Development of human IgE biosensor using Sezawa-mode SAW devices

This paper reports Sezawa-mode surface acoustic wave (SAW) devices with via-isolated cavity to construct the allergy biosensor. To fabricate Sezawa-mode SAW devices, the RF magnetron sputtering method for the growth of piezoelectric ZnO thin films are adopted and influences of the sputtering parameters are investigated. The optimal substrate temperature of 300 °C, RF power of 120 W and sputtering pressure of 2 Pa were used to deposit piezoelectric ZnO films with a smooth surface, uniform grain size and strongly c-axis-orientated crystallization. A back-etched SAW resonator is used in this study. The wet etching of (100)-oriented silicon wafers is used to form a back-side cavity which is critical to the formation of a hopper cavity for holding bio-analytes. The remaining membrane structure silicon thickness was 25 μm. In this report, the chrome (Cr, 12 nm)/gold (Au, 66 nm) layer was initially deposited onto the sensing area of SAW devices as the binding layer for biochemical sensor. The resonance frequency of the Sezawa-mode SAW device is 1.497 GHz. The maximum sensitivity of the Sezawa-mode is calculated to be 4.44 × 10⁶ cm²/g for human immunoglobulin-E (IgE) detection. The stability for human IgE detection is calculated to be 80% and the variation of the stability ±3% was obtained after several tests.     

Site-specific conjugation of ScFvs antibodies to nanoparticles by bioorthogonal strain-promoted alkyne-nitrone cycloaddition

Particularly suitable: An N-terminal serine mutant of anti-HER2 scFv antibody was conjugated to polymer-coated magnetofluorescent nanoparticles by strain-promoted alkyne-nitrone cycloaddition. The resulting nanoparticles (see scheme) proved effective in targeting and labeling HER2-positive breast cancer cells.     

胶体金免疫层析法快速检测三聚氰胺

采用戊二醛法制备三聚氰胺-BSA偶联抗原,胶体金标记三聚氰胺单克隆抗体,建立了检测三聚氰胺的胶体金免疫层析试验。结果显示,该试验具有良好的敏感性,胶体金免疫层析试验对鲜奶、奶粉和饲料样品中三聚氰胺的最低检测量分别为1.0、2.0和2.5μg/g,该法适合现场快速检测三聚氰胺。     

水溶性量子点标记狂犬病P蛋白单克隆抗体的研究

利用共价偶联的方式,在水溶性缩合剂1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)和N-羟基硫代琥珀酰亚胺(Sulfo-NHS)促进作用下,将400 μL的2 g/L狂犬病P蛋白抗体与适量的聚丙烯酸修饰后的水溶性硫脲修饰ZnO掺Cd量子点进行共价偶联反应,经磷酸盐缓冲液(PBS,0.01 mol/L,pH 7.4)透析纯化得到目标偶联物,采用荧光发射光谱、生物质谱、酶联免疫法等对偶联物进行表征.结果表明:偶联后的量子点荧光最大发射波长红移了10 nm,荧光强度随着狂犬病P蛋白抗原浓度的增加而逐渐增强;量子点标记狂犬病P蛋白抗体后的分子离子峰在m/z 67580处,比狂犬病P蛋白抗体分子离子峰增大了1453.由此证实狂犬病P蛋白抗体成功偶联到水溶性量子点上,且结构未受破坏.     

Selective response of antigen‐antibody reactions on chiral surfaces modified with 1,2‐diphenylethylenediamine enantiomers

This work reported a comparative analysis of the amperometric responses of antigen‐antibody reactions on two stable chiral surfaces which were modified with 1,2‐diphenylethylenediamine enantiomers. Alpha‐fetoprotein antibody and antigen (anti‐AFP and AFP) were selected as model systems. First, (1R,2R)‐1,2‐diphenylethylenediamine or (1S,2S)‐1,2‐diphenylethylenediamine was modified on the gold surface of the electrode through amide linkage to construct chiral surfaces. Then, anti‐AFP was immobilized on the chiral electrode surface by electrostatic and hydrogen bonding interactions. The electrochemical characteristics of the modified electrodes were studied via cyclic voltammetry. The selective current responses of antigen‐antibody reactions on chiral electrode surfaces for different incubation time and varying AFP concentrations were monitored. The antigen‐antibody reactions were greatly influenced by the chirality of 1,2‐diphenylethylenediamine enantiomers, and the amperometric responses obtained from the (1S,2S)‐1,2‐diphenylethylenediamine modified electrode was obviously stronger than that from the (1R,2R)‐1,2‐diphenylethylenediamine modified electrode. Such work may not only offer valuable reference to the research of chiral drugs, but also help to comprehend the high selectivity of chiral molecular species in biosystems. Copyright © 2011 John Wiley & Sons, Ltd.     

Generation of anti-azoxystrobin monoclonal antibodies from regioisomeric haptens functionalized at selected sites and development of indirect competitive immunoassays

Azoxystrobin is a modern strobilurin fungicide used around the world to combat prime diseases affecting highly valuable crops. Accordingly, residues of this chemical are frequently found in food, even though mostly under maximum tolerated levels. We herein describe the development of an indirect competitive immunoassay for the determination of azoxystrobin residues. A panel of monoclonal antibodies displaying subnanomolar affinity to azoxystrobin was generated using, as immunizing haptens in mice, four functionalized derivatives carrying the same spacer arm located at different rationally chosen positions. This collection of antibodies was thoroughly characterized with homologous and heterologous antigens, and the immunoassay consisting of monoclonal antibody AZo6#49 and the coating conjugate OVA–AZb6, which displayed an IC₅₀ value of 0.102 μg L⁻¹ and a LOD of 0.017 μg L⁻¹, was eventually optimized. The response to different pH and ionic strength conditions of the specific assay was studied using a biparametric approach. In addition, the influence of Tween 20 and organic solvents over the assay parameters was also evaluated. After optimization, the developed immunochemical assay was applied to the analysis of azoxystrobin in spiked juices of relevant fruits and vegetables, showing excellent recoveries between 2 and 500 μg L⁻¹.     

Amperometric Immunoassay of Azinphos‐Methyl in Water and Honeybees Based on Indirect Competitive ELISA

Abstract

An electrochemical immunosensor based on indirect competitive ELISA technique has been developed and tested for the detection of azinphos‐methyl in aqueous solutions and spiked honeybee extracts. The detection of the pesticide was based on competition for binding to monoclonal antibodies with an ovalbumin (OVA) conjugate, followed by the incubation with anti‐mouse IgG labeled with horseradish peroxidase, whose activity was measured amperometrically with hydroquinone as the substrate. The sensitivity of the azinphos‐methyl assay, estimated as the IC₅₀ value, was found to be 1.2 nmol L^?1 (60 min incubation), with a linear range of 0.6–500 nmol L^?1 in optimal conditions. The matrix effect on the detection of azinphos‐methyl in honeybee extract was found negligible, with the recovery values in the range 92–105%.     

An Aptamer-Based Competitive Fluorescence Quenching Assay for IgE

A simple competitive fluorescence quenching assay based on aptamer was developed for IgE detection. Two DNA probes were used. One is 5′-end fluorescein-labeled IgE aptamer; the other is 3′-end DABCYL-labeled short DNA, which would hybridize with IgE aptamer to quench the fluorescence. In the presence of IgE, the aptamer-IgE complex formed is strong enough to prevent the short DNA probes hybridizing with the bounded aptamer probes, which results in the less decrease of fluorescence intensity. The signal change was found to be proportional to the concentration of IgE from 0.35 to 35 nM with a detection limit of 0.17 nM.     

Preparation of Anti-Cadmium-EDTA Complex Polyclonal Antibody and Its Application for Determination of Cadmium in Aqueous Solution

Abstract

A polyclonal antibody that can recognize cadmium-ethylenediaminetetraacetic acid (CD-EDTA) complex was prepared via the injection of New Zealand white rabbits with Cd-1-(4-isothiocyanatobenzyl) ethylenediamine-N,N,N′,N′-tetraacetic acid–BSA (Cd-ITCBE-BSA). The polyclonal antibody displayed high levels of affinity for Cd-1-(4-isothiocyanatobenzyl) ethylenediamine-N,N,N′,N′-tetraacetic acid-OVA (Cd-ITCBE-OVA) with favorable titer of 1.28 × 10⁶. A simple, reliable, and economical indirect competitive immunoassay based on this polyclonal antibody was developed and validated for detection of cadmium. Assay optimization was performed with respect to chelator concentration, ionic strength, blocking solution, pH, and reaction time. The detection limit of the assay was 0.21 µg L^?1, and the effective linear range was from 10^?1 to 10³µg L^?1. The coefficient of variation (CV) of intra- and interassay were 1.0–8.2% and 2.3–6.9%, respectively. Results yielded low cross-reactivity of the assay to other tested metals such as Pb²⁺, Ni²⁺, Mg²⁺, Ca²⁺, Cu²⁺, Mn²⁺, Zn²⁺, Co²⁺, Cr³⁺, and Fe³⁺, except Hg²⁺, which showed a cross-reactivity of 7.4%. Spike recoveries of ultrapure water, tap water, and samples of the Yangtze River were 85.5–116.3%. These results show that this assay is suitable for quantitative detection of cadmium at trace levels in water samples.     

Construction of Three Single‐Chain Antibody Fragment Variable Fusion Structures for Sensitive Detection of Nucleocapsid Protein of Hantaan Virus

Abstract

Three single‐chain fragment variable (scFv) fusion structures were constructed for use in rapid and sensitive detection of nucleocapsid protein (NP) of Hantaan virus. The detection of NPs on glass chips was signalized by enzyme labeling or fluorescence dye Cy3, or Cy5 cluster nanoparticles. The sensitivity of the methods with different signal systems was evaluated and compared. The detection limits of scFv‐alkaline phosphatase fusion, fluorescence labeling (scFv‐Cy3), and nanoparticles labeling (scFv‐SBP‐streptavidin‐nanoparticle) were 0.1 µg/mL, 1 ng/mL, and 0.1 ng/mL NP, respectively, which were all lower than that in a conventional enzyme‐linked immunosorbent assay (ELISA) (1 µg/mL). Twenty Hantaan virus isolates were detected using the proposed methods.