Improvement of an Enzyme Immunoassay for the Determination of Mercury(II)

Three systems were tested for the optimization of a heterogenous non-competitive enzyme immunoassay (EIA) for the determination of Hg (II). The sensitivity of the non optimized Hg-EIA with a detection limit of 2.1 μg/L Hg (II) was improved by an avidin-biotin-complex (ABC) amplification system to a 2-fold lower detection limit (1.1 μg/L Hg (II)). A conventional competitive ElA with the competition reaction between bound and free Hg (II) for antibody (ab) binding sites yielded a detection limit of 1.0 μg/L Hg (II). Further improvement of sensitivity could be achieved by a competitive displacement EIA. In this case ab molecules bound to immobilized haptens are displaced in the next step by free Hg (II). The detection limit of the displacement approach is 0.4 μg/L Hg (II).     

Rapid extraction and quantitative detection of the herbicide diuron in surface water by a hapten-functionalized carbon nanotubes based electrochemical analyzer

A solid phase extraction micro-cartridge containing a non-polar polystyrene absorbent matrix was coupled with an electrochemical immunoassay analyzer (EIA) and used for the ultra-sensitive detection of the phenyl urea herbicide diuron in real samples. The EIA was fabricated by using carboxylated carbon nanotubes (CNTs) functionalized with a hapten molecule (an amine functionalized diuron derivative). Screen printed electrodes (SPE) were modified with these haptenized CNTs and specific in-house generated anti diuron antibodies were used for bio-interface development. The immunodetection was realized in a competitive electrochemical immunoassay format using alkaline phosphatase labeled secondary anti-IgG antibody. The addition of 1-naphthyl phosphate substrate resulted in the production of an electrochemically active product, 1-naphthol, which was monitored by using differential pulse voltammetry (DPV). The assay exhibited excellent sensitivity and specificity having a dynamic response range of 0.01 pg mL(-1) to 10 μg mL(-1) for diuron with a limit of detection of around 0.1 pg mL(-1) (n = 3) in standard water samples. The micro-cartridge coupled hapten-CNTs modified SPE provided an effective and efficient electrochemical immunoassay for the real-time monitoring of pesticides samples with a very high degree of sensitivity.     

Immunoassay for the detection of lead ions in environmental water samples

A rapid, simple, and reliable competitive immunoassay was developed for measurement of lead ions Pb(II) in environmental samples. Avian antibodies were produced against Pb(II). Since lead ions are too small to elicit an immune response, the metal was coupled to protein carrier Bovine serum albumin (BSA) using a bifunctional chelator 1-(4-isothiocyanobenzyl) ethylenediamine N,N,N′,N′-tetra acetic acid (ITCBE). Poultry birds (layers) were immunised with this Pb(II)–ITCBE–BSA immunoconjugate and the avian antibodies (IgY) isolated from egg yolk recognised Pb(II)-ITCBE complexes as capture reagent and a Pb(II)–ITCBE conjugate of Alkaline phosphatase as an enzyme label. Antibody reaction was optimised for different concentrations of antigen and antibody dilutions. Cross reactivity with other metals were below 1% in competitive ELISA. The IC₅₀ value of this avian antibody was 0.19?µg?mL^?1. The detection range and the detection limit were 0.02–1000?µg?mL^?1and 0.2?µg?mL^?1, respectively.     

Differential-pulse anodic stripping voltammetry of mercury with gold-film micro-electrodes

Cylindrical gold film micro-electrodes are easily produced by plasma-sputtering of gold onto carbon fiber electrodes. The micro-electrodes produced were found to maintain their cylindrical geometry indefinitely, unlike gold wire electrodes of similar dimensions. Application of these electrodes in differential-pulse anodic stripping voltammetry provides a method for quantifying trace levels of mercury(II). Up to 100 μg l^?1 Hg(II) the area of the mercury stripping peak varied linearly with mercury concentration; the detection limit was 3.7 μg l^?1. With more than 100 μg l^?1 Hg(II) a new mercury stripping peak grows in at less positive potentials; its peak height is linear with Hg(II) concentration.     

Development of Enzyme Immunoassays for the Detection of Pyrethroid Insecticides

Abstract

Three monoclonal antibodies (mab) for the determination of the pyrethroid insecticide allethrin were generated using a trans-permethric acid-bovine serum albumin conjugate as immunogen. Spleen cells of immunized BALB/c mice were fused with myeloma cells applying polyethylene glycol. Hybridoma cells were selected by HAT selection and screened for cell lines producing mab. Three stable cell lines could be isolated for further characterization and development of an indirect competitive enzyme immunoassay. The mab 3/B4-1/F8 showed a preference for S-bioallethrin, the most efficient insecticidal isomer of allethrin. The detection range lay between 1 μg/L and 250 μg/L; 50 % inhibition occurred at 46 μg/L. Cross-reactivities were 43 % for the natural pyrethrins. The mab 1/E2-5/B5 and 1/A5-7/C10 preferred bioallethrin, which is a mixture of two allethrin enantiomers. 50 % inhibition was found at 60 μg/L or 54 μg/L, respectively. The tests ranged from 9 μg/L to 170 μg/L (1/E2-5/B5) and from 6 μg/L to 160 μg/L (1/A5-7/C10). Cross-reactivities occurred for phenothrin, resmethrin and bioresmethrin.     

Preparation of Anti—Cardiac Troponin I Monoclonal Antibodies and Their Characterization with Surface Plasmon Resonance Biosensor

IntroductionIn fast and slow skeletal and cardiac muscles,troponin I,the inhibitory protein of the troponin-tropomyosin complex,exists in three isotype formsencoded by three separated genes.The amino acidsequences of the two skeletal and one cardiac Tn Iforms( s Tn I and c Tn I,respectively) exhibit40 %dissimilarity[1] .Moreover,human cardiac Tn I has31 additional residues on the N - terminal end,which do not exist in skeletal forms,thus it pro-vides a high potential for obtaining cardiac-…     

Anodic‐Stripping Voltammetric Immunoassay for Ultrasensitive Detection of Low‐Abundance Proteins Using Quantum Dot Aggregated Hollow Microspheres

A new anodic‐stripping voltammetric immunoassay protocol for detection of IgG1, as a model protein, was designed by using CdS quantum dot (QD) layer‐by‐layer assembled hollow microspheres (QDHMS) as molecular tags. Initially, monoclonal anti‐human IgG1 specific antibodies were anchored on amorphous magnetic beads preferably selective to capture F_ab of IgG1 analyte from the sample. For detection, monoclonal anti‐human IgG1 (F_c‐specific) antibodies were covalently coupled to the synthesized QDHMS. In a sandwich‐type immunoassay format, subsequent anodic‐stripping voltammetric detection of cadmium released under acidic conditions from the coupled QDs was conducted at an in situ prepared mercury film electrode. The immunoassay combines highly efficient magnetic separation with signal amplification by the multilayered QD labels. The dynamic concentration range spanned from 1.0 fg mL^?1 to 1.0 μg mL^?1 of IgG1 with a detection limit of 0.1 fg mL^?1. The electrochemical immunoassay showed good reproducibility, selectivity, and stability. The analysis of clinical serum specimens revealed good accordance with the results obtained by an enzyme‐linked immunosorbent assay method. The new immunoassay is promising for enzyme‐free, and cost‐effective analysis of low‐abundance biomarkers.     

Enzyme immunoassay of angiotensin II in human plasma.

We established a highly sensitive double-antibody enzyme immunoassay (EIA) for angiotensin (ANG) II. For competitive reactions, the ANG II-antibody was incubated with ANG II standard (or sample) and beta-D-galactosidase-labeled ANG III (delayed addition). Free and antibody-bound labeled antigen were separated using an anti-rabbit immunoglobulin G (IgG) coated immunoplate. The enzyme activity on the plate was fluorometrically determined. The present immunoassay allows detection of 0.4 to 72 fmol/well of ANG II. Using the present EIA, ANG II-like immunoreactivity (-LI) in human plasma was determined. The level of ANG II-LI in human plasma from 10 healthy volunteers was 33.3 +/- 10.4 pmol/l.     

Indirect Determination of Trace Copper(II) by Adsorptive Stripping Voltammetry with Zincon at a Carbon Paste Electrode

Traces of copper(II) can be determined by adsorptive stripping voltammetry using 2‐carboxy‐2′‐hydroxy‐5′‐sulfoformazyl benzene (Zincon) as complex forming reagent. First in phosphate buffer pH 6.4, copper(II)‐Zincon complex was adsorbed on carbon paste electrode (CPE) with an accumulation potential of 0.6 V. Following this, adsorbed complex was oxidized and detected by differential pulse voltammetric (DPV) scan from 0.6 to 1.0 V. The effective parameters in sensor response were examined. The detection limit (DL) of copper(II) was 1.1 μg/L and relative standard deviations (RSDs) for 10 and 200 μg/L Cu(II) were 1.81 and 1.03%, respectively. The calibration curve was linear for 2–220 μg/L copper(II). The resulting CPE does not use mercury and therefore, has a positive environmental benefit. The method, which is reasonably sensitive and selective, has been successfully applied to the determination of trace amount of copper in water and human hair samples.     

Ultrasensitive determination of mercury by solution anode glow discharge atomic emission spectrometry coupled with hydride generation

An innovative method for the ultrasensitive detection of mercury by solution anode glow discharge atomic emission spectroscopy (SAGD-AES) coupled with hydride generation (HG) was first investigated. In this method, the mercury vapor generated by the HG was transmitted to the SAGD through the miniature hollow tungsten tube for excitation and detected by a miniaturized spectrograph. A thorough parametric evaluation of the HG and SAGD system was performed, including the type and concentration of carrier acid, He flow rate, concentrations of NaBH₄, discharge current and discharge gap. Under optimal operating conditions, the detection limit for Hg²⁺ achieved 0.03 μg/L, with a relative standard deviation of 1.1% at the Hg²⁺ concentration of 5 μg/L. Moreover, the correlation coefficient of the calibration curve was 0.9996 in the range between 0.1 and 10 μg/L. The accuracy and practicability of HG-SAGD-AES were verified by measuring GBW09101b (human hair), GBW10029 (fish), soil and rice samples. The results showed good agreement with the certified values and values from direct mercury analyzer (DMA).     

Characteristics of a liposome immunoassay on a poly(methyl methacrylate) surface

Liposome immunoassay (LIA) is based on enzyme immunoassay (EIA) but the detection sensitivity could be significantly enhanced by using antibody-coupled immunoliposomes encapsulating HRP (horse radish peroxidase). Here, we applied LIA to non-porous poly(methyl methacrylate) (PMMA) and polystyrene (PS) surfaces to compare its detection sensitivity with that of EIA, using rabbit IgG (a ligand molecule) and anti-rabbit IgG antibody (a capture molecule) as the model system. LIA developed much stronger color signals than EIA, especially at a lower concentration range (< ca. 1 μg mL⁻¹). PMMA showed higher affinity toward rabbit IgG than the PS surface, and the anti-rabbit IgG antibody adsorbed on PMMA was more stable than that on PS. Furthermore, the effects of spot volume and antibody concentration on the signal density were analyzed. The signal density increased as the antibody concentration increased, but it was not significantly affected by the spot volume (2.5–20 μL). In conclusion, LIA on PMMA as a solid support is a very useful, highly sensitive microarray detection system. Sang Youn Hwang and Yoichi Kumada have same rights on this paper.     

Trace Detection of Mercury(II) Using Gold Ultra‐Microelectrode Arrays

The electroanalytical detection of trace mercury(II) at gold ultra‐microelectrode arrays is reported. The arrays consist of 256 gold microelectrodes of 5 μm in diameter in cubic arrangements which are separated from their nearest neighbor by 100 μm. The array was utilized in nitric acid using linear sweep voltammetry where a linear response from mercury additions over the range 10 μg L^?1?200 μg L^?1 (10^?8?10^?6 M) was observed with a sensitivity and detection limit of 0.11 nC/μg L^?1 and 3.2 μg L^?1 (16 nM) respectively from using a deposition time of 30 s at ?0.2 V (vs. SCE). This methodology was explored in 0.1 and 1 M chloride media over the mercury range 10 μg L^?1?200 μg L^?1 (5×10^?8?10^?6 M) where similar sensitivities of 0.087 nC/μg L^?1 and 0.078 nC/μg L^?1 were observed with an identical detection limit. The protocol is demonstrated to be useful for the determination of mercury for analysis of environmental water samples.     

Preparation and characterization of digoxin antibody and its application to immunoassays: comparison of performance characteristics between enzyme immunoassay and immunostrip test

After characterizing digoxin immunogens and antibodies, we investigated the assay performances of enzyme immunoassay (EIA), biotin–streptavidin mediated enzyme immunoassay (B-Av EIA), and immunostrip test and compared the effectiveness of these tests for detecting the presence of digoxin and its analogues. The clinical use of digoxin is very complicated due to its narrow therapeutic range. Thus, the development of a rapid and simple detection device has been needed. In our study, we determined digoxin levels by EIA and B-Av EIA, and compared these tests with the immunotest strip to determine the possibility of personal monitoring at the bedside. Three kinds of digoxin antibodies were produced from various digoxin–BSA immunogens (digoxin/BSA molar ratio of 15:1, 50:1, and 200:1 in their preparation step). The antibodies produced were purified by the immunoaffinity chromatography using digoxin–KLH or digoxin–BSA as the affinity ligand. Antibody #7 and its matching coating ligand of digoxin–KLH were selected by titration and sensitivity studies for use in EIA and B-Av EIA. Colloidal gold-labeled antibody #7 on a glass fiber membrane and digoxin–BSA on a nitrocellulose membrane were selected for the immunostrip preparation. The EIA and B-Av EIA could detect 0.01 ppb digoxin within 2.5 h, but the immunostrip could detect 0.01 ppm within 3 min, which was three orders less sensitive than EIA or B-Av EIA.     

Reduction of mercury(II) by electrons contained in carbon dots: An environmentally friendly cold vapor generation for mercury analysis

In this work, the reduction of mercury ions (Hg²⁺) to elemental mercury (Hg⁰) was easily achieved using highly reductive carbon dots (r-CDs), which synthesized from sucrose by a simple and cost-effective method. After a careful mechanistic study, the reduction was probably accomplished with the large numbers of electrons contained in r-CDs rather than the oxidation of its functional groups. Additionally, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the r-CDs were nontoxic to wildlife and human beings. Consequently, the r-CDs were used as an alternative to toxic reductants (SnCl₂ or NaBH₄) for the sensitive and in situ determination of mercury by cold vapor generation (CVG) coupled to a miniature point discharge optical emission spectrometer (μPD-OES). Limit of detection of 0.05 μg/L was obtained for Hg²⁺, with relative standard deviation (RSD) less than 5.4% at a concentration of 5 μg/L. The accuracy of r-CDs induced CVG-μPD-OES was validated by the determination of mercury in a certified reference material (DOLT-5, dogfish liver) and five natural water samples collected from different rivers and lakes in Chengdu City. Since r-CDs are nontoxic and prepared from abundant and inexpensive sucrose, the r-CDs induced CVG-μPD-OES retains the great potential for the inexpensive and environmentally friendly field analysis of mercury in natural water. The accuracy of the proposed method was validated by the analysis of a certified reference material and several water samples with satisfactory results.     

呋喃唑酮代谢物单克隆抗体制备及酶联免疫吸附分析方法

本研究针对呋喃唑酮代谢物(AOZ),设计合成了系列半抗原,进一步通过偶联牛血清白蛋白(BSA)免疫Balb/c小鼠、细胞融合、筛选和亚克隆等过程成功获得了源于新颖半抗原H3的具有高亲和力(亲和力常数6.68× 1010L/mol)和高特异性(与其它功能类似物交叉反应小于0.1％)抗AOZ单克隆抗体.同时,基于设计合成的系列同/异源半抗原/包被抗原,考察了不同结构包被原对ELISA方法灵敏度的影响.另外,采用最佳的特征结构异源包被原H5 -OVA,建立了以对硝基苯甲醛(p-NP)为衍生剂的AOZ间接竞争ELISA(icELISA)和直接竞争ELISA(deELISA)检测方法.结果表明:icELISA模式的AOZ检测IC50为0.503 μg/L,定量检测线性范围(IC20～IG80)为0.06～14.0 μg/L,检出限(IC10)达0.017 μg/L; dcELISA模式的AOZ检测IC50为1.19 μg/L,定量检测线性范围为0.14～23.6 μg/L,检出限为0.056 μg/L.两种方法对AOZ的检测灵敏度和定量线性范围均达到相关检测限量要求,可满足不同需求的实际样品检测.     

Determination of Mercury(II) in Drinking Water by Total Reflection X-ray Fluorescence Spectrometry and Liquid–Liquid Microextraction

The total reflection X-ray fluorescence determination of mercury(II) in drinking water at concentrations of 7?×?10^?2 to 3.0?µg/L is reported. The mercury(II) preconcentration protocol includes directly suspended droplet microextraction with benzene as a molecular iodine complex. The proposed approach is highly selective. The elements Cr, Mn, Fe, Co, Ni, Zn, and Pb at concentrations up to 0.1?g/L did not interfere with the extraction of trace mercury(II). The method is characterized by high sensitivity (limit of detection of 21?ng/L) and suitable reproducibility (relative standard deviation of 0.12 for 100?ng/L). The accuracy of the results was confirmed by recovery and the method of standard addition.     

Determination of capsaicinoids in salsa by liquid chromatography and enzyme immunoassay

Two simple and rapid methods were developed to monitor pungency of salsa in production. Capsaicin (C) and dihydrocapsaicin (DHC) were quantitated in 17 commercially available tomato-based salsas by enzyme immunoassay (EIA) and liquid chromatography (LC) with fluorescent detection. Samples were extracted with methanol and the extracts were subjected to solid-phase extraction (SPE) using polystyrene-divinylbenzene columns. Analysis of SPE eluates showed good correlation (r2 = 0.953) between LC and EIA, with a slightly high bias for EIA. Salsa fortified with C and DHC from 0.118 to 103.2 microg/g resulted in recoveries of 90-112% (C) and 76-97% (DHC). Limits of detection by LC were 0.1 microg/g for each capsaicinoid and 0.1 microg/g by EIA for total capsaicinoids. The LC on-column response was linear from 0.2 to 100 ng for both C and DHC, whereas the working range for EIA was 0.1-2.0 ppm. Pungency varied between different salsa brands labeled mild, medium, and hot.     

Disposable Bismuth Oxide Screen Printed Electrodes for the High Throughput Screening of Heavy Metals

We present a simplified approach for the trace screening of toxic heavy metals utilizing bismuth oxide screen printed electrodes. The use of bismuth oxide instead of toxic mercury films facilitates the reliable sensing of lead(II), cadmium(II) and zinc(II). A linear range over 5 to 150 μg L^?1 with detection limits of 2.5 and 5 μg L^?1 are readily observed for cadmium and lead in 0.1 M HCl, respectively. Conducting a simultaneous multi‐elemental voltammetric detection of zinc, cadmium and lead in a higher pH medium (0.1 M sodium acetate solution) exhibited a linear range between 10 and 150 μg L^?1 with detection limits of 5, 10 and 30 μg L^?1 for cadmium, lead and zinc respectively. The sensor is greatly simplified over those recently reported such as bismuth nanoparticle modified electrodes and bismuth film coated screen printed electrodes. The scope of applications of this sensor with the inherent advances in electroanalysis coupled with the negliable toxicity of bismuth is extensive allowing high throughput electroanalysis.     

原子荧光法测定饮用水中汞

汞含量是生活用水中的重要毒理学指标.将水中汞在酸性介质中消解,被硼氢化钠还原成原子态,用原子荧光法测定.方法的检出限为1.1×10-3μg/L,线性范围为1.2×10-2μg/L~1.20μg/L,相对标准偏差为6.3%,加标回收率87.6%~112.5%.     

Nanoparticle labelling-based magnetic immunoassay on chip combined with electrothermal vaporization-inductively coupled plasma mass spectrometry for the determination of carcinoembryonic antigen in human serum

A sensitive and selective on chip magnetic immunoassay method, based on a sandwich-type immunoreaction with PbS nanoparticle (NPs) labels in combination with electrothermal vaporization-inductively coupled plasma mass spectrometry (ETV-ICP-MS), was proposed for the determination of carcinoembryonic antigen (CEA). We designed and fabricated a microfluidic chip for magnetic immunoassay, and the prepared iminodiacetic acid modified silica coated magnetic nanoparticles (IDA-SCMNPs) were packed into the central microchannel to form a solid phase column by self-assembly under the magnetic field. After completion of the immunoreaction involving a primary antibody, CEA and a secondary antibody labeled with PbS NPs on a magnetic solid phase packed-column, ETV-ICP-MS was used to determine the concentration of Pb that was released from the captured PbS NPs using an acid-dissolution step. The concentrations of CEA can be correlated with that of Pb. The established method demonstrated a limit of detection of 0.058 μg L(-1) for CEA, with a relative standard deviation (RSD) of 6.7% (c = 10 μg L(-1), n = 7). A linearity ranging from 0.2 μg L(-1) to 50 μg L(-1) and a 2-fold enrichment factor (from 60 μL sample solution to 30 μL eluent) were achieved. The proposed method was further validated by analyzing CEA in human serum. The results were in good agreement with those obtained by chemiluminescent immunoassay, which is currently used as a clinical method. Overall, this method offers the advantages of high speed, high sensitivity, high selectivity, low sample/reagents consumption, high integrity and versatility. Moreover, it can be easily applied to other biological and medical assays.