is enough for the extraction, and the volume of eluting solvent is reduced greatly, thus the evaporation step could be eliminated. Nevertheless, the PFSPE was still including activation, loading, leaching, elution and other processes. Several types of reagents were also need. At the same time, the the detection of biomarkers such as saliva amylase which was based on catalytic requires strict time control. Flow injection assay system (FIA) can just solve these problems. FIA is an automated method of chemical analysis in which a sample is injected into a flowing carrier solution that mixes with reagents before reaching a detector. The principle is similar to that of segmented flow analysis (SFA) but no air is injected into the sample or reagent streams. A wide variety of reagents can be injected into the system sequential or simultaneously. In this study, FIA was usd for the detection of noninvasive biomarkers for accuracy and precision. In this paper, the following five aspects of research were studied: (1) a method for the determination of 5-hydroxytryptamine in samples by HPLC followed with FIA and chemiluminescence was established; (2) development of an online-PFSPE- spectrophotometry system for the quantification of urine sample; (3) establishment of an accurate determination method for heavy metals based on FIA and PFSPE; (4) development and optimizition a rapid, sensitive method for determination of salivary alpha-amylase; (5) study on the effects of obesity on salivary alpha-amylase in preschool children; (6) tested the associations of salivary alpha-amylase stress responses with personality in college students. The research projects will offer knowledge and technical supports for physiology, psychiatry, cognitive science and psychology research. The main aspects of this paper were summarized as follows: 1. Establishment of a method for the determination of 5-hydroxytryptamine in samples by HPLC followed with FIA and chemiluminescence after the separation and enrichment of target compound by use of nanofibers 5-hydroxytryptamine (5-HT or serotonin) is a monoamine neurotransmitter widely distributed in biological systems and plays an important role in a number of pharmacological, physical and fundamental biological processes. A method for the determination of 5- hydroxytryptamine in samples by HPLC followed with FIA and chemiluminescence was established and the solid-phase extraction conditions, chemiluminescence conditions were optimized. Under the optimized conditions, a good linearity of wide range between 20-1000 ng/mL was achieved. The LOD was 5 ng/mL-1. 2. Development of an online-PFSPE- spectrophotometry system A sample pretreatment step prior to analysis is usually necessary, even though it is one of the most tedious and time-consuming steps involved in aqueous sample analysis. PFSPE is a new technique which uses electrospun nanofiber as the SPE sorbent. Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength, which commonly used for the measurement of biomarkers. In this study, spectrophotometry was applied in the detection of rhodamine B which is a fluorescent dye applied in industry, analytical chemistry followed by online PFSPE. A novel PFSPE cartridge packed with electrospun nanofiber was designed to adsorb rhodamine B from water media in an online manner. Under the optimized conditions, a good linearity of wide range between 25-1000 ng/mL was achieved. The LOD was 7.5 ng/mL. The method was applied to analysis RB in water samples with satisfactory results. 3. Establishment of an online-PFSPE-Electrochemical method for the quantification of lead Heavy metals, like lead, cadmium and copper are the cause of one of the most serious pollution problems of our time. They can threaten environment and human health even in small amounts, because they are not biodegradable and therefore retaine indefinitely in the ecological systems and accumulate in food chain. Therefore monitoring of lead in the sample has become essential. The direct determination of heavy metal ions in complex matrices is limited due to the low concentrations and matrix interferences. Thus, to improve the sensitivity and selectivity of the determination of heavy metal ions, a preconcentration and separation procedure, such as liquid–liquid extraction, coprecipitation, and cloud point extraction, is essential and necessary. In this work, an online-PFSPE method was developed. The concentrated lead eluated from the solid phase was impelled toward the electrolytic cell by the pump, and analyzed by anodic stripping voltammetry. Under the optimized conditions, the stripping peak area of lead showed a good linearity of wide range between 5-100 μg/L. The LOD was 0.38 μg/L. The method was applied to analysis of lead in samples with satisfactory results. 4. Development and optimizition a flow injection assay system for online quantification and calibration of salivary alpha-amylase Salivary alpha-amylase as a non-invasive marker for sympathetic nervous system (SNS) activity has drawn more attention in recent years. The determination of salivary alpha-amylase activity is very useful in the mental health assessment. A completely automated analytical system was developed for the determination of salivary alpha-amylase activity using a flow injection-spectrophotometric analysis system basing on an enzyme degradation reaction of starch and the color reaction of starch with iodine. Without any pretreatment, the system can monitor the samples sequentially with high precision and accuracy. When the equipment is functioning, the starch and iodine solution were brought to the confluence point from each line and then dispersed toward the detector. Enzymatic reaction started in the starch solution line at the moment of the sample being injected through the injection valve. An online method was established for calibrating standard salivary alpha-amylase activity.The salivary alpha-amylase standard and starch solution at a known concentration was mixed and then brought into the line, met with iodine and reacted at the detector. The absorbance of the reaction solution was reduced continuously until the starch degraded completely. The accurate value of salivary alpha-amylase activity can be calculated by the time periods of the absorbance reducing. A comprehensive study was initiated to optimize FIA step, such as the concentration of starch and iodine, the speed of the peristaltic pump. The optimal analytical performance was achieved including a wide dynamic range of 1607~19284U/ mL, detection limits of 200 U/mL and precision (as RSD%) lower than 5% for both intra-day and inter-day assays. This method was applied for the determination of salivary alpha-amylase activity in human saliva sampled from the subjects at different ages. The result show that the saliva α-amylase activity of volunteers at age of 20 (7557±3799, n=49) is lower (P<0.05) than that of 5 (9656±5782, n=61). 5. Study on the effects of obesity on salivary alpha-amylase in preschool children Obesity and associated diseases, such as type 2 diabetes, have reached worldwide epidemicproportions. Childhood obesity has become a major public health problem both in westernised and more recently in developing countries which is a focus of many public health efforts. Traditional and cultural perceptions regarding body size, urbanization, poor diet, low socioeconomic status, and lack of physical activity are a few of the suggested contributing factors. There are a number of studies that have examined the role of the hypothalamic–pituitary adrenal axis in obesity, using salivary cortisol as a marker. However, research on the other component of the stress system, for example, the autonomic nervous system (ANS) is limited. An ideal way to study the functioning of the ANS of an individual, is by using salivary alpha-amylase as a biomarker of stress-related ANS activity. In this study, we focos on the effects of obesity on salivary alpha-amylase in preschool children. 30 obese preschool children and 30 cases of control were emloy to measure the body mass index and salivary alpha-amylase activity. And the body mass index of their parents were also asked. The salivary alpha-amylase activity of the children whose mothers with higher body mass index were also significantly higher than the group with lower body mass index. A strong association was shown between the children body mass index, salivary alpha-amylase activity and the body mass index of their mothers. 6. Tested the associations of salivary alpha-amylase stress responses with personality in college students Since stress is a multi-faceted phenomenon, it requires a multidimensional measurement approach. As a consequence, research can gain from additions to the canon of psychobiological parameters. One parameter that has been suggested to reflect stress-related changes in the body is the salivary enzyme alpha-amylase which has been used as a non-invasive salivary marker of sympathetic nervous system activity. Several studies have shown that the activity of sAA is increased by acute stress. While the reports focos on the associations of salivary alpha-amylase stress responses with personality were limited. In this study, 40 college students whose salivary sample were collected before and 0, 15, 20 minutes after speech stress were recruited. Meanwhile, a Eysenck Personality Questionnaire was asked to finished by every subject. A significant difference of the salivary alpha-amylase stress responses were detected between the subjects grouped by the extraversion score. And the difference of salivary alpha-amylase activity between before and after speech stress immediately, 15 and 20 minutes after speech stress was further found.
学术讨论
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儿童发展与学习科学教育部重点实验室 2012年11月23日 14:30-15:30 李文正楼北312大会议室 Prof. Keiji Iramina Measurement and control of Brain Dynamics- Aplication to the disabled person
儿童发展与学习科学教育部重点实验室 2014年5月18日 15:00 李文正楼北335报告厅 Prof. Hideaki Koisumi Research and Development toward Human Security and Well-Being
儿童发展与学习科学教育部重点实验室 2016年2月25日上午8:45 榴园宾馆新华厅 李德毅院士 脑认知形式化
儿童发展与学习科学教育部重点实验室 2016年4月20日9:30 李文正楼北312会议室 崔兴然 面向转化医学的动态生医指标体系
东南大学学习科学中心康学军教授科研组 2013年10月28日 9:00 李文正楼北312会议室 王羽 呼吸气体的氧化还原研究现状
东南大学学习科学中心康学军教授科研组 2013年11月25日 9:00 李文正楼北312会议室 王羽 皮质醇化学发光反应研究现状
东南大学学习科学中心康学军教授科研组 2014年5月29日 9:00 李文正楼北312会议室 王羽 苏州市婴幼儿健康促进工程介绍
东南大学学习科学中心康学军教授科研组 2015年3月16日 9:00 李文正楼北312会议室 王羽 The developmental course of salivaryalpha-amylase and cortisol from 12 to 36months: Relations with early poverty and later behavior problems
东南大学学习科学中心康学军教授科研组 2015年3月30日 9:00 李文正楼北312会议室 王羽 Exploring the Relationship of Autonomic and Endocrine Activity with Social Functioning in Adults with Autism Spectrum Disorders
     
学术会议
会议名称时间地点本人报告本人报告题目
The 3rd International Conference on Emerging Trends in Scientific Research 2015年4 月 25日 马来西亚、吉隆坡 A Flow Injection Assay System for Online Quantification and Calibration of Saliva α-Amylase Activity
17th Intermational Symposium on Advances in Extraction Technologies 2015年11月7-11日 中国 广州 A Polite Study of Exhaled Breath Responses to Psychological Stress Using Electrospun Nanofibers Trapping Combined with Gas Chromatography
The 2012 International Conference on Nanotechnology and Precision Engineering 2012年12月24日 中国 桂林
2012年全国生物与医学纳米技术博士生学术论坛 2012年7月10 日 中国苏州
     
代表作
论文名称
An Ion-Pair Reagent Incorporated Polystyrene Nanofiber Applied to Solid Phase Extraction of 5-Hydrox
Development of an online-PFSPE- spectrophotometry system for the quantification of environmental pol
Development of an online-solid phase extraction-electrochemical method for the quantification of lea
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
孙桂菊 正高 教授 博导 东南大学
卢青 正高 教授 博导 东南大学
葛卫红 正高 主任药师 硕导 南京鼓楼医院
苏二正 正高 教授 博导 南京林业大学
祁争健 正高 教授 博导 东南大学
      
答辩秘书信息
姓名职称工作单位备注
张亚贤 其他 讲师 东南大学