Colloidal Gold Immunochromatographic Assay for Rapid On-Site Detection of Tetracycline in Seawater

Recently increasing concerns from the scientists and public have been paid for seawater pollution due to tetracycline(TC)overuse in maricultural area.However,there are few methods or instruments that can be used for specific and rapid detection of this antibiotic in seawater.In this study,the colloidal gold immunochromatographic assay(CG-ICA)was used to achieve this goal.A commercialized monoclonal antibody against TC(anti-TC mAb)was selected because of its higher sensitivity(half-maximal inhibitory concentration of 2.38μgL^(-1)).The prepared CG particles(average diameter of 20 nm)were used to label anti-TC mAb at pH 8.0.The conjugate pad was formed by spraying the CG-labeled anti-TC mAb on a glass fibre membrane followed by proper dryness.The test pad was made by immobilizing artificial antigen and anti-mouse mAb in the test line and the control line,respectively,in a nitrocellulose membrane.The test strip,assembled with sample pad,conjugate pad,test pad and absorbent pad,could be used to detect TC during seawater sample flowing through these components in turn.The results could be observed by the naked eye in 10min.The visible limit of detection(vLOD)was 20μgL^(-1) for TC in seawater.The CG-ICA test results were in good agreement with those of liquid chromatography-tandem mass spectrometry(LC-MS/MS).The assay also showed that,oxytetracycline(OTC)and chlortetracycline(CTC),as the structural analogues of TC,did not interfere with TC determination.Furthermore,the TC concentration given by test strip could not be affected by the fluctuation of temperature(10℃–30℃),pH(7–9)and salinity(0–40)of seawater.Therefore,CG-ICA is a suitable tool for rapid,on-site,and semi-quantitative detection of TC in seawater.

A Simple and Rapid Colloidal Gold-based Immunochromatogarpic Strip Test for Detection of FMDV Serotype A

A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test,affinity purified polyclonal antibodies from Guinea pigs which were immunized with sucking-mouse adapted FMD virus (A/AV88(L) strain) were conjugated to colloidal gold beads and used as the capture antibody,and affinity purified polyclonal antibodies from rabbits which were immunized with cell-culture adapted FMD virus (A/CHA/09 strain) were used as detector antibody. On the nitrocellulose membrane of the immunochromatographic strip,the capture antibody was laid on a sample pad,the detector antibody was printed at the test line(T) and goat anti-guinea pigs IgG antibodies were immobilized to the control line(C). The lower detection limit of the test for a FMDV 146S antigen is 11.7ng/ml as determined in serial tests after the strip device was assembled and the assay condition optimization. No cross reactions were found with FMDV serotype C,Swine vesicular disease (SVD),Vesicular stomatiti svirus (VSV) and vesicular exanthema of swine virus (VES) viral antigens with this rapid test. Clinically,the diagnostic sensitivity of this test for FMDV serotypes A was 88.7% which is as same as an indirect-sandwich ELISA. The specificity of this strip test was 98.2% and is comparable to the 98.7% obtained with indirect-sandwich ELISA. This rapid strip test is simple,easy and fast for clinical testing on field sites; no special instruments and skills are required,and the result can be obtained within 15 min. To our knowledge,this is the first rapid immunochromatogarpic assay for serotype A of FMDV.

Preparation of colloidal gold immunochromatography strip for detection of methamidophos residue

Methamidophos (Met) is a broad spectrum organophosphorus insecticide and acaricide.Even a trace of its residue is harmful to humans and many animals.In this study,the synthesis and identification of colloidal gold particles and antibody-colloidal gold conjugates were performed,and the preparation of colloidal gold immunochromatography strip was conducted for detection of Met residue.The size of colloidal gold particles was checked using a transmission electron microscope (TEM).The formation of antibody-coll...

Application of Colloidal Gold to Fluorophotometric Determination of Human IgG

A method for the determination of human immunoglobulin G（IgG） based on a colloidal gold label by fluorospectrophotometry was developed. The sandwich immunoreaction among goat-anti-human IgG, human IgG and goat-anti-human IgG labeled with colloidal gold nanoparticles was applied in this experiment. First, a sandwich im- munocomplex was formed on the surface of 96 well clear polystyrene high bind stripwellTM microplate. After the formation of the sandwich immunocomplex, a solution was added to dissociate the immunocomplex at room temperature. Then the solution of each well was transferred into the corresponding test tube. Thirdly, the rhodamine B solution was injected into each test tube. The rhodamine B chloraurate was extracted into ether that was measured with a spectrofluorometer. The experimental results indicate that the fluorescence intensity increased with the increase of human IgG concentration. The fluorescence intensity of rhodamine B chloraurate at 570 nm was proportional to the logarithm of human IgG concentration in a range from 10 to 5 × 10^5 ng/mL. It was shown that the determination of human IgG was easily made with the proposed fluorospectrophotometry.

Effects of 105 traditional Chinese medicines on the detection ofβ-agonists in medicine extracts and swine urine based on colloidal gold immunochromatographic assay

Colloidal gold immunochromatographic assay(CGIA)is commonly used for the on-site detection ofβ-agonists that are sometimes used illegally as feed additives in swine diets.However,few studies have evaluated the causes of false-positive results that sometimes occur when applying CGIA in agricultural settings.In this study,we investigated if this false-positive phenomenon is related to the addition of certain traditional Chinese medicines(TCMs)to swine feed.We established and verified an extraction method for TCMs,and then applied CGIA to detectβ-agonists in the extracts of 105 TCMs and in the urine of swine dosed with TCMs,respectively.Liquid chromatography-tandem mass spectrometry was used to validate the results of the urine samples tested positive forβ-agonists using CGIA.The results were also verified using TCMs and colloidal gold test strips produced by different manufacturers.The extracts of Citri Reticulatae Pericarpium Viride,Citri Reticulatae Pericarpium,Magnoliae Officinalis Cortex,Chaenomelis Fructus,and Rhodiolae Crenulatae Radix Et Rhizoma were tested positive forβ-agonists.Meanwhile,the addition of Citri Reticulatae Pericarpium Viride and Citri Reticulatae Pericarpium to swine feed resulted in false-positive results forβ-agonists in swine urine.The results provide a new way to explain false-positive CGIA results and provide valuable information for livestock feeding programs.

PREPARATION OF POLYMER MICROSPHERES WITH PYRIDYL GROUP AND THEIR STABILIZED GOLD METALLIC COLLOIDS

Narrow disperse poly（ethyleneglycol dimethacrylate-co-4-vinylpyridine） （poly（EGDMA-co-4-VPy）） microspheres were prepared by distillation-precipitation copolymerization of ethyleneglycol dimethacrylate （EGDMA） and 4-vinylpyridine （4-VPy） with 2,2＇-azobisisobutyronitrile （AIBN） as initiator in neat acetonitrile. The polymer microspheres containing pyridyl group were then utilized as stabilizer for gold metallic colloids with the diameter around 7 nm, which were prepared by the in situ reduction of gold chloride trihydrate with sodium borohydride through the coordination of the pyridyl group on the gel layer and surface of the microsphere with the gold metallic nano-particles. The catalytic properties of the pyridyl- functionalized microsphere-stabilized gold metallic colloids and the behavior of the stabilized-catalyst for the recycling were investigated with reduction of 4-nitrophenol to 4-aminophenol as a model reaction.

Observation of Reciprocal Induced CD between Colloidal Gold Nanoparticles and Chiral Schiff Base Zn （11） Complexes with Parallel Dipole Moments

The authors have prepared suprameolecular systems of chiral Schiffbase ZnAZSB （Zn（II） complexes with azo-groups） or without ZnSB （azo-groups） and colloidal AuNP （gold nanoparticles） of 10, 40 and 80 nm diameters. They exhibited gradual shifts of surface plasmon bands as well as fluorescence bands. The authors observed and discussed induced CD bands on gold nanoparticles from chiral ZnAZSB or ZnSB. Absence of cis-trans photoisomerization of ZnAZSB with AuNP also supported direct contact near the surface of AuNP. Quenching and splitting of fluorescence bands of ZnSB （λex = 550 nm and λem = 400 nm） depending on concentration of ZnSB and size of AuNP also suggested intermolecular （electric） interaction on the surface of AuNE Decrease of the intensity of the CD band around 380 nm resulted from reciprocal induced CD effect due to parallel arrangement of electric transition moments of ZnAZSB or ZnSB and surface of AuNP.

Colloidal Gold Strip-NASBA Technique for Rapid Detection of Shigella

An isothermal amplification assay for the detection of Shigella based colloidal gold strip detection is developed. The primers designed corresponded to the ipaH gene of Shigella .The sensitivity of the colloidal gold strip-NASBA method in this study was 6.3 x 101cfu/ml. The specificity of the detection system was detected and the result showed that the NASBA method could distinguish Shigella from other germs.

Interface Characteristics Between Colloidal Gold and Kaolinite Surface by XPS

The distribution of gold colloids in kaolinite and the interaction between gold and kaolinite surface were investigated by transmission electron mieroscotgy （TEM） and X-ray photoelectron spectroscopy （ XPS ）. There is strong interaction between the gold particles and the edge surfaces of kaolinite, in low pH solution, the edge surface of kaolinite is positively charged and electrostutic attrcactive force between colloide gold panicles and the positive edge surface of kaolinite woald facilitate the adsorption of colloidal gold particles onto the suface. TEM observation shows that the aggregate morphology of gold particles was dominated by particle-particle interaction and gold particles were adsorbed on the edge surface of kaolinite crystals , resulting from the electrostatic attractive force between colloidal gold particles and the positice surfaces of kaolinite. XPS data show that in Au4 f electron spectra there are four energy peaks related to gold, 83.8 eV, 85.7 eV, 87.5 eV, and 89.4 eV, respectively, which suggests that in chemical states there are metallic gold and Au bonded to O, similar to the form of Au2O3 , and composite Au2O3 is formed between the edge surface of kaolinite and colloidal gold surface.

Anion Adsorption on an Au Colloid Monolayer Based Cysteamine-Modified Gold Electrode

Anion adsorption behavior on Au colloid surface was investigated in virture of depositing monolayers of Au colloid on the self-assembled monolayers of cysteamine on a gold electrode. Po- tential-dependent anion adsorption-desorption waves via the nonfaradaic current were obtained by means of cyclic voltammetry at Au coltoid-modified gold electrodes in the potential range of -2 00-600 mV. The adsorption sequence in the order of adsorption peak potentials (Epa) is OH- >citrate3 ->H2PO4- >Cl->SO42->ClO4->NO3-. Among them, citrate3- exhibited an en- tirely irreversible adsorption. A rise in temperature can increase the rates of adsorption-desorp- tion and improve the reversibility of the adsorption-desorption of Cl-, SO42-, ClO4-, NO3- and H2PO-4. The adsorption peak potentials shifted more negatively for Ca. 63 mV as the anion con- centrations were increased by a decade factor. The change of pH from 7 to 1 slightly affected the adsorption peak potentials of Cl- and NO3-. Au colloids with a smaller size (16 nm) gave rise to a better reversibility of the adsorption-desorption process and lower adsorption currents. The ex- perimental results of citrate ions adsorption on Au colloid surface show that Au colloids with a smaller size prepared by sodium citrate method exhibited a higher stability in the solution in com- parison to those with larger sizes because of its higher ratio of charge/mass. In other words, the smaller gold nanoparticles are covered with citrate ions monolayer that can also be formed at larg- er gold nanoparticles by means of electrochemical scan.

Observation on the specific binding site of p195 protein on human erythrocytes using colloidal gold labelling

Objective: p195, the major protein on the surface of Plasmodium falciparum merozoites, has been found to have ability to bind sialic acid residues on the surface of human erythrocytes, and this binding is thought to be a prerequisite for recognition of human erythrocyte by merozoite- This study attempted to map out the binding site of p195, thus providing a theoretical clue to developing an antimalaria vaccine which blockades invasion of merozoites into human erythrocytes. Methods: Eight proteins derived from pl95 were expressed in E. coli, and purified by Ni-chelate affinity chromatography. The re folded proteins were labelled with colloidal gold. The labelled protein complexes were co-incubated with human erythrocytes separately and simultaneously, and the proteins were put into the culture supernatant of P- falciParum to observe their effect on invasion of merozoites into erythrocytes. Results: A fragment of p195, M6 (amino acid sequence: 384 - 595), was found to have the ability to bind human erythrocytes. M6 gold complexes showed no ability to bind erythrocytes treated with trypsin or neuraminidase. M6 was also found to have the ability to inhibit invasion of merozoites of P. falciparym into human erythrocytes. Conclusiou: A fragment of p195, M6, has the ability to bind human erythrocytes. The binding is dependent on sialic acid residues, and may be a prerequisite to recognition of erythrocytes by merozoites.

Colloidal Gold Test Strip for the Detection of Fluoroquinolones in Foods Using an Analyzer

A test strip was developed to rapidly detect fluoroquinolones in foods by colloidal gold immunochromatography method in combination with a food safety analyzer. The results showed that the test strip has the detection sensitivity of 20 μg/L for enrofloxacin, sarafloxacin, difloxacin, ofloxacin, norfloxacin, ciprofloxacin, pefloxacin, flumequine and danofloxacin, and the detection sensitivity of 40 μg/L for enoxacin and oxolinic acid. The test strip consumes only 10 min for detection, and the false positive rate and the false negative rate are both zero. The test strip can accurately, reliably and easily detect residual fluoroquinolones in foods, and is suitable for on-site detection of a large number of samples.

Colloid Thin-Layer Chromatography: A Tool for Gold Sols Validation before Used in Application

Gold nanoparticles have been increasingly used in catalysis, biomedical imaging, biological and chemical sensing, drug delivery, etc. In this study, a straightforward method that allows one to monitor the synthesis of gold sols and their aging, before their fine characterization by sophisticated techniques and before their use is described. Indeed, the “Colloid Thin-Layer Chromatography” method allows one to check the quality of gold colloidal sols during the synthesis. It is also well adapted for monitoring the aging of the sol before the visual observation of its degradation.

吡虫啉和啶虫脒胶体金免疫层析法试纸条性能评估及应用效果探究

目的研究吡虫啉和啶虫脒胶体金免疫层析法试纸条在不同基质中残留量检测的应用效果。方法实验考察了市场上主要流通的5种不同品牌的胶体金免疫层析法试纸条在蔬菜、水果和茶鲜叶基质中的检出限、灵敏度、交叉反应率、特异性、假阳性和假阴性率。结果各品牌胶体金试纸条在3种不同类型基质(白菜、苹果和茶鲜叶)中,检出限浓度水平有假阴性出现,其余浓度水平灵敏度较好;特异性和交叉反应性,结果均为阴性;假阳性率和假阴性率考察中,假阳性率为0%,但有假阴性情况出现。结论胶体金免疫层析法在检测吡虫啉、啶虫脒农药残留时具有简便快捷的特点,在蔬菜、水果基质的检测中,判定结果较为准确可靠;在茶叶基质中,由于基质效应的影响,出现假阴性的机率较高。

Influence of Single Use and Combination of Reductants on the Size, Morphology and Growth Steps of Gold Nanoparticles in Colloidal Mixture

A comprehensive investigation on the formation mechanism of gold nanoparticles (AuNPs) in colloidal mixture obtained from the reduction of chloroauric acid (HAuCl4) solution using a single reducing agent (sodium citrate;process-I), (tannic acid;process-II), and a combination of two reducing agents (sodium citrate plus tannic acid;process-III) is reported. The growth steps at different time intervals during synthesis of colloidal AuNPs were monitored in situ and ex situ using various methods for all the three processes. The measurement of changes in the surface plasmon band position of colloidal AuNPs, along with dynamic light scattering results gave important information for the first assessing of particle size, shape and distribution. Besides, the size and morphological changes at different stages during different processes were also analyzed by transmission electron microscopy. The final Au particles of processes-I & II exhibited different shapes (spherical and nanowires) with particle size and nano wire diameter of 12 nm and 17 nm, respectively. Nevertheless, combination of two reductants (process-III) surprisingly leads to drastically reduced size (ca. 3 nm) with spherical morphology compared to their parent solutions with either of single reducing agent. This result clearly indicates that the combination of reductants has a significant influence on the particle size, morphology and formation mechanism.

不同品牌胶体金免疫层析试纸条/卡检测牛羊布鲁氏菌病的效果分析

为比较不同品牌胶体金免疫层析试纸条/卡(简称试纸条/卡)检测牛羊布鲁氏菌病的效果,利用抗体布鲁氏菌阴阳性血清,对6种品牌试纸条/卡的准确性及灵敏度进行观察比较;同时按不同试纸条/卡操作方法对经虎红平板凝集试验(RBPT)初筛和竞争酶联免疫吸附试验(cELISA)复核的临床牛羊血清样品进行检测比较。结果显示:不同试纸条/卡对布鲁氏菌企业标准阴阳性血清检出率均为100%,其中2种试纸条/卡敏感性高,对不同稀释倍数阳性血清均可检出;试纸条/卡对临床阴性血清均100%检出,但对临床阳性血清样品的检测结果差异较大,其中对羊阳性血清样品的检出率为5.88%~70.59%,对牛阳性血清样品为50.0%~100.0%。结果表明,不同品牌试纸条/卡在布病检测时存在一定差异。建议使用试纸条/卡进行布鲁氏菌病初筛后,应结合国家相关布病诊断技术进行复检,以确保检测结果的准确性。

早期香蕉枯萎病Foc4双探针核酸纸基检测传感器研制

为实现对早期香蕉枯萎病4号生理小种(fusarium oxysporum f.sp.cubense 4, Foc4)的准确检测,该研究提出一种基于胶体金的双探针纸基传感器。该传感器将2种不同粒径的胶体金分别与检测探针和信号增强探针结合,利用DNA探针代替抗原和抗体,形成双探针体系,通过增加信号增强探针降低检测限。基于目标序列与双探针体系的碱基互补配对形成“金标探针-目标序列-T线探针”复合物并在传感器的测试区被捕获,10 min内形成肉眼可见的目标产物,通过分析测试条带得到光强度峰面积并代入标准曲线中,实现Foc4定量检测。试验结果表明,双探针纸基传感器的检测限达到0.001 nmol/L,是传统纸基传感器的100倍,提高了检测灵敏度;在0.001~1 000.000 nmol/L范围内,Foc4浓度与测试线光强度峰面积呈线性关系;使用高浓度非互补探针进行试验干扰,发现非互补序列对检测效果基本无影响,表明该传感器具有较好的特异性;香蕉叶片Foc4检测的平均回收率为77.6%~102.3%,相对标准偏差为7.4%~7.7%。与传统形态学观察等检测方法相比,该研究提出的双探针核酸纸基检测传感器可以及时、快速、准确地判断早期Foc4的存在,具有良好的推广性和实际应用价值。

不同预处理对生姜中5种农药胶体金快速检测的影响

蔬菜中的农药残留对人体的危害一直是老百姓十分关注的问题,职能部门非常重视快检在蔬菜中的应用,然而由于国家标准采用的酶抑制率法对部分农药检测容易受干扰,因此开发免疫金快检方法十分重要。以生姜为研究对象,开展了不同预处理方式对生姜中的克百威、灭多威、丙溴磷、甲基异柳磷和辛硫磷快检方法及分析。实验结果表明:相较于酶抑制率法,生姜预处理会对有机磷和氨基甲酸酯类检测试剂产生干扰,尤其是生姜在匀浆、剪碎状态下会产生干扰,且影响最大;在匀浆、剪碎和整株状态下,对生姜中的克百威、灭多威、丙溴磷、甲基异柳磷和辛硫磷采用胶体金5联快速检测无干扰,说明胶体金方法较酶抑制率法对生姜更具备实用性。

酶联免疫吸附法与胶体金免疫层析法对人类免疫缺陷病毒抗体的检测价值分析

目的探析酶联免疫吸附法(ELISA)与胶体金免疫层析法(GICA)对人类免疫缺陷病毒(HIV)抗体的检测价值。方法选取2020年8月至2021年8月于枣庄市立医院接受HIV抗体检测的320例疑似艾滋病(AIDS)患者作为研究对象,均实施ELISA及GICA检测。以实验室病理结果为金标准,比较两种检测方法HIV阳性病例检出情况及诊断准确度、灵敏度、特异度。结果实验室病例组织活检结果显示,阴性285例,阳性35例;GICA检测阴性298例,阳性22例,与实验室病理组织活检结果相比,假阳性5例,假阴性18例;ELISA检测阴性282例,阳性38例,与实验室病例组组活检结果相比,假阳性4例,假阴性1例。ELISA诊断准确度为98.44%、灵敏度为97.14%,高于GICA的92.81%、48.57%,差异有统计学意义(P<0.05),两组特异度比较差异无统计学意义。结论与GICA相比,ELISA检测HIV抗体的准确度、灵敏度较高,因此,可将ELISA作为检测HIV抗体的主要方法。

胶体金免疫层析法快速检测蔬菜中3种农药残留

基于胶体金免疫层析原理研制吡虫啉、丙溴磷及克百威快速检测试纸条,评价其在大蒜、白菜检测中的应用效果。采用柠檬酸三钠还原法制备胶体金,用胶体金颗粒标记吡虫啉、丙溴磷及克百威单克隆抗体作为检测探针,以硝酸纤维素膜为固相载体,包被吡虫啉-BSA、丙溴磷-BSA及克百威-BSA偶联物为检测线,羊抗鼠IgG抗体为质控线,制备胶体金免疫层析试纸条,并对金标抗体用量、标记抗体浓度、检测线包被浓度等参数条件进行优化,评价试纸条的灵敏度、特异性、稳定性和准确性。以大蒜、白菜为对象,优化了样品前处理方法,并对这2种蔬菜样品进行阴性检测及添加标准品农药检测。结果表明,优化后的工艺参数中吡虫啉、丙溴磷、克百威的最适标记抗体质量浓度分别为9.6、8.4、9.6μg/mL,抗原的包被质量浓度均是0.3 mg/mL,检测用时约15 min。吡虫啉、丙溴磷及克百威的检出限分别为20、3000、20 ng/mL,符合国标对2种蔬菜的限量规定。检测与吡虫啉、丙溴磷及克百威功能和结构相似的其他药物时无交叉反应,特异性较好。克百威和吡虫啉农药的最适提取剂分别为4 mL乙酸乙酯和4 mL甲醇,样品用量均为2 g,上清液3 mL;丙溴磷农药的最适提取剂是9 mL乙酸乙酯,样品用量3 g,上清液8 mL,上清液用氮气吹干。50份蔬菜阴性样品和加标样品的检测结果显示,假阳性率为6%,假阴性率为0。可见,研制的试纸条适用于大蒜、白菜现场快速检测以及基层实验室中大量样本的筛查,可为大蒜、白菜中农药残留控制提供有效监管手段。