基于胶体金标记的阳极溶出伏安免疫分析用于免疫球蛋白G的测定

提出了一种基于胶体金标记的阳极溶出伏安免疫分析方法。免疫反应在聚苯乙烯微孔板中以夹心分析模式进行,通过物理吸附将兔抗人免疫球蛋白G(IgG)抗体固定于微孔板上,与相应抗原IgG发生免疫反应后,再通过夹心模式捕获相应的纳米金标记的羊抗人IgG抗体,然后再与金标羊抗人IgG抗体和金标兔抗羊二抗形成的免疫复合物反应,在微孔板上进一步引入大量的纳米金,将金溶解后,在碳糊电极上用阳极溶出伏安法(ASV)对金离子进行检测,溶出峰电流的大小间接与待分析物IgG的浓度成正比。对免疫分析的一些实验条件进行了优化。阳极溶出峰电流与IgG的对数浓度在1.1~1 143 ng/mL范围内呈良好的线性关系,检出限为1 ng/mL。将该方法应用于人血清中IgG浓度的测定,取得了满意结果。     

基于酶催化银沉积质量放大的血吸虫压电免疫传感器的研究

发展了一种基于酶催化金属银沉积信号放大的新型高灵敏气相压电免疫传感检测技术.先将血吸虫抗原(SjAg)共价固定在石英晶体表面,制备得到血吸虫压电免疫传感器.检测时,在晶振上滴加不同浓度的待测血吸虫抗体,再将碱性磷酸酶标记的二抗通过夹心方式结合到传感器表面.然后利用碱性磷酸酶催化磷酸化的抗坏血酸酯水解从而还原硝酸银,使金属银沉积在晶振表面上,放大传感器的质量响应信号.实验结果表明该传感检测方法可显著提高气相压电免疫传感器的检测灵敏度,传感器对血吸虫抗体的响应线性范围在1～225 ng/mL,检测下限为1 ng/mL.     

白血病细胞酶联免疫酶催化银沉积于插指电极阵列电化学免疫分析新方法

建立了一种检测白血病细胞表面抗原的细胞酶联免疫电化学分析新方法. 该方法兼有细胞酶联免疫分析抗原、抗体结合的特异性和插指电极阵列酶催化银沉积电化学分析的灵敏性. 在聚苯乙烯微孔板中包被白血病细胞, 先后加入鼠抗人抗体及碱性磷酸酶(ALP)标记的马抗鼠抗体, ALP催化抗坏血酸磷酸酯(AAP)水解成抗坏血酸(AA), AA使银离子还原成银单质并沉积到插指电极阵列表面, 导致插指电极阵列上相邻两个梳齿导通. 通过对电导率的测定, 可实现对细胞表面抗原的高灵敏分析. 此分析方法灵敏度高(可检测出50个左右的HL-60细胞)、特异性好, 且可用于大量样品的分析, 为白血病等肿瘤疾病的早期诊断和免疫分型提供了新技术. 此外, 该方法也可用于细胞表面分子基因工程抗体活性的检测.     

基于金纳米链标记抗体及HRP信号增强的电流型免疫传感器

酶联免疫分析将抗原-抗体反应的特异性与酶的高效催化放大作用相结合,因而极大地提高了免疫分析的灵敏度~([1]).本研究将均匀分散的碳纳米管修饰到预处理的电极表面,通过电化学还原氯金酸制成碳纳米管-纳米金复合基质用以固载抗体.同时制备了金纳米链(Au NCs)并标记二抗,结合辣根过氧化物酶(HRP),采用双抗体夹心分析模式,以心血管疾病标志物血清人N末端脑钠尿肽前体(NT-proBNP)为免疫蛋白模型,构建了高灵敏电流型酶免疫传感器.     

基于丝网印刷电极的甲胎蛋白电化学免疫传感器

报道了一种基于金纳米粒子(AuNPs)双重信号放大的高灵敏电化学免疫传感器,并应用于肝癌标志物甲胎蛋白(AFP)的检测。通过在丝网印刷电极(SPE)表面电沉积AuNPs提高电极的重现性,利用AuNPs的吸附作用固定AFP抗体,用于捕获样品中的待测AFP抗原,并进一步与固定了辣根过氧化物酶(HRP)标记检测抗体的纳米金免疫探针发生特异性结合,所形成的夹心免疫复合物可以催化底物得到响应电流。用扫描电镜(SEM)和微分脉冲伏安法(DPV)等技术研究电极组装过程以及电极的化学性质,讨论了影响免疫传感器性能的因素。在最优实验条件下,传感器的峰电流信号与AFP浓度在2.5~30ng/mL范围内呈良好的线性关系,检出限为0.16ng/mL。该传感器具有灵敏度高、成本低、仪器体积小的优点,具有较好的应用前景。     

爆米花型金纳米用于信号放大构建夹心型免疫传感器测定甲胎蛋白

通过原位合成法以金纳米种子作为母核诱导纳米金在其表面原位生长生成爆米花型金纳米粒子,标记甲胎蛋白抗体(anti-AFP)和辣根过氧化物酶(HRP)结合形成免疫复合物(antiAFP@popcorn-shaped GNP@HRP)作为检测抗体。氧化石墨烯-亚甲基蓝-金纳米粒子(GOM B-AuNPs)纳米复合材料滴涂到玻碳电极(GCE)表面,形成纳米复合物用于捕获甲胎蛋白抗体构建了免疫传感平台(anti-AFP/GO-MB-AuNPs/GCE)。实验采用夹心型免疫分析模式,功能化爆米花型金纳米材料与石墨烯纳米复合物呈现出良好的性能,建立了一种可行的电流型免疫分析法用于灵敏分析血清样品中甲胎蛋白AFP。在优化的实验条件下,免疫传感器DPV电流响应与AFP浓度的对数值呈正比,线性范围为0.001~20 ng/mL,检测限为0.31 pg/mL(S/N=3)。制备的免疫传感器有良好的精密度,选择性和稳定性,可初步应用于临床检验中AFP测定。     

基于二氧化硅-硫堇纳米复合物构建高灵敏癌胚抗原电流型免疫传感器

利用电沉积纳米金(AuNPs)修饰玻碳电极(GCE)表面并通过AuNPs固定癌胚抗原(CEA)的捕获抗体(Ab1),以牛血清白蛋白(BSA)封闭非特异性吸附位点;以γ-(2,3环氧丙氧)丙基三甲氧基硅烷(GPMS)作交联剂,将单分散的SiO_2纳米粒子与电子媒介体硫堇(Thi)结合成SiO_2-Thi纳米复合物,偶联辣根过氧化物酶(HRP)标记的CEA二抗(HRP-Ab2)作为电化学免疫检测信号,构建了具有信号放大效应的电流型免疫传感器并用于CEA的高灵敏检测。在CEA存在下,进行电化学酶联夹心免疫反应。在含有H2O2的磷酸盐缓冲溶液(PBS)中,标记在SiO_2-Thi纳米复合物上的HRP能催化H_2O_2氧化电子媒介体Thi,产生增强的还原峰电流,从而提高检测CEA的峰电流响应信号,进而实现对CEA的高灵敏电化学酶联夹心免疫分析。在最优实验条件下,该免疫传感器的差分脉冲伏安(DPV)还原峰电流与CEA质量浓度的对数在0.01~20ng/mL范围内呈良好的线性关系,检出限为3pg/mL(S/N=3)。该传感器对血清样品进行加标回收实验,回收率为97.3%~105.7%,可初步用于临床对CEA的检测。     

单颗粒计数策略定量检测癌胚抗原

该文提出了一种基于单颗粒电感耦合等离子体质谱(spICP-MS)的计数策略,用于癌胚抗原(CEA)的灵敏检测.该策略采用50 nm的金纳米颗粒(AuNPs)作为免疫识别和单颗粒计数的探针,以磁性纳米颗粒(MBs)作为免疫识别的捕获中心,基于夹心免疫反应,应用抗体标记的MBs捕获CEA和AuNPs探针.通过磁铁分离后,捕...     

基于银沉积电化学溶出分析的超灵敏甲胎蛋白传感器

制备了基于金标记物诱导银沉积电化学溶出分析的超灵敏甲胎蛋白传感器。通过壳聚糖将多壁碳纳米管修饰在玻碳电极上,利用戊二醛共价固定捕获抗体制得传感器。通过夹心免疫反应,抗体功能化的纳米金被捕获到传感器表面并进一步诱导银沉积。沉积的纳米银可在KCl溶液中直接通过阳极溶出法进行分析,从而实现对α-甲胎蛋白的检测。利用甲胎蛋白作为模型分析物,在0.02～200μg/L范围内,电流强度随着甲胎蛋白的质量浓度呈线性降低(r=0.992 8),检出限(S/N=3)为8 ng/L。将该传感器用于3个血样中甲胎蛋白的测定,所得结果与电致化学发光法测定结果一致。该免疫传感器显示了良好的稳定性、重复性和准确度,在临床诊断中具有较好的应用前景。     

Ultrasensitive α-Fetoprotein Immunosensor Based on Electrochemical Stripping Analysis of Silver Deposition

制备了基于金标记物诱导银沉积电化学溶出分析的超灵敏甲胎蛋白传感器.通过壳聚糖将多壁碳纳米管修饰在玻碳电极上,利用戊二醛共价固定捕获抗体制得传感器.通过夹心免疫反应,抗体功能化的纳米金被捕获到传感器表面并进一步诱导银沉积.沉积的纳米银可在KCl溶液中直接通过阳极溶出法进行分析,从而实现对α-甲胎蛋白的检测.利用甲胎蛋白作为模型分析物,在0.02 ～ 200 μg/L范围内,电流强度随着甲胎蛋白的质量浓度呈线性降低(r=0.992 8),检出限(S/N=3)为8 ng/L.将该传感器用于3个血样中甲胎蛋白的测定,所得结果与电致化学发光法测定结果一致.该免疫传感器显示了良好的稳定性、重复性和准确度,在临床诊断中具有较好的应用前景.     

Gold nanoparticle-based immunoassay by using non-stripping chemiluminescence detection

A novel microplate-compatible chemiluminescence (CL) immunoassay has been developed for the determination of human immunoglobulin G (IgG) based on the luminol-AgNO(3)-gold nanoparticles CL system. Polystyrene microtiter plates were used for both immunoreactions and CL measurements. The primary antibody, goat-anti-human IgG, was first immobilized on polystyrene microwells. Then the antigen (human IgG) and the gold-labeled second antibody were connected to the microwells successively to form a sandwich-type immunocomplex. The gold label could trigger the reaction between luminol and AgNO(3), accompanied by light emission. Under the optimized conditions, the CL intensity of the system was linear with the logarithm of the concentration of human IgG in the range from 25 to 5000 ng mL(-1), with a detection limit of 12.8 ng mL(-1) ( approximately 80 pM) at a signal to noise ratio of three (S/N = 3). Compared with other reported CL immunoassay method based on gold labels, the proposed CL protocol avoids a strict stripping procedure or difficult to control synthesis processes, making the method more simple, time-saving and easily automated. The present CL method is promising for the determination of clinically important bioactive analytes.     

Immunoassay by detecting enhanced resonance light scattering signals of immunocomplex using a common spectrofluorometer

A sensitive, highly specific immunoassay method has been developed by measuring the enhanced resonance light scattering (RLS) signals of immunoreactions with simultaneously scanning both the excitation and the emission monochromators of a common spectrofluorometer. For a given content of antibody (Ab), the RLS signals of an immunoreaction follow Gaussian distribution with antigen (Ag) concentration. The central position of the Gaussian curve represents the concentration of given Ab, and the half bandwidth has proved to be a characteristic constant of a given Ab-Ag immunoreaction. With the RLS signals, the limit of detection for human immunoglobulin G (HIgG) in serum samples could reach 10 ng ml⁻¹, and the concentration of HIgG in blood serum samples could be detected with the recovery of 90.2-107.7% and R.S.D. of 0.8-2.7%. The results of determination for three human serum samples are identical to those obtained by immunoturbidimetry.     

Determination of trace amounts of thallium by adsorptive cathodic stripping voltammetry with xylenol orange.

Trace amounts of thallium(I) can be determined using adsorptive cathodic stripping voltammetry in the presence of Xylenol Orange (XO). The reduction current of the thallium(I)-XO complex ion was measured by square-wave cathodic stripping voltammetry. The peak potential was at -0.44 V vs. Ag/AgCl. The effect of various parameters (pH, ligand concentration, accumulation potential and collection time) on the response are discussed. The response was linearly related to the thallium concentration in the range 0.5-110 ng ml(-1) and 110-2000 ng ml(-1). The limit of detection was 0.2 ng ml(-1). The relative standard deviation for the determination of 80 ng ml(-1) thallium was 2.8%. Many common anions and cations did not interfere with the determination of thallium. The interference of lead was reduced by the addition of 0.003 M sodium carbonate. The voltammetric procedure was then successfully applied to the determination of thallium in various complex samples.     

A new strategy for electrochemical immunoassay based on enzymatic silver deposition on agarose beads

A novel, sensitive electrochemical immunoassay in a homogeneously dispersed medium is described herein based on the unique features of agarose beads and the special amplified properties of biometallization. The immunochemical recognition event between human immunoglobulin G (IgG) and goat anti-human IgG antibody is chosen as the model system to demonstrate the proposed immunoassay approach. Avidin-agarose beads rapidly react with the biotinylated goat anti-human IgG antibody to form agarose beads-goat anti-human IgG conjugate (agarose bead-Ab). Agarose bead-Ab, alkaline phosphatase conjugated goat anti-human IgG antibody (ALP-Ab) and the human IgG analyte are mixed to form sandwich-type immunocomplex followed by the addition of the enzymatic silver deposition solution to deposit silver onto the surface of proteins and agarose beads. The silver deposited are dissolved and quantified by anodic stripping voltammetry. The influence of relevant experimental variables was examined and optimized. The logarithm of the anodic stripping peak current depended linearly on the logarithm of the concentration of human IgG in the range from 1 to 1000 ng/ml. A detection limit as low as 0.5 ng/ml human IgG was attained by 3σ-rule. The R.S.D. of the approach is 9.65% for eight times determination of 10 ng/ml human IgG under same conditions. Optical microscope and TEM graphs were also utilized to characterize agarose beads and silver nanoparticles formed.     

Electrochemical immunoassay of hepatitis B surface antigen by the amplification of gold nanoparticles based on the nanoporous gold electrode

We describe herein the combination of electrochemical immunoassay using nanoporous gold (NPG) electrode with horseradish peroxidase (HRP) labeled secondary antibody-gold nanoparticles (AuNPs) bioconjugates for highly sensitive detection of protein in serum. The electroactive product of o-phenylenediamine (OPD) oxidized with H₂O₂ catalyzed by HRP was reduced in the Britton-Robinson (BR) buffer and the peak current of which was used to determine the concentration of antigen (Ag) in the analyte. The active surface area of NPG electrode was larger than that of a bare flat one. The presence of AuNPs enhanced the immobilized amount of HRP labeled antibody (Ab), which improved the sensitivity of the immunoassay when used as the secondary antibodies. As a result of these two combined effects, the sensitivity of the immunoassay for the determination of target protein was increased significantly. Using hepatitis B surface antigen (HBsAg) as a model, we demonstrate a dose response in the range of 0.01-1.0 ng/mL with a detection limit of 2.3 pg/mL. Analytical results of several human serum samples obtained using the developing technique are in satisfactory agreement with those given by enzyme-linked immune-absorbent assays (ELISA). In addition, the technique was about 100 times more sensitive in the detection of HBsAg than ELISA. All these demonstrated the feasibility of the present immunoassay method for clinical diagnosis.     

Determination of physiological noble metals in human urine using liquid-liquid extraction and Zeeman electrothermal atomic absorption spectrometry

An extremely sensitive, reliable and simple procedure is described for the determination of physiological palladium, platinum and gold in human urine. The urine samples were adjusted to pH 4 (Pd, Au) or pH 5 (Pt), followed by conversion of the analytes to their pyrrolidinedithiocarbamate complexes. These complexes were separated from the matrix by liquid-liquid extraction into 4-methyl-2-pentanone resulting in a 25-fold enrichment. Determination was by electrothermal atomic absorption spectrometry (ET-AAS) using longitudinal inverse alternating current Zeeman-effect background correction. The limits of detection calculated from three standard deviations of the blank values were 20 ng l⁻¹ for Pd and Au and 70 ng l⁻¹ Pt. Within-day precision (n = 10, 5 μg l⁻¹) ranged 5.2%–7.7%. The procedure is successfully applied to determine urinary palladium, platinum and gold in nine unexposed persons. Palladium levels in urine ranged < 20–80 ng l⁻¹ (arithmetical MEAN=38.7 ng l⁻¹), while gold levels ranged < 20–130 ng l⁻¹ (36.0 ng l⁻¹). Physiological platinum levels in urine were all < 70 ng l⁻¹. The accuracy of the procedure was checked by analyzing a series of urine samples by a second independent method (magnetic sector field inductively-coupled plasma-mass spectrometry) in combination with UV photolysis.     

Electrochemical Metalloimmunoassay Based on Enlargement of Gold Nanoparticle Label Treated with Ag Enhancement System

A novel sensitive electrochemical immunoassay with colloidal gold as the antibody labeling tag and subse-quent signal amplification by silver enhancement is described. Colloidal gold was treated by a light-sensitive silver enhancement system which made silver deposit on the surface of colloidal gold(form Au/Ag core-shell structure), followed by the release of the metallic silver atoms anchored on the antibody by oxidative dissolu-tion of them in an acidic solution and the indirect determination of the dissolved Ag ions by anodic stripping voltamrnetry(ASV) at a carbon fiber microelectrode. The electrochemical signal is directly proportional to the amount of analyte(goat IgG) in the standard or a sample, The method was evaluated by means of a non-competitive heterogeneous immunoassay of immunoglobulin G(IgG) with a concentration as low as 0. 2 ng/mL. The high performance of the method is related to the sensitive ASV determination of silver( I ) at a car-bon fiber microelectrode and to the release of a large number of Ag^ ions from each silver shell anchored on the analyte (goat IgG).     

Determination of lead in preserved egg by flame atomic absorption spectrometry after chemically modified preconcentration

A sensitive method for the determination of lead in preserved egg by flame absorption spectrometry using ammonium pyrrolidine dithiocarbamate-polystyrene chemically modified platinum wire matrix is presented. The modified platinum wire matrix, after preconcentrating the lead, is placed in a flame burner for direct atomization and measurement. The concentration range is linear between 5 and 500 ng/ml lead in solution and the detection limit is 0.65 ng/ml. This new technique is sensitive and convenient.     

Multichannel homogeneous immunoassay for detection of 2,4,6-trinitrotoluene (TNT) using a microfabricated capillary array electrophoresis chip

A high-throughput homogeneous immunoassay for the sensitive detection of 2,4,6-trinitrotoluene (TNT) has been developed using radial capillary array electrophoresis microdevices. Samples consisting of equilibrium mixtures of anti-TNT antibody (Ab), fluorescein-labeled TNT, and various concentrations of unlabeled TNT were electrokinetically injected into 48 channels of a radial capillary array electrophoresis microchannel plate. The rapid electrophoretic separation allows us to analyze the equilibrium ratio formed by the competition between the labeled and the unlabeled TNT for Ab binding. The simultaneous parallel TNT separations facilitate determination of a calibration curve for the TNT assay, which has high sensitivity (LOD, 1 ng/mL) and a wide dynamic range (1-300 ng/mL).