响应曲面优化-毛细管电泳-激光诱导荧光快速检测粪便中诺如病毒

建立了粪便中诺如病毒逆转录PCR-毛细管电泳-激光诱导荧光快速检测方法。根据诺如病毒核酸保守序列选择引物,扩增出特异的PCR产物;采用响应曲面法进行毛细管电泳条件优化,以含有DNA荧光染料SYBR Gold的0.5%甲基纤维素为筛分介质,通过激光诱导荧光法检测诺如病毒的PCR产物。在优化的毛细管电泳条件下,9 min内可完成诺如病毒PCR产物的检测。扩增产物测序后,与基因库中诺如病毒的序列进行同源性比对,一致性达99%。迁移时间的日内和日间相对标准偏差分别为1.1%~1.3%和1.8%~2.6%,已用于粪便中诺如病毒的快速检测。     

多重聚合酶链反应-毛细管电泳-激光诱导荧光法检测三种食源性致病菌

建立了食品中常见致病菌大肠杆菌O157:H7的uidA基因、沙门菌的invA基因和志贺菌的ipaH基因的多重聚合酶链反应（PCR）产物的毛细管电泳快速检测方法。根据这3种致病菌的特异性基因序列设计多重PCR引物,优化PCR扩增反应体系,采用7.0 g/L 甲基纤维素为筛分介质,毛细管电泳-激光诱导荧光检测法同时检测了3种常见致病菌的PCR扩增产物。在优化的多重PCR反应和毛细管筛分电泳条件下,该方法可以同时检测沙门菌、志贺菌和大肠杆菌O157:H7基因的多重PCR扩增产物,22 min内即可完成3种常见致病菌的毛细管电泳检测。迁移时间的相对标准偏差为1.47%~2.07%。与凝胶电泳法比较,该法简便快速,灵敏度高,可用于多种致病菌脱氧核糖核酸的检测,为食品安全提供了一种可靠的快速检测方法。     

微流控芯片-激光诱导荧光快速检测4种食源性致病菌

建立了食品中4种常见食源性致病菌的微流控芯片快速检测方法。根据副溶血弧菌的Vpara(16S-23S rDNA IGS)基因、沙门菌的invA基因、大肠杆菌O157:H7的rfbO157基因和志贺菌的ipaH基因序列设计了4对特异性引物,对上述致病菌进行四重PCR扩增,采用微流控芯片-激光诱导荧光检测食品中4种常见致病菌的多重PCR扩增产物。优化了多重PCR扩增和微流控芯片电泳分离的实验条件。当芯片电泳的筛分介质HPMC-50浓度为2.2%、溴乙锭(EB)含量为3.75μmol/L、电场强度为120 V/cm时,pUC Mix DNAMarker-8和待测致病菌的多重PCR扩增产物可以实现基线分离,600 s内即可完成上述4种致病菌的同时检测,迁移时间的日内相对标准偏差为0.74%~2.09%。本方法能够检出1×102cfu/mL的副溶血弧菌、沙门菌、大肠杆菌O157:H7和志贺菌。方法特异性高,所设计的引物在10种非目的菌株体系中均未见扩增的片段。将本法应用于食品中上述致病菌的测定,获得了满意的结果,为常见食源性致病菌的快速检测提供了一种新的可靠分析手段,对保障食品安全具有重要的现实意义。     

转基因玉米的多重PCR-毛细管电泳-激光诱导荧光检测方法研究

采用三重PCR反应, 同时扩增CaMV 35S启动子、 hsp70 intron1和CryIA(b)基因之间序列以及Invertase基因, 扩增产物用无胶筛分毛细管电泳-激光诱导荧光检测, 从而建立了多重PCR-毛细管电泳-激光诱导荧光快速检测转基因玉米的新方法. 对影响多重PCR扩增和毛细管电泳的因素进行了优化. 在优化的条件下, 本方法可以同时检测转基因玉米样品中3种外源基因. 经序列测试证实, 三重PCR 扩增产物的序列与原基因完全一致, 表明扩增结果可靠. 该方法能检出0.05% MON810转基因玉米成分, 远低于欧盟对转基因食品规定标识的质量分数阈值(1％). 该方法对玉米及其制品的检测结果与实时荧光PCR方法的检测结果一致, 与传统的琼脂糖凝胶电泳法相比, 具有特异性高\, 快速及灵敏等优点, 适用于玉米中转基因成分以及转基因玉米MON810品系的快速筛选、 鉴定和检测, 能满足我国实施转基因食品标签法规的要求.     

毛细管电泳-激光诱导荧光检测肌腱和肌腱细胞中的羟脯氨酸

建立毛细管电泳-激光诱导荧光检测(CE-LIF)分析羟脯氨酸的方法。肌腱和肌腱细胞中的胶原蛋白碱水解生成氨基酸,经异硫氰酸荧光素(FITC)衍生,采用LIF-CE分离测定胶原蛋白特异性氨基酸-羟脯氨酸。羟脯氨酸在0.5μg/L~8×103μg/L浓度范围内线性关系良好;检出限为0.5μg/L。相对迁移率和相对峰高的相对标准偏差(RSD)分别为5.0%和6.1%。测定了60份肌腱和9份细胞样品,加标回收率为95%~110%。将所建立的毛细管电泳方法与高效液相色谱法(HPLC)进行比较,两者测定结果相对误差为-1.9%~2.0%。本法仅需一次荧光标记,操作简单、快速灵敏,12min内完成一个分析周期,适于测定肌腱和细胞样品。     

芯片毛细管电泳-激光诱导荧光-电荷耦合器件检测系统

采用自组建的芯片毛细管电泳－激光诱导荧光－电荷耦合器件（CCD）检测系统在数十秒内满意地分离了曙红和荧光素。设计了一种进样、分 离电路,可以有效地消除进样通道的样品溶液向分离通道的渗漏。解决了由这种渗漏所引起的电泳峰变宽、拖尾等问题。提高了芯片毛细管电泳的分辨率和分离效率。     

毛细管电泳-473 nm固体激光诱导荧光检测系统的建立

以发射波长473nm的半导体激光泵浦固体激光器(LD DPSSL)为激发光源,研制了一种小型模块化激光诱导荧光检测器。以异硫氰酸荧光素(FITC)为荧光探针,毛细管电泳柱上检测(0.05mmi.d)评价了该体系,得到了5×10-12mol L的浓度检出限。利用该系统考察了氨基酸、实际样品中B族维生素的检测。     

高效毛细管电泳-激光诱导荧光检测生物活性肽的研究

使用毛细管电泳 -激光诱导荧光 -增强型电荷耦合器件(CE -LIF -ICCD)系统 ,用异硫氰酸荧光素(FITC)柱前衍生了亮脑啡肽、甲硫脑啡肽、血管紧张肽、P物质4种生物活性肽 ,优化了实验条件 ,在8min内 ,对它们进行了快速分离检测 ,血管紧张肽的质量检出限达到2.8×10-18 mol。     

基于适配体亲和毛细管电泳-激光诱导荧光检测的凝血酶分析

以一种高亲和力适配体作为亲和荧光探针,以自建的毛细管电泳-激光诱导荧光(CE-LIF)检测装置为基础,建立了一种高灵敏、快速测定人凝血酶的方法。荧光标记的凝血酶适配体特异性地与凝血酶结合并形成稳定的凝血酶-适配体复合物,采用CE-LIF对复合物进行分离检测,从而测定凝血酶浓度。探讨了盐离子种类及浓度对适配体与凝血酶结合的影响,并在选定的电泳条件下对凝血酶检测的线性范围、检出限和重现性进行了测定。结果表明,盐离子存在的条件下适配体与凝血酶的亲和力降低,不利于两者的结合;人血清溶液中,凝血酶浓度在0.25～10 nmol/L范围内与复合物峰面积具有良好的线性相关性(r20.991),检出限(S/N3)为55.6 pmol/L;精密度和回收率测定结果均能满足分析的要求。     

毛细管电泳-激光诱导荧光检测分枝杆菌脱氧核糖核酸限制性内切酶谱

建立了毛细管电泳分离-激光诱导荧光检测（CE-LIFD）分析分枝杆菌脱氧核糖核酸（DNA）限制性内切酶谱的新方法。用聚合酶链反应(PCR)扩增分枝杆菌hsp65基因的长度为439 bp的片段,该扩增片段经限制性内切酶BstEⅡ和 HaeⅢ酶切后,分别用CE-LIFD装置和常规琼脂糖电泳（AGE）对比检测酶切片段。对PCR扩增片段的酶切样品的预处理和CE条件进行了优化,获得了8种分枝杆菌DNA的限制性内切酶谱图。 DNA片段相对迁移时间的相对标准偏差（RSD）≤3.6%。结果表明,CE的分离效能明显高于AGE,是研究DNA限制性内切酶谱的更有效的检测手段。     

Determination of Yersinia enterocolitica in Food by Capillary Electrophoresis with Laser Induced Fluorescence Detection

A duplex polymerase chain reaction (PCR)-capillary electrophoresis-laser induced fluorescence (CE-LIF) method was developed to determine Yersinia enterocolitica in food sensitively, rapidly, and reliably. Two sets of primers were selected to amplify the genus-specific 16 S ribosomal RNA gene and ail gene associated with the pathogenicity of Yersinia enterocolitica. The parameters of duplex PCR and the conditions for CE-LIF were optimized. Under the optimum conditions, the PCR products of Yersinia enterocolitica were determined within twenty minutes. Alignment analysis showed favorable agreement with published sequences from GenBank, indicating that the primers were specific and the PCR results were reliable. The method detected 16 colony forming units per milliliter pathogenic Yersinia enterocolitica. The intraday precision of migration time of the DNA marker and the PCR products were between 1.13 and 1.81 percent. In summary, a new method combining duplex PCR and CE-LIF is reported for specific, sensitive, and reproducible detection of Yersinia enterocolitica in food with low sample consumption and cost.     

Construction of Post-column Micro-membrane Reactor for Protein Analysis in Capillary Electrophoresis with Laser Induced Fluorescence Detection

Proteomics is becoming more and more mature,but the detection of low abundance proteins is still a difficult task.Laser-induced fluorescence(LIF) detection is one of the most sensitive detection methods in a capillary electrophoresis(CE)system[1-3].However,most proteins do not exhibit favourable native fluorescence,a derivatization procedure is necessary for LIF detection of proteins.Since the derivatization reaction between protein and fluorescent reagent takes place after the separation of protein,the separation cannot be compromised by multiple derivatization products,the post-column derivatization becomes an attractive method for derivatization in CE-LIF system[4'5].The gap reactor,coaxial reactor and sheath flow cuvette reactor have been widely used for post-column derivatization in CE-LIF system[6].The gap reactor is constructed by aligning separation and reaction capillaries with a small gap[7,8].     

Detection of Copper Ion with Laser-Induced Fluorescence in a Capillary Electrophoresis Microchip

A capillary electrophoresis microchip coupled with a confocal laser-induced fluorescence (LIF) detector was successfully constructed for the analysis of trace amounts of heavy metals in environmental sources. A new fluorescence dye, RBPhOH, synthesized from rhodamine B, was utilized in a glass microchip to selectively determine copper with high sensitivity. A series of factors including running buffer concentration, detection voltage, and sample loading time were optimized for maximum LIF detector response and, hence, method sensitivity.     

Determination of Riboflavin in Cereal Grains by Capillary Electrophoresis with Laser-Induced Fluorescence Detection with On-Line Concentration

A very simple, sensitive, and reliable method for the routine determination of riboflavin in cereal grains by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was established. Two on-line concentration techniques, namely, stacking in reverse migrating micelles (SRMM) and sweeping, were examined to enhance the detection signal. The optimum separation buffer consisted of 20 mM phosphoric acid and 140 mM sodium dodecyl sulfate (SDS), and a hydrodynamic injection of 30 s at 0.5 psi was confirmed for the introduction of a sample. In addition, it was found that salt in sample matrix influenced the performance of SRMM, showing the standard addition method was required for the quantitative analysis. The linearity of the method was good with a range of 0.5–500 ng/mL (r = 0.9990). The limit of detection (LOD) was 0.29 ng/mL at a signal-to-noise ratio of 3. This procedure presented was successfully applied to determine riboflavin in 18 samples of 9 types of cereal grains. The recovery rates varied from 94% to 98%, and the relative standard deviation (RSDs) was less than 4.1%.     

蛋白质样品的非竞争性毛细管电泳免疫分析激光诱导荧光检测

以牛血清白蛋白与其单克隆抗体为样品,初步研究了蛋白质的非竞争性毛细管电泳免疫分析方法。混合温育可以在２０ｍｉｎ内达到平衡,电泳分离在１２ｍｉｎ内完成。当示踪物浓度为３２０ｎｍｏｌ／Ｌ时,所得到的标准曲线的线性范围为８￣１５０ｎｍｏｌ／Ｌ,检出限为５ｎｍｏｌ／Ｌ,并考察了缓冲溶液的浓度、ｐＨ、分离电压等因素的影响。