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.