糖蛋白抗原抗体蚕丝丝素膜免疫传感器

蚕丝丝素膜由具有两性离子交换基的聚氨基酸构成,是一种具有广泛应用前景的天然生物材料。该文研究了蚕丝丝素膜的制备、蚕丝丝素膜固定乳腺癌抗原CA15－3抗体的各种方法及原理,以及用于CA15－3非标记免疫传感器的结果及比较。在各种固定方法中,以盐酸活化法、叠氮法、重氮法的效果较好。作者用盐酸活化法制作抗体膜并组装免疫电极,制作了测定CA15－3的标准曲线,讨论了重复性、干扰情况以及传感器的响应机理。     

溶胶-凝胶非标记 CA15-3免疫传感器的研制与应用

采用溶胶-凝胶技术将乳腺癌抗体固定于电极表面,研制成用于检测乳腺癌抗原的非标记型溶胶-凝胶-抗体膜免疫传感器;用循环伏安法对电极的修饰过程进行表征,同时对乳腺癌抗原定量检测的可行性进行了探讨;随着抗体与抗原特异性反应的进行,形成的抗体-抗原免疫复合物使膜电位发生变化(△V),该变化的大小与溶胶-凝胶-抗体膜表面免疫反应进行的程度相关;本文以此为依据对CA15-3进行检测,在5～240U／mL范围内．△V／与lgCCA15-3呈良好的线性关系,线性相关系数r=O.998;该传感器响应迅速,灵敏度高,稳定性好,于4℃干态保存30d,其响应信号基本不变。     

基于功能化溶胶-凝胶膜的乳腺癌抗原免疫传感器

结合溶胶-凝胶(sol-gel)和交联技术,将乳腺癌抗体固定在铂盘电极表面的氨基化sol-gel功能膜上,制备出用于检测乳腺癌抗原(CA15-3)的免疫传感器.用红外光谱验证了该传感器功能膜的形成过程和组成结构,采用循环伏安法对传感器逐层修饰过程进行了表征,并对传感器功能膜的作用机理进行了探讨.该法检测CA15-3的线性范围为8～240U/mL,线性回归方程为ΔE=75.75lgc-56.36,r=0.998.结果表明,该方法很好地保持了被固定抗体的活性,增强了传感器的稳定性,所制备的传感器于4℃干态保存30d,其响应信号基本不变.且该传感器响应迅速、灵敏度高、选择性好,血清中常见抗原不干扰测定.     

甲基睾酮的控温相分离荧光免疫分析

本文报道一种新的甲基睾酮（ＭＴ）测定的免疫分析法。通过将ＭＴ与牛血清白蛋白（ＢＳＡ）偶联制得了ＭＴ全抗原ＭＴ－３－ＢＳＡ（ＡｇＭＴ）,将ＡｇＭＴ经兔子免疫获得了针对ＭＴ的多克隆抗体。与温度敏感性水凝胶偶联的抗原和游离半抗原ＭＴ竞争性地与有限量的异硫氰基荧光素标记抗体反应。根据临界析相温度以上免疫复合物沉淀的特性进行分离,降低背景干扰。该法对ＭＴ测定的线性范围在２７．５ｎｇ／ｍＬ￣１．１μｇ／ｍＬ,     

基于酶标抗体和媒介体共吸附于金胶壳聚糖膜的PSA免疫传感器的制备

本文研制了一种用金胶壳聚糖仿生膜来同时固定四甲基联苯胺（TMB）和酶标抗体的新型电化学免疫传感器,用于检测血清肿瘤标志物前列腺特异性抗原（PSA）的含量。固定的TMB作为电子传递媒介体,在扫速小于45 mV/s时,电极表现为一个表面控制过程,而在扫速大于45 mV/s时则表现为一个扩散控制过程。将固定有酶标抗体和TMB的免疫传感器与待测PSA抗原一起培育,在该传感器上形成的免疫复合物通过TMB-H₂O₂-HRP电化学体系进行了测定。在优化实验条件下,PSA的线性检测范围为5-30 ng·mL^-1,检测限为1.0 ng·mL^-1。该PSA免疫传感器制备方法简单,成本低廉,具有较好的稳定性和重现性。     

新型免疫电极法测定乙型肝炎表面抗原

本文报道一种新型的乙型肝炎抗体膜片。测试时,仅需将25μl被检血清滴注于该膜上,经孵育与洗涤即可制得抗原与抗体复合物膜。用复合物膜组装成免疫电极,测定血清样品中乙型肝炎表面抗原含量。电极的线性范围是20～320ng/ml,方法的日间相对标准偏差为10.78％(n=4,m=5),对血清中可能存在的一些其它抗原具有较好的选择性。105例临床血清样本,本法与酶联免疫吸附分析法测定结果符合率为86％。乙肝抗体膜片系一次性使用,4℃可保存半年以上。     

纳米金修饰电流型CA19-9免疫传感器的制备及应用

通过固定辣根过氧化酶（HRP）标记CA19-9抗体在纳米金修饰的碳糊电极制备了CA19-9安培免疫传感器。该免疫传感器在舍有CA19-9抗原的磷酸盐缓冲溶液中培育后,溶液中CA19-9抗原分子和HRP标记CA19-9抗体分子免疫结合导致了传感器电流的降低。在优化的实验条件下,样品中CA19-9浓度在2～30U／mL范围内与电流降低成线性关系,方法的检出限为1．4U／mL。该免疫传感器表现出较好的稳定性、准确性和重现性,为临床免疫分析提供了一种快速便捷方法。     

基于金微粒修饰的聚苯胺乙脑疫苗传感器的研究

采用聚合物掺杂法将金微粒固定在聚苯胺膜中,再通过静电吸附固定抗体,制成非标记电流型乙脑疫苗传感器。根据抗原抗体特异性结合形成的免疫复合物使电极敏感膜有效扩散面积减小,电流减少的特性,提出以嵌入金微粒的聚苯胺膜作为电子媒介体,用0.1mol/L磷酸盐缓冲液(pH7.4)为测试底液检测乙脑疫苗效价的新方法。用该传感器检测乙脑疫苗效价,线性范围6.1×10-8～4.1×10-6lgpfu/mL,相关系数r=0.994,检测限为3.0×10-8lgpfu/mL(S/N=3)。该传感器可用于生物制品的测定。     

基于聚吡咯修饰膨胀石墨电极的甲胎蛋白电化学免疫传感器

构建了测定人血清中甲胎蛋白(AFP)的电化学免疫传感器。此免疫传感器的制备采用恒电位法在膨胀石墨(EG)电极表面合成聚吡咯(PPy),再以戊二醛(GA)作为交联剂,固定辣根过氧化酶标记的AFP抗体(HRP-anti-AFP)。此免疫传感器在含AFP的溶液中于35℃温育50 min后,再在传感器表面修饰普鲁士蓝(PB)作为电子介体,抗原抗体免疫结合产生的免疫复合物会导致HRP对PB催化氧化的效率降低。在优化条件下,AFP的浓度在0.01～300μg/L范围内呈线性关系,检出限为6.25 pg/mL(S/N=3)。这种基于PPy修饰EG电极的免疫传感器制备简单,灵敏度高且价格低廉,有望成为一次性电化学免疫传感器。     

测定人血清白蛋白的荧光光纤流动免疫分析系统

用新型荧光光纤免疫传感流动分析测试系统,对人血清白蛋白（HSA）进行测试;将偶联于微晶纤维素表面形成的HSA固相抗体与待测HSA（抗原）、荧光标记抗原（FTTC－HSA）竞争性结合。经稀碱液处理,固相载体与抗原抗体复合物分离,用新型荧光光纤免疫传感流动分析系统测定解析液的荧光值,以求得待测HSA的含量;该法检出限为0．1g/L,日内RSD0．91％－6．4％,日间RSD1．8％－8．0％,加标回收率91％－120％;用该法测定注射用HSA,与临床检验常用的溴甲酚绿化对照,具有良好的相关性（r=0.9808)。     

Enzyme immunoelectrode for insulin incorporating a membrane partially treated with water vapour plasma

An enzyme immunoelectrode for the amperometric determination of serum insulin is described. The device consists of an immunoreactive membrane combined with a hydrogen peroxide electrode. The surface of a microporous hydrophobic polypropylene membrane is modified by water vapour plasma treatment to make it partially hydrophilic. Subsequent treatment with octamethylenediamine and glutaraldehyde enables the surface of this membrane to interact with various proteins. Anchoring of the antibody to Protein A immobilized on the membrane was effective for immunoreactivity.     

Development of An Amperometric Immunoassay Based on An Highly Dispersed Immunoelectrode

Abstract

An amperometric immunoassay technique utilizing a highly dispersed immunoelectrode was developed. The assay method is based on the ‘sandwich’ scheme of immunointeraction. Rabbit IgG has been used as model analyte. Horseradish peroxidase labelled conjugates were employed coupled with amperometric detection of iodine reduction. Highly dispersed ULTI carbon serves as an immunosorbent and at the same time as an electrode material. It was found that the dependence of immunoelectrode response on the time of incubation during the second stage of ‘sandwich’ -based assay was complex. This is probably due to non-equilibrium immuno-agglomeration of sub-micron sized particles of immunosorbent. The improvement in the sensitivity of immunoelectrode by increasing the amount of immunosorbent was demonstrated. The increase in the magnitude of the immunoelectrode response is proportional to the increase of the amount of immunosorbent. A detection limit of 10 pM has been achieved with an overall assay time of less than 20 minutes.     

Sensitive detection of tumor marker CA15-3 in human serum by capillary electrophoretic immunoassay with chemiluminescence detection

A new and sensitive non-competitive immunoassay (IA) for tumor marker carbohydrate antigen 15-3 (CA15-3) by CE coupling with ECL detection has been developed. This method is based on luminol-H(2)O(2 )reaction catalyzed by horseradish peroxidase (HRP). The optimum CE separation and CL detection conditions were investigated. After the non-competitive immunoreaction, the free HRP-labeled CA15-3 antibody (Ab*) and the bound Ab*-antigen (Ab*-Ag) complex were separated in a separation capillary and then catalyzed the CL reaction of luminol and H(2)O(2 )in a reaction capillary following the separation capillary. The calibration curve based on the peak areas of Ab*-Ag complex plotted against the concentrations of CA15-3 is in the range of 0-250 U/mL with a correlation coefficient of 0.9983 and the detection limit is 0.035 U/mL (S/N = 3). The response for five consecutive injections of 125 U/mL CA15-3 resulted in RSDs of 0.83% and 3.1% for the migration time and the peak area, respectively. The method was successfully used for the quantification of CA15-3 in human sera obtained from healthy persons and from patients with breast cancer.     

Amperometric carbohydrate antigen 19-9 immunosensor based on three dimensional ordered macroporous magnetic Au film coupling direct electrochemistry of horseradish peroxidase

A sandwich-type electrochemical immunosensor for the detection of carbohydrate antigen 19-9 (CA 19-9) antigen based on the immobilization of primary antibody (Ab₁) on three dimensional ordered macroporous magnetic (3DOMM) electrode, and the direct electrochemistry of horseradish peroxidase (HRP) that was used as both the label of secondary antibody (Ab₂) and the blocking reagent. The 3DOMM electrode was fabricated by introducing core–shell Au–SiO₂@Fe₃O₄ nanospheres onto the surface of three dimensional ordered macroporous (3DOM) Au electrode via the application of an external magnet. Au nanoparticles functionalized SBA-15 (Au@SBA-15) was conjugated to the HRP labeled secondary antibody (HRP-Ab₂) through the Au–SH or Au–NH₃⁺ interaction, and HRP was also used as the block reagent. The formation of antigen–antibody complex made the combination of Au@SBA-15 and 3DOMM exhibit remarkable synergistic effects for accelerating direct electron transfer (DET) between HRP and the electrode. Under the optimal conditions, the DET current signal increased proportionally to CA 19-9 concentration in the range of 0.05 to 15.65 U mL⁻¹ with a detection limit of 0.01 U mL⁻¹. Moreover, the immunosensor showed high selectivity, good stability, satisfactory reproducibility and regeneration. Importantly, the developed method was used to assay clinical serum specimens, achieving a good relation with those obtained from the commercialized electrochemiluminescent method.     

An electrochemical amplification immunoassay using bi-electrode signal transduction system

An electrochemical immunoassay technique has been developed based on the sensitive detection of the enzyme-generated product with a bi-electrode signal transduction system. The system uses two separate electrodes, an immunoelectrode and a detection electrode to form a galvanic cell to implement the redox reactions on two different electrodes, that is the enzyme-generated reductant in the anode region is electrochemically oxidized by an oxidant (silver ions) in the cathode apartment. Based on a sandwich procedure, after immunoelectrode with antibody immobilized on its surface bound with the corresponding antigen and alkaline phosphatase conjugated antibody successively, the immunoelectrode was placed in enzyme reaction solution and wired to the detection electrode which was immerged into a silver deposition solution. These two solutions are connected with a salt bridge. Thus a bi-electrode signal transduction system device is constructed in which the immunoelectrode acts as anode and the detection electrode serves as cathode. The enzyme bound on the anode surface initiates the hydrolysis of ascorbic acid 2-phosphate to produce ascorbic acid in the anode region. The ascorbic acid produced in the anodic apartment is electrochemically oxidized by silver ions coupled with the deposition of silver metal on the cathode. Via a period of 30 min deposition, silver will deposited on the detection electrode in an amount corresponding to the quantity of ascorbic acid produced, leading to a great enhancement in the electrochemical stripping signal due to the accumulation of metallic silver by enzyme-generated product. Compared with the method using chemical deposition of silver, the electrochemical deposition of silver on a separate detection electrode apartment avoids the possible influence of silver deposition on the enzyme activity.     

Electrochemical immunosensor for the breast cancer marker CA 15–3 based on the catalytic activity of a CuS/reduced graphene oxide nanocomposite towards the electrooxidation of catechol

The authors report on an electrochemical immunosensor for the tumor marker carbohydrate antigen 15–3 (CA15–3). It is based on the use of a composite consisting of reduced graphene oxide (RGO) and copper sulfide (CuS) that was placed on a screen-printed graphite electrode. The electrode shows excellent activity towards the oxidation of catechol acting as an electrochemical probe, best at a working potential of 0.16 V. The electrode was modified with antibody against CA15–3. Once the analyte (CA15–3) binds to the surface of the electrode, the response to catechol is reduced. The assay has a linear response in the 1.0–150 U mL^?1 CA15–3 concentration range, with a 0.3 U mL^?1 lower detection limit and a sensitivity of 1.88 μA μM^?1 cm^?2. The immunosensor also shows good reproducibility (2.7%), stability (95% of the initial values after storing for four weeks). The method was successfully applied to the determination of CA15–3 in serum samples, and results were found to compare well to those obtained by an ELISA. Conceivably, this nanocomposite based detection scheme has a wider scope and may be applied to numerous other immunoassays.

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Graphical abstract A label-free electrochemical immunosensor based on copper sulfides/graphene nanocomposites was developed for enzyme-free determination of CA15–3 biomarker. This immunosensor can be utilized as a tool to detect of CA15–3 in real samples.

     

Electrochemical Immunoassay for Tumor Marker CA19-9 Detection Based on Self-Assembled Monolayer

A CA19-9 electrochemical immunosensor was constructed using a hybrid self-assembled membrane modified with a gold electrode and applied to detect real samples. Hybrid self-assembled membranes were selected for electrode modification and used to detect antigens. First, the pretreated working electrodes were placed in a 3-mercaptopropionic acid (MPA)/β-mercaptoethanol (ME) mixture for 24 h for self-assembly. The electrodes were then placed in an EDC/NHS mixture for 1 h. Layer modification was performed by stepwise dropwise addition of CA19-9 antibody, BSA, and antigen. Differential pulse voltammetry was used to characterize this immunosensor preparation process. The assembled electrochemical immunosensor enables linear detection in the concentration range of 0.05–500 U/mL of CA19-9, and the detection limit was calculated as 0.01 U/mL. The results of the specificity measurement test showed that the signal change of the interfering substance was much lower than the response value of the detected antigen, indicating that the sensor has good specificity and strong anti-interference ability. The repeatability test results showed that the relative standard deviations were less than 5%, showing good accuracy and precision. The CA19-9 electrochemical immunosensor was used for the actual sample detection, and the experimental results of the standard serum addition method showed that the RSD values of the test concentrations were all less than 10%. The recoveries were 102.4–115.0%, indicating that the assay has high precision, good accuracy, and high potential application value.     

Direct potentiometric immunoelectrodes

Summary TK pencil fine mines type Faber Castell Super polymer from Staedtler are used as graphite based material for an atrazine-sensitive immunoelectrode. Eighteen procedures are tested to immobilize antibodies against atrazine but only one turned out to be useful for the preparation of a graphite based immunoelectrode. It incorporates binding of glutardialdehyde to active sites of the graphite surface and subsequent cross-linking of the antibodies with glutardialdehyde. The method has been carefully optimized with respect to selectivity and the cross-sensitivity is determined for such an electrode based on atrazine-rabbit and atrazine-sheep antibodies as well. Due to the structural difference of the used immunogens, the sign of the potential response and the cross-sensitivities are different. The application of such an electrode to a natural water sample is demonstrated and shows that the useful concentration range in a final solution is between 20 and 250 ng/l. Of course, interference from ions must be excluded in future analytical applications.Dedicated to Professor Dr. Wilhelm Fresenius on the occasion of his 80th birthdayPart II: Fresenius J Anal Chem