The sandwich-type electrochemiluminescence immunosensor for α-fetoprotein based on enrichment by Fe3O4-Au magnetic nano probes and signal amplification by CdS-Au composite nanoparticles labeled anti-AFP

A novel and sensitive sandwich-type electrochemiluminescence (ECL) immunosensor was fabricated on a glassy carbon electrode (GCE) for ultra trace levels of α-fetoprotein (AFP) based on sandwich immunoreaction strategy by enrichment using magnetic capture probes and quantum dots coated with Au shell (CdS-Au) as the signal tag. The capture probe was prepared by immobilizing the primary antibody of AFP (Ab1) on the core/shell Fe₃O₄-Au nanoparticles, which was first employed to capture AFP antigens to form Fe₃O₄-Au/Ab1/AFP complex from the serum after incubation. The product can be separated from the background solution through the magnetic separation. Then the CdS-Au labeled secondary antibody (Ab2) as signal tag (CdS-Au/Ab2) was conjugated successfully with Fe₃O₄-Au/Ab1/AFP complex to form a sandwich-type immunocomplex (Fe₃O₄-Au/Ab1/AFP/Ab2/CdS-Au), which can be further separated by an external magnetic field and produce ECL signals at a fixed voltage. The signal was proportional to a certain concentration range of AFP for quantification. Thus, an easy-to-use immunosensor with magnetic probes and a quantum dots signal tag was obtained. The immunosensor performed at a level of high sensitivity and a broad concentration range for AFP between 0.0005 and 5.0 ng mL⁻¹ with a detection limit of 0.2 pg mL⁻¹. The use of magnetic probes was combined with pre-concentration and separation for trace levels of tumor markers in the serum. Due to the amplification of the signal tag, the immunosensor is highly sensitive, which can offer great promise for rapid, simple, selective and cost-effective detection of effective biomonitoring for clinical application.     

A Novel Amperometric Immunosensor for Phytohormone Abscisic Acid Based on In Situ Chemical Reductive Growth of Gold Nanoparticles on Glassy Carbon Electrode

Abstract

An amperometric immunosensor for phytohormone abscisic acid was developed based on in situ chemical reductive growth of gold nanoparticles on glassy carbon electrode. First, an approximate 10 nm gold layer was sputtered uniformly onto the electrode surface, and then gold nanoparticles were grown directly on the gold layer for antibody adsorption by immersing the electrode into the H₂AuCl₄ solution. Determination was based on an enzyme-linked competitive immunoreaction between free and enzyme-labeled abscisic acid to bind on immobilized antibody on electrode. The linear response was from 10 ng/ml to 10 µg/ml with a detection limit of 5 ng/ml.     

玉米kn-1同源盒的高效表达

同源盒基因(homeoboxgenes)在植物,动物,真菌的广泛存在说明这种结构在真核生物进化过程中高度保守,并暗示其具有重要功能.本文将玉米kn-1同源盒插入表达载体pGEM△XbaI,并将形成的重组克隆pGEM△MK转化BL21(DE3)而获得高效表达;表达产物的分子量为29×103,并主要以可溶性蛋白形式存在.Kn-1,Quox-1,OSIH-1间的交叉免疫反应证实三者在蛋白质水平具同源性.而Kn-1同源结构域(Homeodomain)高效表达有助于进一步寻找其靶基因.     

一种简便灵敏测定C反应蛋白的免疫共振散射光谱分析新方法

在pH 6.0的柠檬酸-Na₂HPO₄缓冲溶液中及PEG-6000存在下, C反应蛋白(CRP)与羊抗人C反应蛋白可聚集形成免疫复合物微粒, 在350, 390, 440 nm处有三个共振散射峰. 激光散射法测得免疫复合物微粒的平均粒径为1720.0 nm. 在最佳实验条件下, CRP浓度在0.03～1.80 μg•mL^－1的范围内与390, 440 nm处共振散射强度都呈良好的线性关系, 其回归方程、相关系数、检出限分别为ΔI_{390 nm}＝306.4c＋17.3, ΔI_{440 nm}＝296.0c＋10.7; 0.9993, 0.9996; 0.011, 0.012 μg•mL^－1. 该方法选择性较好, 操作简便, 用于人血清中C反应蛋白的测定, 结果与免疫透射比浊法结果一致, 相对标准偏差在0.90%～4.12%.     

Preparation of TiO2 nanosheet-carbon nanotube composite as immobilization platform for both primary and secondary antibodies in electrochemical immunoassay

TiO₂ nanosheets (TNSs) were synthesized and deposited on multi-wall carbon nanotubes (MWCNTs) to form a nano-composite through a hydrothermal method, followed by the characterization with various spectroscopic and microscopic techniques. The TNS-MWCNT composite was then applied as not only an electrode scaffold to immobilize primary antibody, but also as a carrier to load secondary antibody and horseradish peroxidase (HRP). In both cases, bis(sulfosuccinimidyl) suberate sodium salt acted as an amino cross-linker to covalently bind the biomolecules on TNS-MWCNT composite through their surface primary amino groups. After the sandwich-type immunoreaction, HPR was quantitatively captured on the electrode surface via the binding between secondary antibody and antigen, and electrochemical response of the immunosensor was then amplified by a H₂O₂ mediated HRP catalytic reaction. Using α-Fetoprotein as a model analyte, a linear range between 0.005 and 320 ng mL⁻¹ with a detection limit of 2.0 pg mL⁻¹ was achieved by differential pulse voltammetry. The improved immunosensor performance could be attributed to the biocompatibility and high specific surface area of TNS, and excellent electrical conductivity of MWCNTs, which accelerated the electron transfer at the electrode surface.     

Surface plasmon resonance-based trace detection of small molecules by competitive and signal enhancement immunoreaction

A surface plasmon resonance (SPR)-immunosensor for detection of the low molecular weight compound 2,4-dinitorophenol (DNP) at ultra-low concentration has been developed. The sensor strategy is based on a competitive immunoreaction between DNP and a DNP-protein conjugate, namely DNP-bovine serum albumin conjugate (DNP-BSA). Anti-DNP monoclonal antibody was immobilized on a gold thin-film coated SPR-sensor chip by means of a chemical coupling process. DNP-BSA, on contact with the anti-DNP antibody immobilized SPR-immunosensor chip causes an increase in the resonance angle of the sensor chip. The optimum concentration of immobilized antibody on the SPR-sensor chip is 100 μg mL⁻¹. The SPR-immunosensor response for free DNP determination using the competitive immunoreaction had a response time of ca. 15 min. Using this method, DNP could be determined in the concentration range 1 ppt to 1 ppb. The SPR signal for ppt levels of DNP was enhanced by a factor of three by subsequently treating immuno-bound DNP-BSA with a secondary anti-DNP antibody.     

A Novel Chemiluminescence Immunoassay Using Solid‐Phase Antigen for Free 17β‐Estradiol in Human Serum

A high‐performance chemiluminescence immunoassay, with long‐term durability, good precision and time‐saving, was proposed for the detection of free 17β‐estradiol (E₂) in human serum. Ninety‐six microplates were coated with bovine serum albumin conjugated E₂ antigen as solid phase for the immunoassay. The E₂‐BSA antigen coated on the microplate and the E₂ antigen in the sample competed for the binding sites on the horseradish peroxidase (HRP) labeled anti‐E₂ antibody. Chemiluminescence reaction was subsequently carried out by HRP catalyzing luminol‐H₂O₂ substrates, and the chemiluminescence intensity was inversely proportional to the amount of analyte in human sera samples. The concentration of immunoreagents, immunoreaction time, and other relevant variable conditions upon the immunoassay were studied and optimized. The proposed method exhibited detection limit as low as 5.94×10^?3 µg·L^?1 in a linear detection range from 0.01 to 1.00 µg·L^?1, good recoveries between 105% and 108%, and high precision with intra‐ and inter‐assay coefficients between 7.9% and 14.3%.     

Pseudo-homogeneous immunoextraction of epitestosterone from human urine samples based on gold-coated magnetic nanoparticles

A pseudo-homogeneous immunoextraction method based on gold-coated magnetic nanoparticles (MNPs) for the specific extraction and quantitative analysis of epitestosterone (17α-hydroxy-4-androsten-3-one, abbreviated as “ET”) from human urine samples by high-performance liquid chromatography (HPLC) has been developed. Half-IgG of anti-ET monoclonal antibodies were covalently immobilized onto (Fe₃O₄)_core-Au_shell (Fe₃O₄@Au) MNPs. An external magnetic field was applied to collect the MNPs which were then rinsed with distilled water followed by elution with absolute methanol to obtain ET as the analyte. The obtained extraction solution was analyzed by HPLC with UV detection (244 nm) within 12 min. The standard calibration curve for ET showed good linearity in the range of 20-200 ng mL⁻¹ in phosphate-buffered saline (PBS) solutions with acceptable accuracy and precision. Limit of detection for ET was 0.06 ng mL⁻¹ due to an enrichment factor of 100-fold was achieved. The results obtained by the present method for spiked human urine samples were in agreement with those from indirect competitive enzyme-linked immunoadsorbent assays (ELISAs). The antibody-conjugated Fe₃O₄@Au MNPs are novel materials for immunoaffinity extraction. Compared with the conventional technique using immunoaffinity column, the method described here for sample pretreatment was fast, highly specific, and easy to operate.     

Evaluation of the molecular recognition of monoclonal and polyclonal antibodies for sensitive detection of 2,4,6-trinitrotoluene (TNT) by indirect competitive surface plasmon resonance immunoassay

Detection of TNT is an important environmental and security concern all over the world. We herein report the performance and comparison of four immunoassays for rapid and label-free detection of 2,4,6-trinitrotoluene (TNT) based on surface plasmon resonance (SPR). The immunosensor surface was constructed by immobilization of a home-made 2,4,6-trinitrophenyl–keyhole limpet hemocyanin (TNPh–KLH) conjugate onto an SPR gold surface by simple physical adsorption within 10 min. The immunoreaction of the TNPh–KLH conjugate with four different antibodies, namely, monoclonal anti-TNT antibody (M-TNT Ab), monoclonal anti-trinitrophenol antibody (M-TNP Ab), polyclonal anti-trinitrophenyl antibody (P-TNPh Ab), and polyclonal anti-TNP antibody (P-TNP Ab), was studied by SPR. The principle of indirect competitive immunoreaction was employed for quantification of TNT. Among the four antibodies, the P-TNPh Ab prepared by our group showed highest sensitivity with a detection limit of 0.002 ng/mL (2 ppt) TNT. The lowest detection limits observed with other commercial antibodies were 0.008 ng/mL (8 ppt), 0.25 ng/mL (250 ppt), and 40 ng/mL (ppb) for M-TNT Ab, P-TNP Ab, and M-TNP Ab, respectively, in the similar assay format. The concentration of the conjugate and the antibodies were optimized for use in the immunoassay. The response time for an immunoreaction was 36 s and a single immunocycle could be done within 2 min, including the sensor surface regeneration using pepsin solution. In addition to the quantification of TNT, all immunoassays were evaluated for robustness and cross-reactivity towards several TNT analogs.      

基于巯基自组装单层膜的植物生长激素吲哚乙酸电化学免疫传感器的研究

提出了一种酶标识抗原与待测样品的竞争免疫反应定量测定IAA的方法．该方 法是基于金基底上形成了均一、稳定、有序的硫基自组装单层膜，能以共价方式固 定抗体．利用循环伏安法探讨了底物在不同条件下的反应特征，并研究了最佳实验 条件．在浓度5．68×10^-7～2.83×10^-5mol/L范围内、-200mV电位下，响应电流 与lg[IAA]呈线性关系，其回归方程；i(nA)=-20.8333×lg(10^6[IAA])+68.9167， 相关系数0.9915。对样品IAA的含量进行测定，结果令人满意．