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。     

铽增敏高效液相色谱柱后衍生荧光检测法同时检测鸡肉中的三种氟喹诺酮类药物残留

基于氟喹诺酮类药物与铽离子形成配合物后的荧光增强作用,建立了同时检测鸡肉中氟喹诺酮类(FQs)药物环丙沙星、诺氟沙星和恩诺沙星残留的Tb3+增敏高效液相色谱（HPLC）柱后衍生荧光检测方法。优化的实验条件如下：流动相为0.05 mol/L 醋酸/醋酸钠缓冲液(pH 6.0)-乙腈(体积比为89∶11),色谱柱为Hypersil BDS-C18,柱温40 ℃,流速0.8 mL/min;Tb3+浓度为8×10-5 mol/L;衍生反应温度40 ℃,衍生泵流速0.5 mL/min;荧光检测激发波长271 nm,发射波长545 nm。实验结果表明,将上述3种药物以1.0,10.0,50.0,100.0 ng/g水平添加到鸡肉后的回收率范围为66.3%~88.0%,相对标准偏差（RSD）均小于15.0%。定量分析的线性范围为0.1~500 ng/mL,方法的日内和日间RSD均小于13.0%;最低检出限分别为0.05(环丙沙星)、0.05(诺氟沙星)和0.08(恩诺沙星)ng/g,比前人报道的非衍生高效液相色谱荧光检测法检测FQs药物的灵敏度有极大的提高。该项研究为FQs药物多残留检测提供了灵敏度更高的新方法。     

高效液相色谱-荧光检测法测定牛奶中氯霉素的残留量

建立了对牛奶中氯霉素的残留量进行检测的高效液相色谱-荧光检测方法。氯霉素还原后在温和条件下与荧光胺发生衍生化反应,采用十八烷基键合硅胶固定相,以乙腈/四氢呋喃/0.02 mol/L醋酸钠-醋酸缓冲液(pH 6.0)(体积比为16∶8∶76)为流动相,流速1.0 mL/min,柱温40 ℃,荧光检测激发波长为410 nm,发射波长为508 nm。在上述实验条件下,氯霉素检测的线性范围为0.4~800 μg/L (r2=0.9999),检出限为0.2　μg/L。当空白样品中氯霉素添加水平为2~40 μg/L时,该方法的回收率为66.6%~92.8%,相对标准偏差为4.5%~9.4%。该方法适用于牛奶中氯霉素痕量残留的监测,具有干扰小、选择性好、灵敏度高等优点。     

呋喃它酮代谢物时间分辨荧光免疫分析法的建立与应用

建立了Eu3+标记的间接竞争时间分辨荧光免疫分析法( Indirect competitive time-resolved fluoroimmu-noassay , ic-TRFIA)用于鱼肉样品中呋喃它酮代谢物AMOZ的检测。通过单因素实验考察了包被抗原浓度、抗体稀释倍数、竞争反应时间等参数对方法灵敏度的影响。结果表明,ic-TRFIA的最佳反应条件为：包被抗原浓度为0.25μg/mL,抗体稀释5×104倍,最佳竞争时间为50 min。在优化的条件下,方法的检出限( LOD, IC10)为0.01 ng/mL,半抑制浓度(IC50)为0.26 ng/mL,线性范围(IC20~IC80)为0.025~2.83 ng/mL,对鱼样中AMOZ回收率在78.0%~86.0%之间,各样品变异系数均小于15%,与HPLC-MS/MS对比检测结果显示相关性良好。本方法灵敏度高、特异性好,能够满足实际样品的检测需求。     

基于金纳米簇探针的荧光猝灭检测铁离子

以D-色氨酸为保护剂和还原剂, 采用水热法快速制备了具有强荧光的金纳米簇(D-Trp@AuNCs); 以其作为荧光探针, 建立了基于荧光猝灭的选择性高灵敏检测Fe³⁺的传感方法. 利用透射电子显微镜(TEM)、 紫外-可见光谱(UV-Vis)和红外光谱(IR)等手段对制备的金纳米簇进行了表征, 并利用荧光光谱研究了D-Trp@AuNCs的荧光性能. 结果表明, D-Trp@AuNCs具有较好的生物相容性, 其最大激发波长为370 nm, 最大发射波长为460 nm; 向金纳米簇溶液中加入Fe³⁺后, D-Trp@AuNCs的荧光发生明显猝灭, 其猝灭程度与Fe³⁺的浓度在0.3~500.0 μmol/L范围内呈现良好的线性关系, 检出限为33.1 nmol/L(S/N=3). 将该荧光探针用于实际水样中Fe³⁺的检测, 回收率为86.6%~106.5%.     

以葛根素为辣根过氧化物酶底物的新体系及其分析应用

以一种天然活性成分葛根素(Puerarin)为辣根过氧化物酶(HRP)底物建立了葛根素-辣根过氧化物酶-过氧化氢反应新体系. 在反应体系中 HRP 催化H₂O₂ 氧化葛根素(弱荧光)形成二聚体产物(强荧光), 该产物在315 nm 的激发光下能发射波长为478 nm的强荧光, 并且反应体系荧光强度增加与HRP量在一定浓度范围内呈线性相关. 根据此关系和竞争型免疫定量原理, 以兔布氏杆菌抗体为分析对象建立了基于葛根素的酶联荧光免疫传感分析新方法. 对葛根素性质的研究结果证实, 葛根素在空气中稳定、对温度稳定, 对H₂O₂＋HRP 敏感性优于传统底物如对羟基苯乙酸、Amplex Red和高香草酸. 优化了酶联荧光免疫传感分析方法的实验条件如HRP-BrAb 用量、温度等. 运用新体系测定了兔血清样品的布氏杆菌抗体, 该方法线性范围为1.3～120 ng/mL, 检测限为1.3 ng/mL (3σ), 相对标准偏差为3.8%.     

日光辐射光还原合成左旋多巴胺功能化的荧光发射的银纳米簇和铁离子检测

以左旋多巴胺(L-3,4-dihydroxyphenylalanine,DOPA)为稳定剂,采用日光辐射光还原法,合成了强荧光发射的银纳米簇(silver nanoclusters,Ag NCs)。透射电镜分析表明,所合成的Ag NCs表现亚纳米非晶态结构。Ag NCs在可见-近红外波长范围内(400~750 nm)有明显光吸收带,最大荧光激发和发射峰分别为550和630 nm,荧光量子产率为2.3%(相对于罗丹明B)。Ag NCs的荧光强度与合成时的日光辐射时间、DOPA浓度以及pH值等因素有关。进一步优化了合成Ag NCs的条件。基于荧光猝灭原理,所合成的DOPA功能化的Ag NCs能选择性地灵敏响应Fe3+。修饰在Ag NCs表面的配体DOPA能够选择性地结合Fe3+,导致Ag NCs显著聚集,伴随荧光猝灭。Ag NCs具有的较高量子产率和红荧光发射特性,有利于提高Fe3+的分析灵敏度。     

荧光检测-高效液相色谱法测定水果、蔬菜中抗蚜威残留量

建立了水果、蔬菜中抗蚜威残留的荧光检测-高效液相色谱法。样品以乙腈提取,固相萃取氨基小柱(LC-NH2)净化,Waterscarbamateanalysis(3.9mm×150mm,4μm),V(甲醇)∶V(水)∶V(乙腈)=16∶68∶16为流动相,柱温30℃,流速为1.5mL/min进行分离,用荧光检测器进行检测,激发波长和发射波长分别为317nm,392nm。回收率在98.5%~105.4%,相对标准偏差为3.0%~4.6%,检出限为0.01mg/kg。     

基于牛血清白蛋白修饰的金纳米簇的荧光光谱法快速测定维生素B12药片中Co2+的含量

采用化学还原法合成牛血清白蛋白(BSA)修饰的金纳米簇(AuNCs),基于Co²⁺对AuNCs的荧光猝灭作用,提出了一种快速、简便、灵敏测定Co²⁺含量的方法。以柠檬酸为还原剂、BSA为保护剂、氯金酸为原料合成AuNCs。分取0.20 mL AuNCs溶液,加入0.6 mL pH 9.0碳酸钠-碳酸氢钠缓冲溶液,用水稀释至1.00 mL,在发射波长630 nm处测量上述体系(空白体系)的荧光强度F₀。取10片维生素B₁₂药片,研磨后,分取0.127 0 g,用水溶解并稀释至100 mL,分取10μL于空白体系中,混匀后静置反应5 min,测量荧光强度F,利用荧光强度的差值ΔF(ΔF=F₀-F)进行定量。结果显示：合成的AuNCs分布均匀,Co²⁺对AuNCs体系有荧光猝灭作用,属于动态猝灭过程。添加10倍Co²⁺浓度的干扰离子,Fe²⁺、Pb²⁺、Al³⁺、Zn     

基于免疫磁分离的荧光微球免疫层析法检测猪霍乱沙门氏菌

建立了基于免疫磁分离的荧光微球免疫层析法,检测猪霍乱沙门氏菌.待检样品经免疫磁分离富集和热洗脱处理后,用荧光微球免疫层析试纸条进行检测.每毫克纳米磁珠标记30μg抗体制备的免疫磁珠,对浓度为102 ～ 106 CFU/mL的猪霍乱沙门氏菌的捕获率均大于90％,特异性好;在pH=6时,以300μ,g/mg猪霍乱沙门氏菌单抗11D8-D4标记荧光微球,制备免疫荧光微球;以2.0 mg/mL猪霍乱沙门氏菌单抗5F11-B11喷涂检测线(T线),以1.0 mg/mL驴抗鼠IgG喷涂质控线(C线),制备免疫层析试纸条.采用建立的基于免疫磁分离的荧光微球免疫层析方法检测猪霍乱沙门氏菌,在PBS缓冲液中检出限为1.5×105 CFU/mL,牛奶中检出限为7.6×105 CFU/mL,与直接采用荧光微球免疫层析方法检测相比,检出限分别降低了10倍和200倍.本方法可有效富集牛奶中的沙门氏菌,避免了基质干扰,灵敏度大大提高,具有较好的应用前景.     

Development of a lateral flow fluorescent microsphere immunoassay for the determination of sulfamethazine in milk

The fluorescent microsphere has been increasingly used as detecting label in immunoassay because of its stable configuration, high fluorescence intensity, and photostability. In this paper, we developed a novel lateral flow fluorescent microsphere immunoassay (FMIA) for the determination of sulfamethazine (SMZ) in milk in a quantitative manner with high sensitivity, selectivity, and rapidity. A monoclonal antibody to SMZ was covalently conjugated with the carboxylate-modified fluorescent microsphere, which is polystyrene with a diameter of 200 nm. Quantitative detection of SMZ in milk was accomplished by recording the fluorescence intensity of microspheres captured on the test line after the milk samples were diluted five times. Under optimal conditions, the FMIA displays a rapid response for SMZ with a limit of detection of as low as 0.025 ng mL^?1 in buffer and 0.11 μg L^?1 in milk samples. The FMIA was then successfully applied on spiked milk samples and the recoveries ranged from 101.1 to 113.6 % in the inter-batch assay with coefficient of variations of 6.0 to 14.3 %. We demonstrate here that the fluorescent microsphere-based lateral flow immunoassay (LFIA) is capable of rapid, sensitive, and quantitative detection of SMZ in milk.

An ultrasensitive immunosensor array for determination of staphylococcal enterotoxin B

Staphylococcal enterotoxin B (SEB) is a potent gastrointestinal toxin and is heat resistant. SEB is also a potential bioterrorism agent. The ability to measure accurately very low amounts of staphylococcal enterotoxin B in food and other samples is very important. A highly sensitive and stable sandwich fluorescence immunoassay based on a pair of monoclonal antibodies against SEB which were produced by us was developed. Classical sandwich immunoassay was adopted and the glass slides were used as the base of the immunologic reaction. The functionalized fluorescent core-shell silica nanoparticles were used as labels. The fluorescence issued from the labels was detected by a laser-induced fluorescence millimeter sensor array detection platform. The fluorescence intensity has a linear relationship with the amount of SEB in the range of 50 pg/mL-5 ng/mL, and the detection limit of SEB was 20 pg/mL (the absolute detection limit was 0.02 pg). The relative standard deviation (RSD) for 5 parallel measurements of SEB (1 ng/mL) was 9.2%.     

Determination of bisphenol A in human breast milk by HPLC with column-switching and fluorescence detection

A highly sensitive HPLC method was developed for the determination of xenoestrogenic compound, bisphenol A (BPA) in human breast milk samples. After a two-step liquid-liquid extraction, BPA was derivatized with fluorescent labeling reagent, 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). The excess fluorescent reagent could be removed effectively using a column-switching system. The separation of DIB-BPA from endogenous materials in milk was carried out on two C(18) columns and fluorescence intensity was monitored at 475 nm with the excitation of 350 nm. A good linearity (r = 0.994) was observed of BPA in the concentration range of 0.2-5.0 ng mL(-1) in breast milk, and the detection limit was 0.11 ng mL(-1) at a signal-to-noise ratio of 3. Intra- and inter-day precision (RSD, %) were less than 8.7 and 10.4, respectively. Twenty-three breast milk samples of healthy lactating women were analyzed for the BPA concentration; the mean value was 0.61 +/- 0.20 ng mL(-1), with no correlation to the lipid content of milk samples.     

镝荧光探针的构建及对牛奶中四环素残留的检测

在乙醇体系中构建三元配合物的镝荧光探针,并利用盐酸四环素(TC)对镝荧光探针具有荧光猝灭作用,提出了一种检测牛奶中四环素残留的新方法.首先确定体系的激发波长为305 nm,发射波长为574 nm.在配比、加料顺序、时间等方面对镝荧光探针进行了条件优化,确定了镝离子(Dy³⁺)、磺基水杨酸(SSA)、三正辛基氧化磷(TOPO)的最佳配比为1:2:0.1和30 min最佳检测时间.其次建立了检测盐酸四环素的线性曲线,并获得检测范围为10^-6~2×10^-5 mol/L.最后,利用此荧光探针对处理的牛奶样品进行加样回收检测,回收率在96.9 %~104.4 %.实验证明建立的方法科学可行,对盐酸四环素具有高选择性.     

Lateral flow immunoassay for progesterone detection

A new express method based on lateral flow immunoassay (LFIA) for progesterone detection was developed. To increase the assay sensitivity an enzyme label (horse-radish peroxidase) was used instead of colloidal gold. An optimal assay format was chosen and the influence of a range of buffer supplements (detergents, proteins and sucrose) was investigated by enzyme-linked immunosorbent assay (ELISA). Linear range of LFIA was between 2 and 40 ng/mL in buffer. Limit of detection was 2 ng/mL, assay time was within 15 min.     

Dye-doped silica nanoparticle labels/protein microarray for detection of protein biomarkers

We report a dye-encapsulated silica nanoparticle as a label, with the advantages of high fluorescence intensity, photostability, and biocompatibility, in conjunction with microarray technology for sensitive immunoassay of a biomarker, interleukin-6 (IL-6), on a microarray format. The tris(2,2'-bipyridyl)ruthenium(ii) chloride hexahydrate (Rubpy) dye was incorporated into silica nanoparticles using a simple one-step microemulsion synthesis. In this synthesis process, Igepal CA520 was used as the surfactant, therefore, no requirement of cosolvent during the synthesis and the particle size was reduced comparing to the commonly used Triton surfactant system. The nanoparticles are uniform in size with a diameter of 50 nm. The microarray fluorescent immunoassay approach based on dye-doped silica nanoparticle labels has high sensitivity for practical applications with a limit of detection for IL-6 down to 0.1 ng mL(-1). The calibration curve is linear over the range from 0.1 ng mL(-1) to 10 ng mL(-1). Furthermore, results illustrated that the assay is highly specific for IL-6 in the presence of range of cytokines or proteins. The RuDS dye-labeled nanoparticles in connection with protein microarrays show the promise for clinical diagnosis of biomarkers.     

Rapid and sensitive lateral flow immunoassay for influenza antigen using fluorescently-doped silica nanoparticles

We report on a lateral flow immunoassay (LFIA) for influenza A antigen using fluorescently-doped silica nanoparticles as reporters. The method is taking advantage of the high brightness and photostability of silica nanoparticles (doped with the dye Cy5) and the simplicity and rapidity of LFIA. The nucleoprotein of influenza A virion (one of its most abundant structural proteins) was used as a model to demonstrate a performance of the LFIA. Under optimized conditions and by using a portable strip reader, the fluorescence-based LFIA is capable of detecting a recombinant nucleoprotein as low as 250 ng?·?mL^-1 using a sample volume of 100 μL, within 30 min, and without interference by other proteins. The successful detection of the nucleoprotein in infected allantoic fluid demonstrated the functionality of the method. By comparison with a commercial influenza A test based on gold nanoparticles as reporters, the system provides an 8-fold better sensitivity.

Electrophoresis-Assisted Multilayer Assembly of Nanoparticles for Sensitive Lateral Flow Immunoassay**

Lateral flow immunoassay (LFIA) is a rapid, simple, and inexpensive point-of-need method. A major limitation of LFIA is a high limit of detection (LOD), which impacts its diagnostic sensitivity. To overcome this limitation, we introduce a signal-enhancement procedure that is performed after completing LFIA and involves controllably moving biotin- and streptavidin-functionalized gold nanoparticles by electrophoresis. The nanoparticles link to immunocomplexes forming multilayer aggregates on the test strip, thus, enhancing the signal. Here, we demonstrate lowering the LOD of hepatitis B surface antigen from approximately 8 to 0.12 ng mL⁻¹, making it clinically acceptable. Testing 118 clinical samples for hepatitis B showed that signal enhancement increased the diagnostic sensitivity of LFIA from 73 % to 98 % while not affecting its 95 % specificity. Electrophoresis-driven enhancement of LFIA is universal (antigen-independent), takes two minutes, and can be performed by an untrained person.     

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.     

Determination of Aflatoxin M1 in Milk by a Magnetic Nanoparticle-Based Fluorescent Immunoassay

A sensitive and rapid magnetic nanoparticle-based fluorescent immunoassay for the determination of aflatoxin M1 in raw milk was developed. Aflatoxin M1 was converted to aflatoxin M1-o-carboxymethyl oxime. The aflatoxin M1-oxime was used for the preparation of aflatoxin M1-oxime-fluoresceinamine conjugate through the carbodiimide reaction. The aflatoxin M1-oxime-fluoresceinamine conjugate was characterized by ultraviolet–visible and infrared spectroscopy. Magnetic nanoparticles (Fe₃O₄) were synthesized and modified by 3-(aminopropyl)triethoxysilane. The size of initial (139?nm) and functionalized magnetic nanoparticles (147?nm) was determined by particle analysis. The optimal mass of immobilized antibody (25?µg) and optimal concentration of aflatoxin M1-oxime-fluoresceinamine conjugate (15?µg?mL^?1) for magnetic nanoparticle-based fluorescent immunoassay were determined. The developed immunoassay provided a linear aflatoxin M1 concentration range from 3.0 to 100?pg?mL^?1 in bovine milk. The detection limit was 2.9?pg?mL^?1. The results of aflatoxin M1 magnetic nanoparticle-based fluorescent immunoassay in heat-treated milk and phosphate-buffered saline at pH 6.6 were compared. The influence of the somatic cell count, pH, and fat concentration in bovine milk on the aflatoxin M1 immunoassay was investigated. The influence of the milk species on the immunoassay was also characterized. The high fat concentration ovine milk depressed the sensitivity of the aflatoxin M1 immunoassay.