基于碳纳米管的脂质体电化学发光免疫传感器检测人免疫球蛋白G

将人免疫球蛋白G(hIgG)抗体固定在多壁碳纳米管修饰的玻碳电极表面,制备了一种电化学发光(ECL)免疫传感器.以hIgG抗体标记的联吡啶钌脂质体为标记物,采用三明治型检测方式,成功建立了hIgG的ECL免疫检测技术.电化学发光强度与hIgG的浓度在0.01～0.8 μg/L范围内呈良好的线性关系; 线性回归方程为y=618.7x(μg/L)-23.9(n=6,r=0.995); 检出限为0.004 μg/L.用于人血清中hIgG的检测,结果令人满意.     

新型流动注射电化学发光分析系统及性能表征

为研发电化学发光体外诊断试剂,设计了一种流动注射池,构建了喷射式流动注射电化学发光分析系统,并运用Ru(bpy)2+3/TPA混合进样模式对此系统进行了评估.在检测电位1.18 V,进样量100 μL,进样速度50 μL/s,TPA浓度10 mmol/L,样品缓冲液100 mmol/L PBS(pH 8.4), 载体缓冲液10 mmol/L PBS(pH 8.4)最优条件下,获得Ru(bpy)2+3的检出限(S/N=3)为0.005 μmol/L; 线性范围为0.01～5 μmol/L; RSD为1.02%.结果表明,此分析系统灵敏度高,线性范围宽,分析速度快,样品用量少,实现了进样、检测及清洗过程自动化.     

毛细管电泳电化学检测法测定马钱子中的士的宁和马钱子碱

用毛细管电泳电化学检测法测定了马钱子中的士的宁和马钱子碱.以BR缓冲溶液(35 mmol/L混酸用180 mmol/L NaOH调节至pH 6.50)为电泳液,分离电压为6 kV,检测电位为0.95 V,士的宁和马钱子碱在30 min内得到了良好分离.士的宁和马钱子碱浓度分别在0.35～35.00 μg/mL和0.40(40.00 μg/mL内具有良好的线性关系,检测限分别为0.08μg/mL和0.15μg/mL.应用于马钱子样品的测定.     

土壤中锑的双阳极电化学氢化物发生-原子荧光光谱法测定

采用双阳极电化学氢化物发生与原子荧光在线联用测定土壤中的锑含量,其中氢化物发生通过以纯铅为阴极,2根钛丝作阳极的双阳极电解池实现.对电解池工作条件及原子荧光参数进行了优化,最佳工作条件为以0.5 mol·L-1硫酸作电解液,流速2.0 mL·min-1,阴极电解电流密度0.5 A·cm-2.同时以去离子水为进样载流,流速设定为1.5 mL· min-1,六通阀进样100 μL得到方法检出限为0.91 μg· L-1 (S/N＝3);相对标准偏差(RSD)为1.58% (50 μg·L-1,n＝11);线性范围为1 ～300 μg·L-1.     

基于Ru(bpy)2+3/AuNPs/SWCNTs/腺苷适配体电化学发光生物传感器用于腺苷的检测

利用AuNPs/Nafion复合膜技术固定Ru(bpy)2+3,采用羧基化碳纳米管固定氨基化腺苷适配体,制备腺甘电化学发光生物传感器.采用循环伏安法和电化学发光法对传感器进行表征.结果表明,此传感器具有良好的稳定性和重现性.腺苷与传感器作用后,腺苷与其适配体形成G四面体结构,Ru(bpy)2+3的电化学发光强度降低.在最佳实验条件下,电化学发光强度降低量与腺苷浓度的负对数在1.0×10-11~1.0×10-7 mol/L范围内呈良好的线性关系,线性方程为ΔIECL=-890lgC-5050,检出限(S/N=3)为5.0 × 10-12 mol/L.对1.0 × 10-10 mol/L腺苷平行测定11次,相对标准偏差为2.7%.用于尿液中腺苷的测定,加标回收率在 97.1%~110.0%之间.     

基于酶催化沉积放大的电化学阻抗法用于人免疫球蛋白M的检测

提出了一种基于酶催化沉积放大的电化学免疫分析方法。先通过免疫夹心反应,将酶标抗体(羊抗人IgM-HRP)固定到电极表面。通过HRP催化双氧水氧化3,3′-二氨基联苯胺在电极表面形成不溶性沉积物,从而放大电化学检测信号。实验通过蛋白A实现抗体的定向固定。考察了包被抗体的浓度和包被溶液的pH对抗体固定的影响,以及酶标抗体的用量和沉积时间对免疫分析的影响。传感器信号响应与人免疫球蛋白M(IgM)浓度在2.1~670μg/L范围内具有良好的线性关系;检出限达0.08μg/L。     

热溶液中Nafion膜电极电化学分析法测定偶氮二甲酰胺

基于偶氮二甲酰胺在热溶液中的高溶解度及其与Nafion膜电极的静电作用,建立了偶氮二甲酰胺的电化学分析法.考察了偶氮二甲酰胺溶解度的温度效应,研究了偶氮二甲酰胺在Nafion膜电极上还原反应的机理.利用热溶液中偶氮二甲酰胺在Nafion膜电极上的伏安响应,用差分脉冲伏安法测定了面粉中的偶氮二甲酰胺含量.在水浴恒温80℃、pH 6.0的实验条件和优化的测试参数下,Nafion膜电极的电流响应与偶氮二甲酰胺的浓度在0.93～ 10.5 μg/L范围内呈线性关系,检出限为0.58 μg/L(S/N=3),对实际样品测定的相对标准偏差小于5.9％,回收率为95.8％ ～ 104.0％,氨基脲和呋喃西林不干扰偶氮二甲酰胺的测定.     

电化学液相微萃取-循环伏安法对海带中溴的测定

将液相微萃取与电化学法相结合,建立了快速测定海带中溴含量的电化学液相微萃取-循环伏安法(ELPME-CV).苯甲酸乙酯作为粘结剂组成碳糊电极,同时充当微液膜萃取相与样品溶液接触.在正电位下,溴离子被氧化成溴分子,溴分子经微液膜萃取富集,在负电位下还原溶出.在0.01 ～0.50 mmol/L范围内,溴的还原峰电流与溶液中溴离子浓度成正比,检出限为0.05 mmol/L.测定市售海带中溴的含量为91 μg/g,相对标准偏差为11%.     

Ru(bpy)_3~(2+)体系电化学发光法测定盐酸左氧氟沙星

基于盐酸左氧氟沙星对联吡啶钌(Ru(bpy)2+3)的电化学发光信号有较强的增敏作用,建立了一种检测盐酸左氧氟沙星的电化学发光分析新方法。结果表明,在0.1mol/L NaHPO4-KH2PO4介质中,以恒电位电解,Ru(bpy)2+3的浓度为1.0×10-5g/mL时,盐酸左氧氟沙星对Ru(bpy)2+3电化学发光信号的增敏效果最好。在此条件下,测定盐酸左氧氟沙星的线性范围为6~400×10-9g/mL,检出限为5.0×10-9g/mL,对8.0×10-9 g/mL盐酸左氟沙星平行测次11次,相对标准偏差(RSD)为1.9%。该方法已应用于人体尿样中盐酸左氧氟沙星的测定,并且用此方法对盐酸左氧氟沙星的国产及进口片剂进行了体外溶出度的测定。     

石墨烯/铋复合膜修饰玻碳电极检测板蓝根中的铅和镉

采用滴涂法制备石墨烯(GR)修饰的玻碳电极(GCE),通过电化学富集Bi沉积在GR表面,得到GR/Bi-GCE修饰电极.用方波溶出伏安法研究了Cd2+和Pb2+在GR/Bi-GCE上的电化学行为.在0.1 mol/LpH 4.5的醋酸缓冲溶液中,在-1.1 V富集0.5 mg/L Bi(NO3)3溶液210 s后,溶出峰电流与Cd2+和Pb2+的浓度在0.01～85.0 μmol/L范围内呈良好的线性关系,检出限均为0.003 μmol/L.实验结果表明,此修饰电极对Cd2+和Pb2+均有较好的电化学活性,可对两种物质实现同时测定,具有较高的灵敏度和稳定性.将此电极用于板蓝根中Cd2+和Pb2+的含量测定,结果令人满意.     

Amplified Immunoassay of Human IgG Using Real—time Biomolecular Interaction ANalysis （BIA） Technology

An automated biomolecular interaction analysis instrument(BIAcore)based on surface plasmon resonance(SPR)has been used to determine human immunoglobulin G(IgG) in real time.Polyclonal anti-human IgG antibody was covalently immobilized to a carboxymethyldextran-modified gold film surface.The samples of human IgG prepared in HBS buffer were poured over the immpbilized surface.The signal amplification antibody was applied to amplify the response signal.After each measurement,the surface was regenerated with 0.1mol/L H3PO4.The assay was rapid,requiring only 30 min for antibody immobilization and 20 min for each subsequent process of immune binding,antibody amplification and regeneration.The antibody immobilized surface had good response to human IgG in the range of 0.12-60 nmol/L with a detection limit of 60 pmol/L.The same antibody immobilized surface could be used for more than 110 cycles of binding,amplification and regeneration.The results demonstrate that the sensitivity,specificity and reproducibility of amplified immunoassay using real-time BIA technology are satisfactory.     

Protein A tangential flow affinity membrane cartridge for extracorporeal immunoadsorption therapy.

Tangential flow affinity membrane cartridge (TFAMC) is a new model of immunoadsorption therapy for hemoperfusion. Recombinant Protein A was immobilized on the membrane cartridge through Schiff base formation for extracorporeal IgG and immune complex removal from blood. Flow characteristics, immunoadsorption capacity and biocompatibility of protein A TFAMC were studied. The results showed that the pressure drop increased with the increasing flow rate of water, plasma and blood, demonstrating reliable strength of membrane at high flow rate. The adsorption capacities of protein A TFAMC for IgG from human plasma and blood were measured. The cartridge with 139 mg protein A immobilized on the matrix (6 mg protein A/g dry matrix) adsorbed 553 mg IgG (23.8 mg IgG/g dry matrix) from human plasma and 499.4 mg IgG (21.5 mg IgG/g dry matrix) from human blood, respectively. The circulation time had a major influence on IgG adsorption capacity, but the flow rate had little influence. Experiments in vitro and in vivo confirmed that protein A TFAMC mainly adsorbed IgG and little of other plasma proteins, and that blood cell damage was negligible. The extracorporeal circulation system is safe and reliable.     

免疫吸附治疗用ProteinA切流膜色谱柱的研究

切流膜色谱柱是免疫吸附治疗中实现全血灌流的一种新的模式。实验研究表明,切流膜色谱柱的结构和血液流动形式对血细胞的损害很小。分别考察了水、血浆、血液等不同流体流速与切流膜色谱柱柱压降之间的关系,柱压降随着流体流速和粘度的升高而增高,血液流速为120mL／min时,柱压降达到93kPa;考察了ProteinA切流膜色谱柱对人血浆中免疫球蛋白IgG的吸附能力,切流膜色谱柱ProteinA健合量为139mg（6mgProteinA／g干介质）,血浆在柱中循环1h可吸附IgG553mg（23．8mgIgG／g平介质）,而血液在柱中循环1h,可吸附IgG499.4mm（21.5msIgG／g卡介质）。     

Gold Nanoparticle-based “Mix and Measure” Fluorimetric Assays to Quantify Antibody Titer

Monoclonal antibodies (mAbs) for treatment of human diseases are typically human or humanized Immunoglobulin G (IgG) produced in mammalian cell lines. A rapid, less tedious, and high throughput method to quantify mAbs is in demand to accelerate mAb production efficiency. To quantify mAb titer, we developed gold nanoparticle (AuNPs)-based “mix and measure” fluorimetric assays by exploiting AuNPs’ fluorescence quenching ability. The AuNPs are functionalized by an Fc binding protein, i. e. protein G, which binds human IgG and fluorescently labeled rat IgG (Alexa Fluor 488-rat IgG) with differential affinity. The assays can be in competition or displacement format. The competitive binding of human IgG drug and the labelled rat IgG to protein G-coated AuNP lead to varied fluorescent intensity that is proportional to the amount of human IgG analte; or the displacement of the labelled rat IgG from protein G-coated AuNP by human IgG can lead to fluorescent recovery that is also proportionally related to human IgG concentration. The assays can quantify therapeutic mAbs in the range of 10–1,000 mg/L, demonstrated for Herceptin, Avastin, and Humira in cell culture media. The assays have fast turn over time (within 15 min). They can be performed in microplates and are suitable for high throughput “on-line” or “at-line” measurement in mAbs production lines.     

Copper-enhanced gold nanoparticle tags for electrochemical stripping detection of human IgG

We demonstrate herein a novel electrochemical protocol for quantification of human IgG based on the precipitation of copper on gold nanoparticle tags and a subsequent electrochemical stripping detection of the dissolved copper. The immunoassay was conducted by following the typical procedure for sandwich-type immunoreaction. Goat anti-human IgG was immobilized on the wells of microtiter plates. The human IgG analyte was first captured by the primary antibody and then sandwiched by secondary antibody labeled with gold nanoparticles. The copper enhancer solution was then added to deposite copper on the gold nanoparticle tags. After dissolved with HNO₃, the released copper ions were then quantified by ASV. The detection limit is 0.5 ng/mL by 3σ-rule. In order to investigate the feasibility of the newly developed technique to be applied for clinical analysis, several standard human IgG serum specimens were also examined by the method. To our knowledge, the copper enhancing procedure is the first time to be developed for immunoassay. The new strategy of using copper-enhanced gold nanoparticle tags for electrochemical stripping detection holds great promise for immunoassay and DNA detection.     

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 immunocomplex 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 5×105 ng/mL. It was shown that the determination of human IgG was easily made with the proposed fluorospectrophotometry.     

Phospho‐l‐tyrosine‐agarose chromatography: Adsorption of human IgG and its proteolytic fragments

The behavior of human immunoglobulin G (IgG) and antigen‐binding fragment (Fab fragment) adsorption onto phospho‐l ‐tyrosine immobilized on agarose (P‐Tyr‐agarose) was evaluated by pseudoaffinity chromatography. The effects of buffer systems MES, MOPS, Bis–Tris, Tris–HCl and sodium phosphate (NaP) and pH on IgG adsorption were studied and high purity values were obtained (96%, based on ELISA analysis of albumin, transferrin and immunoglobulins A, G and M) when IgG was purified from human plasma diluted in 10 mmol L^?1 NaP buffer at pH 6.0. The capture of IgG by the P‐Tyr‐agarose was also promising, since 91% of the IgG was adsorbed when plasma was diluted in 25 mmol L^?1 MES buffer at pH 5.5, recommending its use for IgG depletion from human plasma under this condition. The experimental data on IgG adsorption kinetics were in agreement with the pseudo‐second‐order model. The adsorption isotherm data were well described by the Langmuir–Freundlich model with the value of parameter n being <1 (0.72), indicating negative cooperativity. Selectivity was achieved on P‐Tyr‐agarose from digested human IgG in HEPES 25 mmol L^?1 buffer at pH 7.0 where Fab fragments were obtained in eluted fractions without Fc fragments (but with uncleaved IgG) with 86.2% recovery.     

Organic-inorganic matrix for electrochemical immunoassay: Detection of human IgG based on ZnO/chitosan composite

A new strategy to construct amperometric immunosensor for human IgG assay based on ZnO/chitosan composite as sensing platform has been described. This material, which combined the advantages of inorganic species, ZnO and organic polymer, chitosan, can maintain biological activity well. A sequential sandwich immunoassay format was performed on the ZnO/chitosan composite supported by glass carbon electrode (GCE) using goat-anti-human IgG antibody (IgG Ab) and human IgG as a model system. Amperometry was used to determine the amount of horse-radish peroxidase (HRP) fixed on the sensor surface, which was related to the content of the desired human IgG. Assay conditions that were optimized included the amount of labeled antibody, the incubation time and temperature, the pH of the substrate solution, etc. Using hydroquinone as a mediator, amperometric detection at −150 mV (versus SCE) resulted in a detection range 2.5-500 ng mL⁻¹, with a detection limit of 1.2 ng mL⁻¹. The simple manipulations of the construction of ZnO/chitosan composite, as well as low-cost and broad linear range, are the main features of the proposed immunosensing method.     

Sensitive amplification immunoassay for human IgG and anti-human IgG by using an enzyme cascade system

A sensitive heterogeneous immunoassay for human IgG and anti-human IgG was developed using an enzyme cascade system in limulus amoebocyte as a signal amplification system. Lipopolysaccharide (LPS) was conjugated to human IgG and anti-human IgG was adsorbed on polystyrene beads. The LPS-labelled human IgG mixed with unlabelled human IgG was allowed to react in a competitive manner with the immobilized anti-IgG on the polystyrene bead surface. After B/F separation, the LPS activity in the supernatant (free) and LPS activity on the bead (bound) were measured by using the chromogenic limulus test. IgG could be measured in the range 10^?7-10^?11 g ml^?1. LPS-labelled anti-IgG and IgG absorbed on polystyrene beads were prepared, and LPS-labeled anti-IgG mixed with unlabelled anti-IgG was allowed to react again in a competitive manner with solid-phase IgG. The LPS activity specifically bound to the bead was then measured. Anti-IgG could be measured in the range 10^?7-10^?11 g ml^?1.     

ProteinA亲合膜色谱柱的性能及从人血浆中纯化免疫球蛋白的研究

以木纤维为基质,与甲基丙烯酸环氧丙酯共聚接枝合成了复合膜介质,用复合膜介质制备了ｐｒｏｔｅｉｎＡ亲合膜色谱柱,考察了ｐｒｏｔｅｉｎＡ亲合膜色谱柱液相流动特性和吸附性能。实验证明：流速与亲合膜色谱柱柱压呈线性关系,当流速为３ｍＬ／ｍｉｎ时,柱压为１６０ｋＰａ。免疫球蛋白（ＩｇＧ）浓度和上样速度是影响ｐｒｏｔｅｉｎＡ键合容量的重要因素,对其进行了优化研究。用动态吸附法确定了对人ＩｇＧ动态最大吸附能力可达２１．７ｍｇ／ｇ（干介质）。