A highly sensitive and rapid biological probe enhanced real-time immuno-polymerase chain reaction for detecting 2,2′,4,4′-tetrabromodiphenyl ether in indoor airborne particles

Polybrominated diphenyl ethers (PBDEs) are bromine flame retardants that are widely distributed in the environment. In this study, 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) was used as a model PBDE compound. Based on the preparation of BDE-47 immunogen, coating antigen and polyclonal anti-BDE-47 antibody, a highly sensitive and rapid real-time immuno-polymerase chain reaction using biological probes (BP-IPCR) was developed to detect BDE-47 in indoor airborne particles. Several physiochemical factors that might influence the assay performance were optimised, such as OVA-BDE-47 and the biological probe concentration, primer concentration and annealing temperature. Under the optimal conditions, a standard curve was constructed with BDE-47 concentrations ranging from 5 pg L^?1 to 50 ng L^?1. The limit of detection was 1.32 pg L^?1. The BP-IPCR assay was highly selective, presenting low cross-reactivity values with BDE-47 analogues (below 5.26%). The recoveries of spiked samples were 92.6–107.9% and the coefficients of variation were 3.7–8.8%. The BP-IPCR data for the detection of BDE-47 in indoor airborne particles was consistent with gas chromatography-mass spectrometry findings (R² = 0.9849). This immunoassay is sensitive and reliable; it can be used for specific and sensitive batch detection of BDE-47 in environmental samples.     

Native vs photoinduced chemiluminescence in dimethoate determination

The determination of dimethoate using either its native chemiluminescent (CL) properties or its photoinduced chemiluminescence obtained by irradiation with a 15 W low-pressure mercury lamp was studied. Thereby, two flow injection systems (FIA) with and without irradiation were exhaustively optimized and their analytical characteristics studied. Better sensitivity and selectivity was found in absence of irradiation, due to the enhancing effect of hexadecylpyridinium chloride (HPC), which acted as a sensitizer. In the developed FIA-CL system, the alkaline hydrolysis of dimethoate with NaOH was performed on-line in presence of HPC. The oxidation of the product of hydrolysis with Ce(IV) in hydrochloric medium induced chemiluminescence. The method provided a limit of detection of only 0.05 ng mL⁻¹ without any pre-treatment. However, the combination with solid phase extraction allowed the removal of some potential interferents as well as the preconcentration of the pesticide. Finally, the developed method was successfully applied to natural waters with recoveries between 95 and 108%.     

Nylon and nylon blend nanotubes and nanorods

Nylon nanorods and nanotubes (200 nm diameter) were fabricated by the membrane wetting technique (solvent and melt wetting) from a range of nylons (6; 6,6; 6,9; 6,10; 6,12; 11; 12, 6(3)T) and nylon blended with different dyes (Nylon Cast Blue, Nylon 6/6 Black) or with molybdenum disulfide (Nylon cast MDS). The 65-μm long nylon nanotubes and nanorods were characterized by scanning electron microscopy. The nanoscale nylon 6,6 served as an effective high surface area alternative to a nylon membrane as a solid support in a chemiluminescent assay for nylon-bound biotinylated nucleic acids based on streptavidin- alkaline phosphatase and chemiluminescent detection of the bound alkaline phosphatase label with the dioxetane substrate, CDP-Star. Layer-by-layer deposition of the cationic polymer (Sapphire-II™; Tropix) onto the nylon 6,6 nanostructures prior to UV-cross-linking with biotinylated DNA resulted in further enhancement of binding and detection of biotinylated DNA.     

Chemiluminescence determination of antibacterial drug trimethoprim by automated sequential injection technique with permanganate and hexametaphosphate as reagents

Antibacterial drug trimethoprim [2,4-diamino-5-(3,4,5-trimethoxybenzyl)-pyrimidine] (I) was determined in pharmaceutical formulations by using a lab-made PC-controlled SIA analyser linked to conventional HPLC fluorimetric detector equipped with a chemiluminescence module. The chemical principle is the oxidation of I by KMnO(4) in acid medium; the reaction is accompanied by the emission of chemiluminescence, which is enhanced in the presence of hexametaphosphate (HMP). The optimum sequence and the flow parameters and concentrations and volumes of reagents aspirated optimised by a computer-aided simplex method were, 100 mul of 5 mM HMP, 40 mul of a test solution of I, 2 mul of 0.5 M H(2)SO(4) and 20 mul of 1 mM KMnO(4); the luminescing zone was pushed into the detector at a flow rate of 49 mul s(-1). The calibration graph relating the intensity of luminescence to concentration of I was parabolic (r=0.9994) in the range 0.5-100 mug ml(-1) of I with rectilinear part (r=0.9999) in the range 20-100 mug ml(-1) of I; the limit of detection was 0.1 mug ml(-1) of I. The method was used for the assay of Triprim(R) tablets (with nominal content 100 or 200 mg of I) for the active substance as well as for content uniformity tests; the R.S.D. values did not exceed 1% (n=5). The SIA results did not show statistical difference from those obtained by pharmacopoeial acidimetric titration in non-aqueous medium; the excipients such as microcrystalline cellulose, maze starch, povidone, talc, magnesium stearate and gelatin did not interfere.     

Peptide mimotopes of phomopsins: Identification, characterization and application in an immunoassay

Peptide mimotopes of plant-associated toxins offer the potential for improving analytical and diagnostic methodologies as well as providing candidates for potential protective vaccines against plant poisoning diseases. Monoclonal antibody (mAb) C₃C₁₁, which recognizes the antimicrotubule phomopsin mycotoxins, was used to isolate peptide mimics of phomopsin A from a random 15-mer phage display peptide library. A total of 46 clones were isolated that showed specific reactivity with the mAb. Amino acid sequence analysis revealed four different types of mimotope sequences, all of which contained a common motif V-A-L/V-C. Of the 46 clones isolated, 44 contained the motif V-A-L-C while 2 contained the V-A-V-C motif. All four types of phage clones inhibited the reactivity of the mAb with phomopsin A in a competition ELISA. The clone with the mimotope sequence CT VALCNMYFGAKLD demonstrated the strongest binding. It was further shown that synthetic peptides containing these mimotope amino acid sequences were able to inhibit the mAb-phomopsin A interaction, indicating that the peptide mimotopes were responsible for the specific binding, independent of the phage framework. The results also suggest that the mimotope peptides bind to mAb C₃C₁₁ at the same site as phomopsin A. The application of recombinant phage particles carrying phomopsin mimotopes in immunoassay was evaluated and the results demonstrated approximately 100-fold increase in sensitivity in comparison with a conventional immunoassay using a chemically linked phomopsin-horseradish peroxidase conjugate.     

Chemiluminescence detection of superoxide anion release and superoxide dismutase activity: modulation effect of<Emphasis Type="Italic"> Pulsatilla chinensis</Emphasis>

A novel flow-injection chemiluminescence-based method has been developed for determination of superoxide dismutase (SOD) activity. An in-vitro superoxide anion generation xanthine/xanthine oxidase stable source was established on line with FIA/CL-detection apparatus, for measuring SOD activity. This method can detect SOD in the linear range of 0.002–2.00 U mL^–1 with a detection limit of 0.001 U mL^–1. Another method for detection of superoxide anion is based on the luminol–FeCl₃ chemiluminescence (CL) reaction. This method was used to evaluate superoxide release and SOD activity in rats treated with the traditional Chinese herb Pulsatilla chinensis, which resulted in high clearance of hepatitis B virus (HBV) after treatment of a hepatitis B patient. Interestingly, we found that treatment with Pulsatilla chinensis can specifically increase superoxide release by liver tissues and, at the same time, slightly increase extracellular SOD (ECSOD) activity in plasma; in particular it can markedly increase MnSOD activity in mitochondria in liver tissue. This work revealed a possible mechanism whereby Pulsatilla chinensis prevents possible infection (for example HBV) by specifically increasing superoxide release in the liver and increasing MnSOD activity to minimize superoxide-mediated toxicity.     

Chemiluminescence microfluidic system sensor on a chip for determination of glucose in human serum with immobilized reagents

A chemiluminescence (CL) biosensor on a chip coupled to microfluidic system is described in this paper. The CL biosensor measured 25×45×5 mm in dimension, was readily produced in analytical laboratory. Glucose oxidase (GOD) was immobilized onto controlled-pore glass (CPG) via glutaraldehyde activation and packed into a reservoir. The analytical reagents, including luminol and ferricyanide, were electrostatically co-immobilized on an anion-exchange resin. The most characteristic of the biosensor was to introduce the air as the carrier flow in stead of the common solution carrier for the first. The glucose was sensed by the CL reaction between hydrogen peroxide produced from the enzymatic reaction and CL reagents, which were released from the anion-exchange resin. The proposed method has been successfully applied to the determination of glucose in human serum. The linear range of the glucose concentration was 1.1-110 mM and the detection limit was 0.1 mM (3σ).     

Dialysis membrane sampler for on-line flow injection analysis/chemiluminescence-detection of peroxynitrite in biological samples

Peroxynitrite, as a derivative of nitric oxide, is a potent oxidant. It reacts with several biological molecules, makes cellular and tissue damages, and is related with many diseases; therefore, it is of major concern in current medical research works. In this work, a special perm-selective cellulose acetate membrane sampler is used to implement flow injection analysis (FIA)/chemiluminscence (CL)-detection method for the detection of peroxynitrite with Luminol CL-reagent. Optimum detection conditions were established, and the permeability of peroxynitrite through cellulose acetate (CA) membrane, as well as the interference from matrix constituents were studied. The proposed method has the high sensitivity of the CL-detection and the selectivity of perm-selective membrane sampler. The obtained detection limit of 1×10⁻¹¹ M (without dialysis membrane) and 1×10⁻¹⁰ M (with dialysis membrane), makes it possible to monitor the elusive peroxynitrite in biological samples. The mechanism of luminol CL-emission generated during oxidation by peroxynitrite and the kinetics of peroxynitrite decomposition were also studied using FIA/CL-detection set-up.     

光泽精与钨（Ⅲ）的流动注射－胶束增敏化学发光反应

采用了Jones柱在线还原钨（Ⅵ）为钨（Ⅲ），详细研究了钨（Ⅲ）与光泽精的化学发光反应，加入乳化剂OP能明显提高发光的信噪比，基于此建立了痕量钨的流动注射化学发光分析法。该法线性范围为1×10－6～1×10－2 g·L－1, 检出限为5×10－7 g·L－1 , 相对标准偏差为2.0％（5×10－5 g·L－1, n=11）。文中还探讨了反应机理。     

Multiplex chemiluminescence microscope imaging of P16INK4A and HPV DNA as biomarker of cervical neoplasia

Classification of cervical intraepithelial neoplasia (CIN) lesions in low-grade (CIN1) or high-grade (CIN2-3) ones is crucial for optimal patient management, but current histological diagnosis on bioptic samples is often hampered by inter-observer variability. To allow objective classification, we have exploited the peculiar characteristics of chemiluminescence detection, such as high sensitivity and easy quantification of the luminescence signal, to perform sequentially in the same tissue section both an immunohistochemical quantitative detection of p16^INK4A (a protein marker of high-grade CIN lesions) and an in situ hybridization for human papillomavirus (generally accepted as a necessary but insufficient cause of cervical carcinoma). Different label enzymes (alkaline phosphatase and horseradish peroxidase) were employed in order to avoid any interference between the two assays, and quantitative chemiluminescence image analysis was used to obtain objective evaluation of sample positivity. The multiplexed method allowed detection of two complementary biomarkers and provided discrimination between different lesions (non-neoplastic, low-grade and high-grade CIN). This assay might thus represent an accurate and objective diagnostic test providing important information for counseling, selection of therapy and follow up after surgical treatment.