基于HRP催化过硫酸盐阴极电致化学发光的非标记型免疫传感器

基于酶催化阴极电致化学发光（ECL）构建灵敏的免疫传感器用于肿瘤标志物检测。利用过氧化氢酶（HRP）催化H2O2底物反应产生大量溶解氧,催化增强S2O82-的阴极ECL信号,构建非标记型免疫传感器。当抗原蛋白在电极表面与抗体形成免疫复合物时,屏蔽了检测液中底物与电极表面固载酶之间的接触并阻碍了电子传递,使ECL信号强度随抗原浓度增加而减小,建立ECL信号与待测物浓度之间的线性依赖关系,实现对血清中肿瘤标志物CEA的快速灵敏检测,检测限达1.04 ng/mL。     

基于甲胎蛋白抗体和巯基丁二酰胺铜(II)共固定修饰电极的人血清中甲胎蛋白免疫传感器

研制基于甲胎蛋白抗体(Ab-AFP)和巯基丁二酰胺铜(Ⅱ)(CuL)共固定修饰玻碳电极(GCE|Ab-AFP/TiO2/CuL)的免疫传感器,用于测定人血清中AFP抗原水平。该免疫传感器是利用溶胶-凝胶气相沉积技术,将AFP抗体分子固定在CuL修饰玻碳电极表面制备而成。电极表面的CuL具有电活性,对H2O2有良好的电化学还原催化。当该免疫传感器在含AFP样品的溶液中于25℃温育30min后,AFP抗原与Ab-AFP抗体分子的免疫结合物导致CuL的电子传递被部分阻碍,使CuL对H2O2电催化还原的效率降低,电流值的下降量和AFP浓度成正比,可用于AFP定量测定。在pH=6的PBS中,25℃温育30min的优化条件下,该传感器对AFP的检测线性范围为0.5～3.9和3.9～100ng/mL,检出下限为0.081ng/mL(3σ)。该免疫传感器制备过程比基于酶标抗体修饰传感器简单,尤其是对H2O2催化响应迅速,稳定性好,可以在磷酸盐缓冲溶液中稳定存放15d。对于构建无试剂的免疫传感器和临床免疫分析提供了一种新颖的方法。     

适用于鱼肉中氯霉素检测的抗体包被金磁纳米微粒修饰安培免疫传感器研究

研制了基于氯霉素抗体包被Fe3O4/Au金磁纳米微粒(GMP)和三乙撑四胺铜(II)(CuL)共固定修饰平面热解石墨电极的安培免疫传感器(PRG|CuL / anti CAP-GMP),用于测定鱼肉中CAP含量.该免疫传感器是利用外加磁场,将anti CAP-GMP吸引到CuL修饰的PRG电极(PRG|CuL)表面制备而成.CuL对H2O2还原具有良好的电催化能力,当该传感器在含CAP样品液中温育后,CAP与电极表面的anti CAP的免疫结合物导致CuL对H2O2的催化还原电流(I)降低,电流下降值(△I)和CAP浓度成正比,可用于CAP定量测定.在25℃的pH=6.5磷酸盐缓冲液(PBS)中温育30 min,该传感器对CAP的检测线性范围为0.6~110 ng/mL,检出下限为0.092 ng/mL(3σ法).应用于鱼肉中CAP检测并与传统的液相色谱法(HPLC)比较,结果一致,其添加回收率在97%~104%之间.该免疫传感器集分离、富集为一体,电极表面可更新,检测灵敏快速,对于水产品中痕量氯霉素分析提供了一种新颖的方法.     

基于纳米金/硫堇修饰金电极的脱落酸安培免疫传感器的研制

提出了一种基于纳米金/硫堇修饰金电极的ABA安培免疫传感器。该传感器基于H2O2-HRP-硫堇催化波体系构建,其中硫堇为传感介质。当HRP存在时,通过加入H2O2,硫堇的还原电流大幅增加,并且电流的增加依赖于HRP活性。HRP活性又由ABA与HRP酶标抗体结合物调控,产生一个减小的催化波。用BSA封闭硫堇单分子层修饰后可能存在的活性位点以避免非特异性吸附。优化了测定条件,包括酶标抗体和硫堇的最佳比例、培育时间、缓冲液的pH值和H2O2浓度。此传感器的还原电流在ABA浓度0.5~1000ng/mL范围内呈线性下降,回归方程为y=0.0209x 17.071,相关系数为0.9922,检测限为0.2ng/mL。     

自组装CA125免疫传感器的研制及其对血清中CA125的分析

利用自组装的方法在金电极上制得巯基丁二胺钌(Ⅲ)/纳米金胶/卵巢癌抗体(抗CA125)免疫修饰电极.用该修饰电极对CA125进行检测,发现溶液中CA125 抗原分子和传感器表面固定化CA125抗体发生免疫反应.结果显示: CA125 抗体导致了该传感器电流的降低.在优化的实验条件下,样品中CA125浓度在2～140 unit/mL 范围内与电流降低成线性关系,检测限为0.8 unit/mL.该免疫传感器表现出良好的准确性和精确性.对40 unit/ mL的CA125重复测定8次,相对标准偏差约为2.9%.该传感器具有较好的制备重复性和稳定性.该方法的提出为卵巢癌临床免疫分析提供了一种有前景的方法.     

碳纳米管组装电化学免疫传感器测定IgG抗体的研究

应用吸附法将IgG抗原固定于多壁碳纳米管修饰的玻碳电极表面,制备用于IgG抗体检测的电化学免疫传感器。以辣根过氧化物酶为标记物,对苯二酚为底物,利用辣根过氧化物酶标记IgG抗体与待测IgG抗体竞争电极表面固定的IgG抗原,建立了免疫竞争法检测IgG抗体的高灵敏度电化学分析方法。碳纳米管的大比表面积和电化学催化作用,提高了分子识别物质的固定量和电化学检测的灵敏度。工作电位为 0.030 V(vs.SCE)时,响应电流与IgG抗体浓度在0.30~10μg/mL范围内呈良好的线性关系,检出限为0.11μg/mL。     

纳米金修饰铂金电极固载白喉抗原免疫传感器的研究

该文报道了一种制作电流型白喉抗原免疫传感器的方法.用金纳米颗粒吸附白喉抗原,采用聚乙烯醇缩丁醛(PVB)为辅助固定抗原膜基质将其固定在铂金电极的表面,制成白喉抗原免疫传感器.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,实现对白喉抗体的定量检测.该传感器对白喉抗体检测的线性范围是2...     

基于纳米磁珠修饰印刷电极的牛奶中三聚氰胺检测安培免疫传感器

共固定氯代邻菲哕啉双核铜配合物(Cu2(phen)2Cl2(μ-Cl)2,简称CuzL)和包被三聚氰胺抗体(anti MA)的FbO4/Au胶(简称纳米磁珠)于丝网印刷电极(SPCE)表面,构建了一种测定牛奶中三聚氰胺(简称MA)含量的安培免疫传感器.当该传感器在含MA溶液中温育时,MA和同时加入的HRP标MA二抗(HRP-anti MA Ⅱ),与电极表面的anti MA形成三元免疫复合物.该复合物上的HRP能催化HzO2还原,并经过Cu2L传导在电极表面形成电流,电流强Ⅰ与MA浓度在0.5～40和60～100ng/mL范围内呈线性关系,检测限为0.25 ng/mL.在pH 6.2的PBS底液中,该传感器制作简单、可抛弃,对MA检测时间小于20 min,灵敏度为1μA·ng-1mL,明显高于酶联免疫吸附(ELISA)法;可实现牛奶中MA免分离现场分析.     

基于囊状银钯铂合金的无标记型癌胚抗原免疫传感器的研制

该文将所合成的中空囊状银钯铂合金和壳聚糖（CHIT）修饰于玻碳电极（GCE）表面,利用中空囊状银钯铂合金比表面积大、反应活性位点多、导电能力良好、催化性能优异和吸附能力强等优点将癌胚抗原的抗体（anti-CEA）固定到电极表面,从而制得高灵敏的无标记型癌胚抗原（CEA）免疫传感器。当抗体与抗原发生免疫反应形成复合物时,会降低银钯铂合金的电催化活性并增加传质阻力。采用示差脉冲伏安法（DPV）检测电化学探针铁氰化钾的响应电流信号的减小程度,实现对CEA的检测。实验考察了电极表面的电化学行为,并对免疫传感器的性能进行了研究。在最优的实验条件下检测癌胚抗原的线性范围为0.5-80.0 ng/mL,线性相关系数为0.989 0,检测下限为0.17 ng/mL。     

基于抗体包被纳米金和铜配合物共固定修饰电极的癌胚抗原安培免疫传感器研究

研制了基于抗体包被化学镀纳米金(AuNPs)和[Cu(bpy)2(ONO)]NO3配合物(CuL)共固定修饰玻碳电极(GCE)的安培免疫传感器,并用于血清中癌胚抗原(CEA)的检测。首先将GCE电极表面氧化形成羧基,进而键合上乙二胺。将此胺化电极浸泡在CuL和化学镀金溶液后,CuL可通过π-π堆积作用吸附到GCE表面,并在电极表面还原成30~50nm的纳米金层(GCE|CuL/AuNPs)。将上述电极浸泡在CEA抗体(anti CEA)溶液中,利用AuNPs固定anti CEA,并通过辣根过氧化物酶(HRP)封闭剩余的AuNPs位点,由此构建了一类快速检测CEA的无试剂安培免疫传感器(GCE|CuL-AuNPs/anti CEA-HRP)。其中CuL作为电子媒介体对过氧化脲(CP)有催化还原作用,而且HRP可增强这种作用。当该传感器在37℃下,含CEA的pH6.5PBS溶液中温育30min后,随着温育液中CEA浓度的增加,电极表面形成的免疫复合物也增加,导致CuL对CP的催化电流下降。电流下降百分比1%与CEA浓度在0.1~80ng/mL之间成线性关系,检测限为0.052ng/mL(3σ)。由于采用化学镀法可...     

基于琼脂糖和纳米金的电流型免疫传感器快速检测副溶血性弧菌

将HRP-anti-VP掺杂于琼脂糖和纳米金中并修饰于丝网印刷电极表面,从而制备副溶血性弧菌免疫电极.采用循环伏安法表征免疫电极和监测酶促反应,根据还原电流的变化来定量测定副溶血性弧菌.在优化的实验条件下,免疫电极线性检测范围为105～109 cfu/mL,检出限为7.4×104 cfu/mL(S/N=3).该免疫电极具备较好的特异性、重现性(RSD＜6%)、稳定性(一周后电流响应为初始值的91%)和准确性(与ELISA符合率为97.5%),可初步用于副溶血性弧菌的快速筛检.     

Sensitive electrochemical immunosensor based on enlarged and surface charged gold nanoparticles mediated electron transfer

A simple and sensitive electrochemical immunosensor for the detection of protein biomarker interleukin-6 (IL-6) based on gold nanoparticles (AuNPs) as label was reported. The signal of the immunosensor was originated from enlarged and positively charged AuNPs mediated electron transfer between insulating self-assemble monolayer (SAM) modified electrode and K₃Fe(CN)₆ solution. The gold electrode was first modified with SAM to block the electron transfer between the electrode and K₃Fe(CN)₆ solution. After the preparation of the immunosensor, the AuNPs attached onto electrode was enlarged and positively charged when treated in solution containing HAuCl₄, ascorbic acid and cetyltrimethylammonium bromide (CTAB). The enlarged and positively charged AuNPs then mediated electron transfer between the electrode and K₃Fe(CN)₆ solution, creating redox current that is proportional to the concentration of IL-6 detected. The reported immunosensor has high sensitivity and wide linear range. This novel signal amplification strategy can be applied to the detection of different kinds of protein biomarkers.     

可再生使用的磁性纳米修饰C反应蛋白电流型免疫传感器

利用Fe3O4(核)/Au(壳)(简称GMPs)标记C反应蛋白酶标抗体(HRP-anti CRP),构建了一类新型的磁性纳米探针(HRP-anti CRP/GMPs),将其修饰在丝网印刷电极(SPCE)表面构建了可再生使用的CRP安培型酶联免疫传感器.首先将多壁碳纳米管(MCNTs) -硫堇(Thi) -Nafion复...     

新的免疫细胞传感器CANARY的研究应用进展

免疫细胞传感器的出现推动了病原体检测的发展.该文综述了B淋巴细胞作为传感器敏感元件的构建,B细胞传感器的检测原理及其在病原体检测中的应用研究进展,并展望了免疫细胞传感器的潜在发展前景.     

A micro amperometric immunosensor for detection of human immunoglobulin

1 Introduction In recent years, electrochemical immunosensors have gained considerable interests as bioanalytical devices[1―3]. They have many attractive features, such as convenience for manipulate, being easy to achieve high sensitivity and excellent d…     

Reliable new immunosensor for atrazine pesticide analysis

The aim of the present research was to develop a highly selective and sensitive amperometric immunosensor for atrazine based on a “competition” assay procedure. An immunosensor device for the determination of triazinic pesticides based on two different competition procedures is therefore described. The immunosensor developed uses an amperometric electrode for hydrogen peroxide as transducer and the peroxidase enzyme as marker. The results demonstrate the full validity of this immunosensor method which was optimized by comparing two different “competition” operating procedures. The results obtained using the new immunosensor were then compared with those found using an inhibition OPEE (Organic Phase Enzyme Electrode) for triazinic pesticide detection, which showed no selectivity toward different classes of triazinic, carbamate or organophosphate pesticides. The immunosensor developed displayed on the contrary an appreciable selectivity both toward different classes of pesticides and toward triazinic or benzotriazinic products. In addition, the K_aff value was evaluated. Lastly, the immunosensor was also used to test triazinic pesticide recovery from common real matrices such as milk and vegetal samples, obtaining good recoveries.     

Conductimetric immunosensor for atrazine detection based on antibodies labelled with gold nanoparticles

A novel conductimetric immunosensor for atrazine detection has been designed and developed. This immunosensor is mainly based on antibodies labelled with gold nanoparticles. Additionally, the immunosensor consists of an array of two coplanar non-passivated interdigitated metallic μ-electrodes (IDμE) and immunoreagents specifically developed to detect this pesticide. The chemical recognition layer was covalent immobilized on the interdigital space. Immunochemical detection of the concentration of atrazine is achieved by a competitive reaction that occurs before the inclusion of the labelled antibodies. It is shown that the gold nanoparticles provide an amplification of the conductive signal and hence makes possible to detect atrazine by means of simple DC measurements.The conductimetric immunosensor and its biofunctionalization steps have been characterized by chemical affinity methods and impedance spectroscopy.This work describes the immunosensor structure, fabrication, physico-chemical and analytical characterization, and the immunosensor response using conductivity measurements. The immunosensor developed detects atrazine with limits of detection in the order of 0.1–1 μg L⁻¹, far below the maximum residue level (MRL) (100 μg L⁻¹) established by European Union (EU) for residues of this herbicide in the wine.Although in this paper the competitive reaction occurs in buffer, an initial study of the wine matrix effect is also described.     

基于蛋白A-纳米金-丝网印刷电极的雌二醇免疫传感器

将蛋白A固定在纳米金修饰的丝网印刷电极表面,纳米金大的比表面积以及较高的表面自由能使蛋白A比较牢固地固定在其上;蛋白A分子可以定向结合17β-雌二醇抗体的Fc片段,使抗体在电极表面有序固定化。利用17β-雌二醇和酶标17β-雌二醇的酶免疫竞争反应,研制了快速测定17β-雌二醇的电化学传感器。用循环伏安法和扫描电子显微镜对修饰过程进行了表征。优化测定条件后,传感器呈现了很高的测定灵敏度,电流响应信号与17β-雌二醇浓度在0.1μg/L～20μg/L范围内呈良好的负线性相关,检出限为0.035μg/L(S/N=3)。应用该免疫传感器测定了正常人群尿样中17β-雌二醇含量,测定范围与报道的高效液相色谱法测定结果相一致,样品加标回收率为96%～114%。     

Development of an immunosensor based on the measurement of fluorescence

Consumers concern about the presence of pesticides in food is increasing. However current techniques for detecting pesticide residues in olive oil are very expensive and slow, which make them difficult to apply in commercial conditions. For this reason, the Instituto Valenciano de Investigaciones Agrarias (IVIA) and Universidad Politécnica de Valencia (UPV) have decided to develop an immunosensor as an alternative, fast and cheap method to measure the concentration of some pesticides in olive oil. This work shows the developed transducer by IVIA based on a photovoltaic sensor that transforms the fluorescent radiation to an electrical signal. The immunosensor is based on an immunologic reaction which causes fluorescence on the sample. The intensity of the fluorescence is related with the concentration of marked antibodies. The performed tests with two types of immunoassays and fluorescent markers (FITC and ELF97) have demonstrated the robustness and reliability of the developed transducer. The immunosensor is able to discriminate different concentrations of marked antibodies. This study has demonstrated the possibility of applying this immunosensor to detect chemical residues from pesticide treatments in olive oil samples. These results validate the sensor for future commercial applications.     

石英晶体微天平传感的酶联免疫分析法测定免疫球蛋白IgM

结合酶联免疫分析技术与石英晶体微天平检测技术,发展了一种基于石英晶体微天平传感的免疫传感器用于测定血液中免疫球蛋白IgM的量.羊抗人IgM抗血清通过氨基硅烷化共键固定在石英晶体表面上,采用竞争吸附免疫分析法,以辣根过氧化物酶(HRP)标记的IgM作标记物,N-四甲联苯胺(TMB)和H2O2作酶的底物.结合在石英晶体微天平上的酶标记物催化TMB氧化形成沉淀,使石英晶体微天平的振荡频率发生改变,据此测定标记物HRP-IgM和IgM的量.实验优化了分析条件下,对IgM的检测范围为0.03μg/mL to 7μg/mL,它具有仪器简单、操作方便、灵敏度高等优点.