量子点荧光微球免疫层析试纸条定量检测恶性疟原虫

以羧基化Cd Te/Zn Se量子点荧光微球为荧光标记物,采用1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)法偶联抗恶性疟原虫富组氨酸蛋白(Pf)单克隆抗体制备荧光探针;以羊抗恶性疟原虫组氨酸多克隆抗体和驴抗鼠二抗分别喷涂硝酸纤维膜,形成试纸条检测线和质控线,建立了免疫层析试纸条定量检测血清中恶性疟原虫的方法。所使用的羧基化量子点荧光微球的荧光强度为单个量子点的2800倍。实验结果表明,该荧光试纸条定量检测血清中恶性疟原虫线性范围为5.8~8010 Parasite/μL,最低灵敏度达到5.8 Parasite/μL,单个样品检测时间只需15 min。加标回收实验显示,试纸条批内回收率为93.0%~111.8%,批间回收率为98.3%~115.1%,且批内、批间的相对标准偏差均小于5%。     

新型“磁包金”金磁纳米粒子免疫层析试纸条定量检测血清中的人绒毛膜促性腺激素

将油酸修饰的氧化铁纳米粒子(Oleic acid coated iron oxide nanoparticles, OC-IONPs)与油胺修饰的金纳米粒子(Oleylamine-coated gold nanoparticles, OA-AuNPs)通过乳液自组装法共封装在聚合物基质中,合成了具有"磁包金"核壳异质结构的新型金磁纳米粒子(Magnetic coated gold nanoparticles, MGNPs)。由于OA-AuNPs聚集在内核, OC-IONPs分布于外壳,有效避免了OA-AuNPs的磁屏蔽效应,相较于传统"金包磁"型纳米结构,此MGNPs具有高的磁饱和强度(为初始氧化铁纳米粒子的80%)和强的吸光度(为传统30 nm胶体金纳米粒子的12.5倍)。进一步以此MGNPs为免疫层析(Immunochromatographic assay, ICA)的新型双功能标记探针,以人绒毛膜促性腺激素(Human chorionic gonadotropin, HCG)为模型检测物,建立了高灵敏检测人血清中HCG的免疫层析方法(MGNPs-ICA)。本研究所构建的试纸条定量...     

氟苯尼考胶体金免疫层析定量检测方法的建立

建立了定量检测氟苯尼考的胶体金免疫层析方法.对胶体金标记抗体时溶液pH和抗体浓度、金标抗体用量、检测线上抗原浓度以及检测时间进行了优化.采用胶体金试纸条读取仪测定试纸条检测线和质控线的信号强度,以标准品的浓度为横坐标,阳性样本和阴性样本的检测线/质控线的信号比值(Bx/B0)为纵坐标建立标准曲线.结果表明,胶体金免疫层析试纸定量检测氟苯尼考的线性范围为0.1~1.5 ng/mL,检出限为0.08 ng/mL,检测时间为15 min.本方法具有简便、快速和可定量等特点,适于大批量样品的现场筛查.     

五氯酚免疫层析检测试纸条的研究

利用胶体金免疫层析技术建立了一种快速检测五氯酚(PCP)残留的方法。采用柠檬酸三钠还原法制备大小一致、分布均匀、粒径为20 nm的胶体金颗粒,以此标记五氯酚抗体,制备金标抗体。将五氯酚包被抗原和羊抗鼠二抗分别结合于硝酸纤维膜上,依次将型号Millipore135硝酸纤维膜、型号VL78金标垫、型号SB06样品垫及吸水纸组装于PVC底板上,组装成胶体金免疫层析检测试纸条。通过试纸条上颜色的深浅,检测样品中PCP的残留量。试纸条检出限为10 ng/mL,检测时间为5 min。该方法检测所需试剂已预先包被在试纸条上,操作简单、重复性好、成本低廉,可用于五氯酚的现场快速检测。     

金纳米棒免疫层析试纸条定量检测前列腺特异性抗原

建立了一种基于金纳米棒(GNR)的免疫层析方法,用于快速定量检测前列腺特异性抗原(PSA)。以GNR为标记探针,通过包裹羧基化的聚合物后,采用共价偶联的方式连接抗PSA单克隆抗体形成偶联物,以鼠抗PSA单克隆抗体和羊抗鼠IgG分别喷涂硝酸纤维素膜,形成试纸条的检测线和质控线,采用三明治夹心法构建GNR免疫层析试纸条,用于PSA的定量检测。结果表明,GNR免疫层析试纸条特异性强,稳定性好,灵敏度高,对PSA检测的线性范围为0.1～50 ng/mL,检出限为0.1 ng/mL,批内和批间变异系数均小于10%,且具有良好的保存稳定性。本方法可用于检测血清中PSA的浓度水平,对于前列腺癌的早期诊断、监测治疗及预后判断具有重要意义。     

菊酯类农药广谱型免疫层析试纸条的研究及应用

建立了菊酯类农药广谱型免疫层析检测方法,可同时检测水果、蔬菜中12种甲氰菊酯、溴氰菊酯、氯氰菊酯、三氟氯氰菊酯农药残留。以粒径20 nm的胶体金标记羊抗小鼠IgG抗体于金标垫上,分别固定包被原(检测线,T线)、兔抗山羊 IgG 抗体(质控线, C 线)于硝酸纤维素膜( NC 膜)上,与吸水垫及聚氯乙烯( PVC)底板组合成层析试纸条。10 g样品经乙腈提取,PBS稀释4倍,取100μL与菊酯类农药的单克隆抗体混合后,直接用于试纸条检测。结果表明,试纸条对甲氰菊酯、溴氰菊酯、氯氰菊酯、三氟氯氰菊酯农药的裸眼观察检测灵敏度为0.5,5.0,5.0和5.0μg/mL,检测时长为8~10 min,采用QuEChERS方法,方法检测灵敏度可提高16倍。对蔬菜和水果的方法验证表明,除辣椒基质干扰呈假阳性外,其它样本的检测结果均与GC方法结果一致。试纸条采用金标羊抗小鼠IgG二抗的方法,使检测结果的重现性更好、更稳定,且抗基质干扰能力强,为菊酯类农药的累积毒性检测提供了新方法。     

基于荧光硅球的克伦特罗快速定量免疫层析试纸条的研制

采用荧光硅球为标记物制备了快速定量检测克伦特罗(CLE)的荧光硅球免疫层析试纸条。通过正交实验得到最优荧光硅球抗体标记量、硅球垫抗体标记物用量以及检测线抗原浓度。在最优条件下,试纸条线性范围为0.28~3.3 mg/L。猪尿样品CLE加标回收率为81.7%~101%,表明此试纸条可实现猪尿中CLE残留的快速定量检测。     

基于BHHCT-Eu3+@SiO2荧光稀土二氧化硅纳米颗粒的免疫层析试纸条检测卡那霉素

采用微波辅助合成的荧光稀土二氧化硅纳米颗粒(BHHCT-Eu3+@SiO2)为标记物,建立了快速定量检测卡那霉素(Kana)残留的荧光免疫层析方法.实验结果表明,微波辅助合成的BHHCT-Eu3+@SiO2纳米颗粒呈球形,粒径约36 nm,具有良好的荧光发射性能,最大吸收波长和最大发射波长分别为343和615 nm.将BHHCT-Eu3+@SiO2与卡那霉素抗体(Kana-ab)通过醛基化葡聚糖交联,合成了荧光标记抗体Eu3+-Kana-ab,结合定量侧向层析读数仪,建立了牛奶中Kana残留的快速定量检测方法,对Kana的检出限(IC10)为0.85 ng/mL,半数抑制浓度(IC50)为12.76 ng/mL,检测范围(IC20-IC80)为3.0~76.0 ng/mL,牛奶中的Kana的加标回收率范围为93.7%~97.4%,RSD为3.1%~4.6%,与Kana类似物的交叉反应均<1%.牛奶中Kana残留的测定结果与ELISA方法相关性良好.     

基于纳米金星的层析试纸条用于检测HIV-DNA

构建了基于纳米金星(AuNSs)的快速、简单且可视化的横向流层析试纸条(LFTS),并用于检测人类免疫缺陷病毒的DNA。采用一步法合成AuNSs,并对其进行生物功能化,目标物与DNA修饰的AuNSs结合。该复合物通过碱基互补配对原则被捕获在测试线上,依据测试线上纳米金星颜色的变化进行定性和半定量分析,使用便携式读条器在最佳实验条件下进行定量分析。该方法的线性范围为0.2~50 nmol/L,检出限为0.14 nmol/L。相同条件下,该方法比传统纳米金试纸条的灵敏度约高5倍。该方法可用于人血清中HIV DNA的检测,结果良好。     

基于上转换发光技术和免疫层析技术对氯霉素定量的检测方法

基于上转换发光技术和免疫层析技术对氯霉素定量的方法,属于纳米生物标记、免疫学及光学检测领域。将200~300 nm的上转换荧光纳米颗粒进行羧基化修饰,使之成为水溶性好、分散性高且易于与生物分子偶联的标记物;将样品垫、上转换标记物处理过的结合垫、抗原抗体点样过的硝酸纤维素膜(NC膜)和吸水纸通过粘性底衬结合起来,抗原为氯霉素–BSA作检测线(T),抗体为羊抗鼠抗体作质控线(C),且相距0.5 cm。用切条机将组装好的试纸切成长6 cm、宽4 mm的试纸条,装备成壳,建立免疫层析试纸;将不同浓度梯度的氯霉素标准抗原加于试纸壳中的样品垫里,经10~15 min静置后,放于上转换检测器里进行检测。本发明制备工艺和操作简单,不需要复杂的仪器设备,快速、灵敏,能准确定量检测食品中氯霉素含量。     

快速诊断疟疾的免疫层析试条的研制

开发成功了一种能快速、灵敏、高特异性检测疟疾的免疫层析试条。选自恶性疟原虫富组蛋白Ⅱ－一级结构中的九肽（ＡＨＨＡＨＨＡＡＤ），经人工合成后作为免疫原，生产了一些单表位单抗；完成了一些单抗的纯化与鉴定；制取了金标单抗并对其吸收光谱进行了分析；选取了包被单抗和金标单抗间的配对；比较了影响检测的各种因子。当用此试条检测６２名已用常规血间检法确证患有疟疾个体的全血，准确率达９３．５４％。     

基于新型量子点荧光微球的氯霉素免疫层析试纸条的制备和应用

以羧基化CdTe/ZnSe量子点荧光微球为标记物,通过1-乙基-(3-二甲基氨基丙基)碳二亚胺/N-羟基琥珀酰亚胺(EDC/NHS)活化法将氯霉素(CAP)单克隆抗体与量子点荧光微球偶联制备荧光探针.氯霉素全抗原(CAP-HS-BSA)及羊抗鼠二抗分别喷涂硝酸纤维素膜,形成检测线(T线)和质控线(C线),组装成新型氯霉素量子点荧光微球免疫层析试纸条,建立了快速、定量检测牛奶中CAP的方法.本研究开发的量子点荧光微球试纸条可在15 min内完成牛奶样品中CAP的定量检测,线性范围为0.1~100.0μg/L,检出限为0.1μg/L.牛奶样品CAP的加标回收率为93.3%~97.9%,相对标准偏差在4.9%~6.9%之间.     

Comparison of Fluorescence Immunochromatographic Assay Strip and Gold Colloidal Immunochromatographic Assay Strip for Detection of Microcystin

Abstract

Microcystins are a family of cyclic polypeptides produced by different species of cyanobacteria (blue green algae), which can form blooms in lakes and water reservoirs. However, it is difficult to detect microcystins directly in the water since the concentration of the toxins in water is usually too low. It is necessary to develop a simple and quick method to detect microcystins. In this paper, different detection characteristics of fluorescence immunochromatography and gold colloidal immunochromatography for analysis of cyanobacterial toxins were studied. These two immunochromatography assays are easy to perform, rapid, sensitive, and their quantitative range is within detectable microcystin concentrations in water samples. The fluorescence immunochromatographic system has the unique advantages of low detection limit, and satisfactory accurate results are obtained. The gold colloidal immunochromatographic system has the strong advantage of direct detection of microcystins at the test site without having to bring the samples back to the laboratory. Therefore, these two techniques supplement each other.     

LC-MS分析饲料中的己烯雌酚

己烯雌酚(diethylstilbestrol)是一种非甾体激素,曾被广泛用于促进动物生长,增加瘦肉率,提高饲料转化率。从而其药物原形或代谢产物不可避免地残留于食品动物的器官、组织或通过排泄物进入外界环境成为环境雌激素,通过食物链进入消费者体内或通过人与污染的外界环境的接触而在体内发挥雌激素的效果而影响消费者健康。2002年农业部第176号公告和第193号公告中规定禁止使用己烯雌酚,但由于己烯雌酚作为畜禽增重剂效果好、经济回报高,仍有一些不法饲养业主受利益驱使而违法使用。本实验建立了高效液相色谱-质谱联用法(LC-MS)测定饲料中己烯雌酚,旨在为政府从源头上监控饲料中己烯雌酚的违法使用提供准确可靠的检测方法。     

Determination of Salmonella typhimurium by a Fluorescence Resonance Energy Transfer Biosensor Using Upconversion Nanoparticles as Labels

A Salmonella typhimurium (S. typhimurium) biosensor based on a fluorescence resonance energy transfer between upconversion and gold nanoparticles is reported. NaYF₄:Yb,Er nanoparticles were synthesized and modified with a S. typhimurium target DNA complementary sequence to form the sensor. Gold nanoparticles were modified with a S. typhimurium target DNA complementary sequence to constitute the quenching probe. In the presence of S. typhimurium target DNA, gold and upconversion nanoparticles formed a sandwich complex, and the upconversion fluorescence resonance energy transfer occurred. Under the optimal conditions, the relative fluorescence was proportional to the concentration of S. typhimurium target DNA in the range of 0.001 pmol/L to 1 pmol/L with a limit of detection of 1 fM. S. typhimurium was detected from 30 cfu/mL to 5150 cfu/mL with a detection limit of 3 cfu/mL. The procedure was successfully applied to determine S. typhimurium in milk and validated by a traditional plate counting method. The developed upconversion fluorescence resonance energy transfer method is simple, fast, sensitive, specific, and incorporates nanomaterials in biosensor design.     

Ultrasonic Synthesis of Gold Nanoparticles and Its Use in Immunochromatographic Assay for Detection of Kanamycin

An ultrasonic method to synthesize gold nanoparticles with uniform size was reported. Effects of ethanol concentration, solution pH, ultrasonic irradiation power, and time on the formation of gold nanoparticles were investigated. The obtained gold nanoparticles were characterized by ultraviolet-visible (UV-vis) absorption spectra and scanning electron microscope (SEM). The method performed in ethanol solution under ultrasonic irradiation is friendly to the environment. Furthermore, a nanogold-labeled probe was used to develop an immunochromatographic method for detection of kanamycin.     

Development of an Up-Conversion Homogenous Immunoassay for the Determination of Diethylstilbestrol in Water

This article describes an up-conversion immunoassay (UCIA) method for the determination of diethylstilbestrol with homogenous and rapid properties. Biotinylated diethylstilbestrol was used as a bridge for the formation of a streptavidinated beads-biotin-diethylstilbestrol‐antibody-acceptor beads complex in the immunoassay. The complex gave out imitation fluorescence at a wavelength of 680 nm when its excitation wavelength was at 615 nm as a result of the generation of singlet oxygen. The optimal test conditions and analytical performance of the method were studied. The optimized reaction time of ≥ 80 min was determined. The best buffer of phosphate buffer containing the surfactant of Tween 20 was established. Additionally, the sound analysis result can be obtained with highest antibody concentration, which was set in the experiments. Calibration curve, fit by 4-parameters formulation, resulted in R² values of 0.9989, a linear range from 0.025 to 12.5 ng/mL and the limit of detection (LOD) of 0.01 ng/mL. The results of trace diethylstilbestrol concentrations in water samples showed recoveries ranging from 92.0% to 95.0%, and coefficients of variation between 3–8%. The data suggest that UCIA method is a good method with sensitivity for the determination of diethylstilbestrol in water.     

Development of an Ultra-sensitive Chemiluminescence Enzyme Immunoassay for the Determination of Diethylstilbestrol in Seafood

An ultra-sensitive indirect competitive chemiluminescence enzyme immunoassay was developed for screening diethylstilbestrol in fish and shrimp samples. The concentration of diethylstilbestrol that caused 50% inhibition of the binding enzyme marker (IC₅₀) was 0.32 ng/mL and the limit of detection was 0.0068 ng/mL; the linear range was from 0.028 ng/mL to 3.60 ng/mL. The assay showed cross-reactivity of 7.1% and 2.8% with dienestrol and hexoestrol, respectively, but negligible cross-reactivity with estradiol, estrone, ethinyloestradiol, and progestin. The recovery from spiked fish and shrimp samples varied from 68.5% to 92.5%, and the mean coefficients of variation within groups and between groups were 6.2% and 8.0%, respectively. Our results indicated that the assay is a simple, sensitive, specific, and accurate method for screening fish and shrimp samples for diethylstilbestrol.     

Recent Advances in Controlled Synthesis of Upconversion Nanoparticles and Semiconductor Heterostructures

It's of great importance for construction of upconversion nanoparticles (UCNPs)/semiconductor heterostructures activated by near infrared light, which have gained worldwide research interests owing to important applications in photocatalysis, solar cells, nanomedicine, and etc. In this review, we highlight the synthetic strategies developed to fabricate upconversion nanoparticles based heterostructures, such as chemical epitaxial growth method, electrospinning technique, self‐assembly method, hydrothermal method, and etc. Numerous examples are given concerning the use of the strategies to fabricate various microstructures/nanostructures incorporated with UCNPs and semiconductors materials. The latest advances and perspectives in the synthetic strategies and preparation of this kind of composite nanostructures are made.     

基于适配体胶体金侧向层析试纸快速检测卡那霉素的方法研究

该文基于适配体以及非巯基化核酸修饰的胶体金纳米探针（AuNPs@polyA－DNA）,建立了一种新型的卡那霉素胶体金侧向层析试纸条。分析了试纸条各组装元件,包括适配体浓度、链霉亲和素（SA）与Biotin－DNAT的比例、SA－生物素（Biotin）－DNAT偶联物浓度以及孵育时间与温度等,对显色反应的影响。最佳实验条件为：缓冲液为4xSSC（0.5% Tween 20）,SA与Biotin－DNAT的最佳摩尔比为1∶6,检测区喷涂偶联物SA－Biotin－DNAT浓度为4 μmol/L,适配体浓度为10 nmol/L,室温孵育时间为20 min。在优化条件下,该试纸条对卡那霉素的肉眼分辨浓度为25 ng/mL,线性范围为5.0～125 ng/mL,检出限为1.5 ng/mL。用于蜂蜜中卡那霉素的检测,其回收率为95.1%～105%,相对标准偏差（RSD）为3.4%～8.5%。该试纸条具有灵敏度高、特异性好、架构简单、重复性高等优点,可用于实际样品中卡那霉素的检测。