生物荧光传感器检测环境水样中氨基甲酸酯类农药残留

建立了简便、灵敏的氨基甲酸酯类农药生物荧光传感器及其检测方法。通过构建氨基甲酸酯类农药降解菌H5基因组文库,筛选出氨基甲酸酯类农药特异性响应功能基因的调控序列,将其与增强型绿色荧光蛋白基因(EGFP)连接,构建了非细胞体系生物荧光传感器H12-E,非细胞体系蛋白浓度为1.0 g/L。在室温条件下,对不同浓度呋喃丹标准液进行检测,30 min即可检测出fg级氨基甲酸酯类农药总量,灵敏度高于国家标准,线性范围1×10!9~1×10!4g/L。采集吉林省松花江流域等水样,检测氨基甲酸酯类农药残留,并向伊通河水样中添加高中低3个水平呋喃丹标准液,方法回收率95%~110%,相对标准偏差(RSD)2.4%~4.9%。本方法可望用于环境水体中氨基甲酸酯类农药残留的快速检测。     

绿色荧光碳点的制备及其在铜检测中的应用

以D-葡萄糖和L-天冬酰胺为原料,通过热解法制备得到绿色荧光碳点。该碳点在Cu2+存在下,碳点荧光发生显著猝灭。基于此,建立了一种可选择性检测Cu2+的方法,在1.0~20.0μmol/L线性范围内对Cu2+呈现良好的响应,检出限为6.3 nmol/L。通过标准加入法将其用于实际水样中Cu2+的检测,回收率为95.2%~105.0%,该碳点可以应用在环境水样中Cu2+的检测。此外,为充分利用该碳点较低的细胞毒性和良好的生物相容性等优点,通过荧光共聚焦成像技术将其成功用于细胞中Cu2+的可视化检测,进一步拓宽了碳点的应用领域。     

一种可用于检测苦味酸的水溶性共轭聚合物荧光传感器的制备及应用

制备了一种高灵敏的水溶性荧光共轭聚合物(PPE-OBS), 将其用于苦味酸的荧光检测. 利用质谱、 核磁共振波谱和红外光谱等方法对PPE-OBS进行了表征, 并对其检测苦味酸的条件进行了优化. 结果表明, 在硼酸-氢氧化钠缓冲溶液(pH=9.5)中作用20 min, 苦味酸对PPE-OBS荧光的猝灭效率最大. 在最优条件下, PPE-OBS检测苦味酸的线性范围为1.0~60 μmol/L(R²=0.999), 检出限为0.831 μmol/L(S/N=3). 将PPE-OBS制成荧光试纸用于检测环境样品中的苦味酸, 获得了较好的检测结果.     

生物基质中雌激素的液相色谱-串联质谱定量分析方法研究进展

雌激素是一类重要的内源性甾体类激素,具有广泛的生物活性.内源性雌激素及其代谢物含量的测定对生理和病理学诊断具有极其重要的意义.然而,雌激素在生物基质中浓度通常极低,复杂基质干扰大,难以准确定量检测.液相色谱-串联质谱法(LC-MS/MS)具有高灵敏度、高准确性以及高通量的优势,已经成为生物基质中雌激素检测的主要技术平台...     

吐温20增敏同步荧光光谱法测定尿液中1-羟基芘

尿液中1-羟基芘作为人体内多环芳烃(PAHs)的代谢产物,是高浓度PAHs职业环境和一般环境中人体接触PAHs的一个灵敏指标,已被广泛用于评价人体和动物与多环芳烃接触的内暴露生物指示物,其含量的测定具有十分重要的意义.本研究建立了人尿中1-羟基芘(1-OHP)的同步荧光测定方法.在Britton-Robinson 缓冲溶液(pH 2.6)中,以同步波长差Δλ=34 nm 进行同步荧光扫描,其同步特征峰的强度与1-OHP的浓度呈线性关系.表面活性剂吐温20对1-OHP的同步荧光具有增敏作用.本方法的线性范围为2.5×10-10～5.0×10-7 mol/L; 检出限为9.5×10-11 mol/L; 相对标准偏差(RSD)为0.78%(1.0×10-7 mol/L, n=11).应用于实际尿样的测定,加标回收率为93.8%～107.4%.     

基于核酸适配体的荧光法检测水胺硫磷和丙溴磷

建立了基于适配体的农药水胺硫磷和丙溴磷的荧光检测方法.采用可特异性识别水胺硫磷和丙溴磷、且5 '端标记荧光基团FAM的核酸适配体(F-ssDNA),与3 '末端标记猝灭基团DABCYL的短链序列(Q-ssDNA)互补杂交形成双链结构,荧光基团的荧光被淬灭,荧光信号很弱;此时加入靶分子,特异性结合核酸适配体,引起互补短链序列从双链结构中解离,使适配体荧光信号增强,基于此可实现水胺硫磷、丙溴磷的定量检测.优化后的检测条件为:将终浓度为25 nmol/L F-ssDNA与50 nmol/L Q-ssDNA在25℃孵育20 min,使二者杂交形成双链适配体探针复合物,加入等体积的农药样品孵育60 min,然后检测体系的荧光信号变化值△I.在最佳条件下,△I与水胺硫磷和丙溴磷的浓度均在50～ 500 μmol/L范围内呈线性关系.水胺硫磷的检出限(LOD,3σ)为11.4 μmol/L,相对标准偏差(RSD)为5.8％(n=10);丙溴磷的检出限为14.0 μmol/L,RSD为4.9％(n=l0).用于实际水样中两种农药的检测,加标回收率为85.8％ ～95.3％.     

利用实时荧光PCR鉴定小体鲟物种的快速方法

从小体鲟线粒体基因筛选出位于COⅠ(细胞色素氧化酶)基因中的一段保守序列,针对其设计引物,优化SYBR Green实时荧光PCR反应体系,建立了一种鉴定小体鲟的实时荧光PCR定性检测方法.该方法检测小体鲟DNA灵敏度为0.04mg/L.通过对采集和市售小体鲟鱼样品检测,该方法可检测出样品中的小体鲟成分.实验证明该方法可对血液样品和组织样品中的小体鲟进行种源鉴别.     

基于富G序列的无标记荧光传感器检测单链核酸

具有特定构象的富G序列（如G-四链体和G-三链体）与荧光染料相互作用可有效增强其荧光信号强度，被广泛应用于构建无标记荧光生物传感。本研究以硫磺素T(Thioflavin T, ThT)为荧光染料，构建了两种基于富G序列的无标记荧光传感器，用于检测阿尔兹海默病标志物β-淀粉样蛋白（β-Amyloid protein, Aβ）的基因序列。实验结果表明，分子发夹茎长为4个碱基时，富G序列以G-三链体的结构存在，以此构建的G-三链体传感器的输出信号随Aβ基因浓度增加而降低，线性检测范围为1～100 nmol/L，检出限为0.3 nmol/L(S/N=3)。分子发夹茎长为8个碱基且5′端添加碱基AATT时，其与Aβ基因结合后，富G序列多以G-四链体结构存在，以此构建的G-四链体传感器的输出信号随Aβ基因浓度增加而增强，线性检测范围为0.1～100 nmol/L，检出限为0.04 nmol/L (S/N=3)。两种传感器制备过程相似但检测原理不同，为富G序列的深入研究与应用提供了参考，同时为单链核酸的无标记荧光检测提供了新的思路。     

在线固相萃取-高效液相色谱法同时测定人尿液中7种多环芳烃代谢物

采用双三元液相色谱系统结合荧光检测器,建立了在线固相萃取-液相色谱法同时测定人尿液中7种多环芳烃代谢物的方法。目标化合物首先在Turboflow Cyclone固相萃取柱上在线富集浓缩,然后通过六通阀转移至Hypersil Green PAH色谱柱,以乙腈-水为流动相进行梯度洗脱分离,流速1.0 mL/min,柱温30℃,荧光检测器检测,分离周期为20 min。在优化的色谱条件下,5~2000 ng/L或50~20000 ng/L范围内,7种多环芳烃代谢物均呈良好的线性关系(r≥0.999),方法检出限为0.5~15 ng/L,加标回收率为80.7％~110.7％。应用本方法对吸烟和非吸烟人群尿液中7种多环芳烃代谢物的含量进行了测定,吸烟者尿液中的2-羟基萘、1-羟基萘、2-羟基菲、2-羟基芴、4-羟基菲、6-羟基显著高于非吸烟者。     

高效液相色谱法测定大气颗粒物中的杂环胺

建立了大气颗粒物中杂环胺的高效液相色谱检测方法.采用ODS C18色谱柱(250 mm×4.6 mm, 5 μm),乙腈-0.01 mol/L三乙胺缓冲溶液(pH 4.0 )为流动相,非线性梯度洗脱,流速1.0 mL/min,柱温30 ℃,紫外检测波长263 nm,并优化荧光激发和发射波长条件,实现了6种杂环胺的基线分离和4种杂环胺的高灵敏度荧光检测.本方法中荧光和紫外检测器的检出限分别为0.0018～0.0084 mg/L和0.093～0.609 mg/L(S/N=3),相关系数在0.9920～0.9999之间,RSD小于5.9%,平均回收率为75.3%～111.6%,回收率相对标准偏差为1.7%～2.3%,具有较高的精确度和准确度.     

A Novel Method for Quality Evaluation of Gardeniae fructus Praeparatus during Heat Processing Based on Sensory Characteristics and Chemical Compositions

The intrinsic chemical components and sensory characteristics of Gardeniae fructus Praeparatus (GFP) directly reflect its quality and subsequently, affect its clinical curative effect. However, there is little research on the correlation between the appearance traits and chemical compositions of GFP during heat processing. In this study, the major components of five typical processed decoction pieces of GFP were determined. With the deepening of processing, the contents of geniposidic acid and 5-HMF gradually increased, while the contents of deacetyl-asperulosidic acid methyl ester, gardenoside, and two pigments declined. Moreover, the electronic eye, electronic tongue, and electronic nose were applied to quantify GFP’s sensory properties. It was found that the chroma values showed a downward trend during the processing of GFP. The results of odor showed that ammonia, alkenes, hydrogen, and aromatic compounds were the material base for aroma characteristics. Complex bitterness in GF was more obvious than that in other GFP processed products. Furthermore, one mathematical model was established to evaluate the correlation between the sensory characteristics and chemical composition of GFP during five different stages. A cluster analysis and neural network analysis contributed to recognizing the processing stage of GFP. This study provided an alternative method for the exterior and interior correlation-based quality evaluation of herbs.     

Rapid determination of corticosteroids in pharmaceuticals by flow injection analysis

The proposed method is based on the reduction of blue tetrazolium by the steroid in an alkaline medium to form a highly colored formazan. The effects of reagent concentration, temperature, flow rate, and manifold design on the reaction are discussed for a typical steroid, methylprednisolone acetate. Analytical readout is obtained within 30 s after sample introduction and up to 100 samples/h can be processed with baseline resolution between peaks. Typical relative standard deviations of 0.5% are obtained with 10-μ1 injection volumes. Results obtained by flow injection analysis are similar to those obtained with the AutoAnalyzer technique.     

Quantification of green fluorescent protein in cellular supernatant by capillary electrophoresis with laser-induced fluorescence detection for measurement of cell death

A common method for quantifying cell death is measuring the concentration of lactate dehydrogenase (LDH) released by cells as their membranes become unstable. In cells expressing green fluorescent protein (GFP), degradation of the cell membrane also results in the release of GFP into the surrounding supernatant. In this study, we used capillary electrophoresis with laser-induced fluorescence detection to measure the levels of GFP in supernatants of UBIGFP/BL6 primary macrophages that had been infected with Salmonella typhimurium, treated with staurosporine, or exposed to H₂O₂, all known inducers of cell death. We also used a standard LDH assay to measure the release of LDH into supernatants. We observed the rate of cell death quantified by release of GFP and LDH into supernatant to be essentially identical, demonstrating that GFP release is at least as good as an indicator of macrophage cell death as the established LDH release method.     

Application of a green fluorescent fusion protein to study protein-protein interactions by electrophoretic methods

A screening procedure for protein-protein interactions in cellular extracts using a green fluorescent protein (GFP) and affinity capillary electrophoresis (ACE) was established. GFP was fused as a fluorescent indicator to the C-terminus of a cyclophilin (rDmCyp20) from Drosophila melanogaster. Cyclophilins (Cyps) belong to the ubiquitously distributed enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases) and are well known as cellular targets of the immunosuppressive drug cyclosporin A (CsA). The PPlase activity of the GFP fused rDmCyp20 as well as the high affinity to CsA remain intact. Using native gel electrophoresis and ACE mobility-shift assays, it was demonstrated that the known moderate affinity of Cyp20 to the capsid protein p24 of HIV-1 was detectable in the case of rDmCyp20 fused to the fluorescent tag. For the p24 / rDmCyp20-GFP binding an ACE method was established which allowed to determine a dissociation constant of Kd = 20+/-1.5 x 10(-6) M. This result was verified by size-exclusion chromatography and is in good agreement with published data for the nonfused protein. Moreover the fusion protein was utilized to screen rDmCyp20-protein interactions by capillary electrophoresis in biological matrices. A putative ligand of rDmCyp20 in crude extracts of embryonic D. melanogaster was discovered by mobility-shift assays using native gel electrophoresis with fluorescence imaging and ACE with laser-induced fluorescence detection. The approach seems applicable to a wide range of proteins and offers new opportunities to screen for moderate protein-protein interactions in biological samples.     

Rapid purification of antisteroid antibodies by high-performance liquid affinity chromatography

An adsorbent for the high-performance affinity chromatography of antisteroid antibodies was prepared, based on a commercial pre-packed column. The column contained activated microparticulate silica beads bearing epoxide functions, on which the steroid dexamethasone was covalently linked. The column was used successfully for the rapid and complete isolation of several hundred microgram amounts of specific antidexamethasone antibodies from rabbit antisera. The practical aspects of the purification procedure, especially the optimization of the washing and of the elution steps, are detailed. Despite non-biospecific elution with 20% acetonitrile in an acidic buffer, the purification yield was very satisfactory and the biological activity of the purified immunoglobulins appeared excellent.     

Rapid and sensitive analysis of phthalate metabolites, bisphenol A, and endogenous steroid hormones in human urine by mixed-mode solid-phase extraction, dansylation, and ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry

Steroid hormone levels in human urine are convenient and sensitive indicators for the impact of phthalates and/or bisphenol A (BPA) exposure on the human steroid hormone endocrine system. In this study, a rapid and sensitive method for determination of 14 phthalate metabolites, BPA, and ten endogenous steroid hormones in urine was developed and validated on the basis of ultra-performance liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry. The optimized mixed-mode solid phase-extraction separated the weakly acidic or neutral BPA and steroid hormones from acidic phthalate metabolites in urine: the former were determined in positive ion mode with a methanol/water mobile phase containing 10 mM ammonium formate; the latter were determined in negative ion mode with a acetonitrile/water mobile phase containing 0.1 % acetic acid, which significantly alleviated matrix effects for the analysis of BPA and steroid hormones. Dansylation of estrogens and BPA realized simultaneous and sensitive analysis of the endogenous steroid hormones and BPA in a single chromatographic run. The limits of detection were less than 0.84 ng/mL for phthalate metabolites and less than 0.22 ng/mL for endogenous steroid hormones and BPA. This proposed method had satisfactory precision and accuracy, and was successfully applied to the analyses of human urine samples. This method could be valuable when investigating the associations among endocrine-disrupting chemicals, endogenous steroid hormones, and relevant adverse outcomes in epidemiological studies.

Synthesis of 19,B-Bisnoranalogs of Steroid Androgens with cis-Fused B and C Rings

Reduction by Birch procedure of 3-methoxy-B-nor-8-isoestra-1,3,5(10)-trienes followed by hydrolysis of reaction products furnished 19,B-bisnor-8,10-isoanalogs of steroid androgens. With the use of the correlation NMR spectroscopy a complete assignment of signals in the ¹H and ¹³C NMR spectra was performed for two representatives of this steroid group, and their prevailing conformations in solution were established.     

Direct monitoring of the expression of the green fluorescent protein-extracellular signal-regulated kinase 2 fusion protein in transfected cells using capillary electrophoresis with laser-induced fluorescence detection

Proper subcellular localization of the extracellular signal-regulated kinases (ERKs) is important in regulating physiological functions such as proliferation and differentiation in the pheochromocytoma cell line (PC12 cells). Thus, a direct visualization method is necessary to observe ERK localization within the cell or in crude cellular extracts. In this paper, a determination method was established for the detection of ERK2 localization in PC12 cells using green fluorescent protein (GFP) and capillary electrophoresis with laser-induced fluorescence (LIF). GFP as a reporter or labeling tag for gene expression in biochemistry and cell biology was used for the detection of ERK2 localization in PC12 cells. PC12 cells were transfected with GFP-ERK2 plasmid construct that was inserted into a variant GFP gene (enhanced green fluorescent protein), and successfully expressed GFP-ERK2 fusion proteins. GFP-ERK2 fusion proteins were detected within 5 min by CE analysis using an uncoated fused-silica capillary with LIF. Optimum conditions for GFP-ERK2 fusion proteins detection were 100 mM 3-(cyclohexylamino)-1-propanesulfonic acid buffer containing 100 mM sodium dodecylsulfate, pH 11, running at 20 degrees C. This result offers new opportunity in screening for the determination of localization of intracellular components, protein-protein interactions and kinase activity within the cells.     

The internal link of serum steroid hormones levels in insomnia,depression, and Alzheimer's disease rats: Is there an effective way to distinguish among these three diseases based on potential biomarkers?

Insomnia, depression, and Alzheimer's disease are all neurodegenerative diseases and are associated with the levels of steroid hormones. To investigate the internal connection and difference of steroid hormones among these three diseases and distinguish them from the perspective of biomarkers, an easy, quick, and efficient high‐performance liquid chromatography with tandem mass spectrometry method was established and validated to determine six steroid hormones simultaneously in rat serum. The separation was accomplished on a SHIM‐PACK XR‐ODS chromatographic column with 0.1% v/v formic acid and methanol as the mobile phase and the detection was performed with electrospray ionization source in the positive ion mode. Based on the concentrations of steroid hormones, all the groups could be distinguished obviously from each other by using partial least square discriminant analysis. Meanwhile, 11‐deoxycortisol, corticosterone, and cortisol were identified as potential biomarkers and 100% of samples were classified correctly by Bayes’ discriminant function. These biomarkers were further screened by one‐way analysis of variance and cortisol was significantly different among all these groups. Bayes’ discriminant function was also built by cortisol and the classification accuracy was 87.2%. This workflow including determination of steroid hormones and discrimination among three neurological diseases would provide a basis for further clinical studies.     

Purification method for recombinant proteins based on a fusion between the target protein and the C-terminus of calmodulin

Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein. The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins. A CaM-GFP fusion protein was expressed in E. coli and purified on a phenothiazine-derivatized silica column. CaM binds to the phenothiazine on the column in a Ca(2+)-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP. The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa. Pure GFP was eluted with a Ca(2+)-containing buffer, while CaM was eluted later with a buffer containing the Ca(2+)-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.