以2-磷酸抗坏血酸酯为底物检测碱性磷酸酯酶的微分脉冲伏安法

提出以2_磷酸抗坏血酸酯为碱性磷酸酯酶(ALP)底物微分脉冲伏安法测定ALP的方法 ;2_磷酸抗坏血酸酯在ALP的催化作用下发生水解反应生成抗坏血酸 ,抗坏血酸在玻碳电极上 +0.40V(vsAg/AgCl)被氧化而产生一个灵敏的氧化峰 ,氧化峰电流随着酶浓度的增大而增大 ,借助此氧化峰电流可以测定ALP ,进而可用于以ALP为标记酶的酶免疫分析 ;用微分脉冲伏安法对酶催化反应条件和酶催化反应产物的测定条件进行了详细的研究 ,建立了以2_磷酸抗坏血酸酯为底物的伏安酶联免疫分析新体系 ,测定游离ALP的线性范围是0.4～2.0×103 U/L,检测限为0.3U/L,对游离的IgG_ALP的测定最大稀释比为1∶200000。     

Investigation of voltammetric enzyme-linked immunoassay based on new system of ODA-H_2O_2-HRP

A voltammetric enzyme-linked immunoassay based on a new system of ODA-H2O2-HRP has first been developed and used in the detection of HRP and labelled HRP. By this method, the enzyme-catalyzing reaction of H2O2 oxidizing odianisidine (ODA) couples the electrode-reduction reaction of the oxidizing product of odianisidine, which produces a sensitive polarographic wave at potential of -0.56V (SCE) in Britton-Robinson buffer solution. In using this polarographic wave, a detection limit to HRP is 3.7×10-12g/mL and a linear range 1.0×10-11-2.0×10-9g/mL. And the mechanisms of the coupling reaction and the process of electro-reduction in the ODA-H2O2-HRP voltammetric enzyme-linked immunoassay system have also been carefully studied.     

Development of a Capillary Electrophoresis-Based Enzyme Immunoassay with Electrochemical Detection for the Determination of Okadaic Acid and Dinophysistoxin2 in Shellfish Samples

A capillary electrophoresis-based enzyme immunoassay (CE-EIA) with electrochemical (EC) detection system was developed for the determination of two diarrheic shellfish poisoning (DSP) toxins okadaic acid (OA) and dinophysistoxin2 (DTX2). In this method, after the competitive immunoreaction in liquid phase, the horseradish peroxidase (HRP)-labeled antigen (Ag*) and the bound enzyme-labeled complex (Ag*-Ab) were separated and then the system of HRP catalyzing H₂O₂/o-aminophenol (OAP) reaction was adopted. The limit of detection (S/N = 3) was determined to be 0.05 and 0.07 ng/mL for OA and DTX2, respectively. The total analysis time was less than 40 min. The developed CE-EIA with EC detection system was capable of quantitatively detecting OA and DTX2 contents in the tested contaminated samples, and the results were compared with the same samples analyzed through enzyme-linked immunosorbent assay (ELISA). Consistent results between CE-EIA with EC detection and ELISA were found in most of the tested samples. The proposed system appeared to be more sensitive and faster than ELISA for determination of OA and DTX2 in shellfish meat extracts. Real shellfish samples were validated in recovery test, and the recoveries tested by the proposed method were 91.7–108.3% and 95.2–112.5% for OA and DTX2, respectively. The CE-EIA with EC detection provides a valid and sensitive analytical approach, not previously available, for the determination of OA and DTX2 in shellfish samples.     

Detection and identification of the designer benzodiazepine flubromazepam and preliminary data on its metabolism and pharmacokinetics

The appearance of pyrazolam in Internet shops selling ‘research chemicals’ in 2012 marked the beginning of designer benzodiazepines being sold as recreational drugs or ‘self medication’. With recent changes in national narcotics laws in many countries, where two uncontrolled benzodiazepines (phenazepam and etizolam), which were marketed by pharmaceutical companies in some countries, were scheduled, clandestine laboratories seem to turn to poorly characterized research drug candidates as legal substitutes. Following the appearance of pyrazolam, it comes with no surprise that recently, flubromazepam (7‐bromo‐5‐(2‐fluorophenyl)‐1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one), a second designer benzodiazepine, was offered on the market. In this article, this new compound was characterized using nuclear magnetic resonance, gas chromatography‐mass spectrometry (GC–MS), liquid chromatography–mass spectrometry (LC–MS/MS) and liquid chromatography quadrupole time‐of‐flight MS (LC–Q–ToF–MS). Additionally, a study was carried out, in which one of the authors consumed 4 mg of flubromazepam to gain preliminary data on the pharmacokinetic properties and the metabolism of this compound. For this purpose, serum as well as urine samples were collected for up to 31 days post‐ingestion and analyzed applying LC–MS/MS and LC–Q‐ToF‐MS techniques. On the basis of this study, flubromazepam appears to have an extremely long elimination half‐life of more than 100 h. One monohydroxylated compound and the debrominated compound could be identified as the predominant metabolites, the first allowing a detection of a consumption for up to 28 days post‐ingestion when analyzing urine samples in our case. Additionally, various immunochemical assays were evaluated, showing that the cross‐reactivity of the used assay seems not to be sufficient for safe detection of the applied dose in urine samples, bearing the risk that it could be misused in drug‐withdrawal settings or in other circumstances requiring regular drug testing. Furthermore, it may be used in drug‐facilitated crimes without being detected. Copyright © 2013 John Wiley & Sons, Ltd.     

硒功能化杯芳烃载体银离子选择性膜电极的研究

合成6个硒功能化杯芳烃为载体,采用双层夹心膜电位法直接测定了溶剂聚合物膜中载体与金属离子生成的络合物生成常数。制备并考察了以硒功能化杯芳烃为载体的银离子选择性电极的性能,发现以载体6为银离子选择性电子的载体,制成的膜电极,用作Ag^ 电位滴定Cl^-的指示电极,效果最佳。     

Surface modification of hydrophobic NaYF_4:Yb,Er upconversion nanophosphors and their applications for immunoassay

β-NaYF4:Yb,Er upconversion nanophosphor (UCNP) is known as one of the most efficient NIR-to-visible upconversion materials, which shows great potential in bioanalytical chemistry and bioimaging. However, its applications are greatly limited due to its low water dispersibility and thus poor biocompatibility. In this paper, poly(acrylic acid) (PAA)-based ligand exchange strategies are carried out to modify oleic acid-capped hydrophobic β-NaYF4:Yb,Er UCNPs into hydrophilic ones. After efficient surface modific...     

Comparative study of three immunoassays based on monoclonal antibodies for detection of the pesticide parathion-methyl in real samples

Three immunoassay systems: indirect, direct competitive enzyme-linked immunosorbent assay (IC-ELISA and DC-ELISA), fluorescence polarization immunoassay (FPIA) based on monoclonal antibodies for the detection of parathion-methyl (PM) were developed and optimized. Several PM derivatives (haptens) were conjugated to proteins and fluoresceinthiocarbamyl ethylenediamine (EDF) to obtain immunogens and competitors. The influence of immunogen and competitor structures on the assay performance was investigated. IC-ELISA was the most sensitive of all techniques developed, with a detection limit of 0.08 ng ml⁻¹, but assay time was the longest (3.5 h per 96-well microtitre plate). DC-ELISA was easier to perform and quicker (1.5 h per 96-well microtitre plate) but less sensitive than IC-ELISA (detection limit was 0.5 ng ml⁻¹). FPIA was the fastest and simplest (7 min per 10 samples) but the least sensitive (detection limit was 15 ng ml⁻¹) technique. The methods were characterized by high specificity and reproducibility. The cross-reactivity for parathion-ethyl was around 30-40% for IC-ELISA and FPIA, but significantly higher (125%) for DC-ELISA. The immunoassays were applied to the analysis of PM residues in different food and environmental matrices. Methanol extracts of vegetable, fruit and soil samples were used for the analysis. Recoveries for most spiked samples averaged between 85 and 110%. The methods developed can be used for screening of food and environmental samples for PM residues without complicated clean-up.     

Determination of human IgG by solid-substrate room-temperature phosphorescence immunoassay based on an antibody labeled with nanoparticles containing rhodamine 6G luminescent molecules

Luminescent 50-nm silicon dioxide nanoparticles containing both types of rhodamine 6G (R; particles denoted R-SiO₂) were synthesized by the sol–gel method. In the presence of Pb(Ac)₂ as a heavy atom perturber the particle can emit the intense and stable room-temperature phosphorescence (RTP) signal of R on a polyamide membrane, with _ex^max/_em^max=470/635 nm for R. Our research indicates that the specific immune reaction between goat-anti-human IgG antibody labeled with R-SiO₂ and human IgG can be carried out quantitatively on a polyamide membrane, and the phosphorescence intensity was enhanced after the immunoreaction. Thus a new method for solid-substrate room-temperature phosphorescence immunoassay (SS-RTP-IA) for determination of human IgG was established on the basis of antibody labeled with the nanoparticles containing binary luminescent molecules. The linear range of this method is 0.0624–20.0 pg spot^–1 of human IgG (corresponding to a concentration range of 0.156–50.0 ng mL^–1, sample volume 0.40 L spot^–1). The regression equations of the working curves are I_p=71.27+7.208m_IgG (pg spot^–1) (r=0.9996). Detection limits calculated as 3S_b/k are 0.022 pg spot^–1. Compared with the same IA using fluorescein isothiocyanate (FITC) as the marker the new method was more sensitive and had a wider linear range. After elevenfold replicate measurement RSD are 4.5 and 3.6% for samples containing 0.156 and 50.0 ng mL^–1 IgG, respectively. This method is sensitive, accurate, and of high precision.     

Express detection of nonylphenol in water samples by fluorescence polarization immunoassay

The development of express method for detection of endocrine-disrupting chemicals (EDC) such as alkylphenols is required for ecological monitoring. Several attempts have been made to produce antibodies against 4-nonylphenol (NP) in recent years. This work describes the production of new antibodies against NP and also summarizes the characterization of antibodies obtained earlier. Three approaches used to produce alkylphenol-specific antibodies are compared; these are based on: 1. omega-(4-hydroxyphenyl)nonanoic or omega-(4-hydroxyphenyl)heptanoic acid NP derivatives designed to mimic the linear NP isomer; 2. 4-aminophenol, which potentially mimics various substituted phenolic compounds with different side-chain structures at position 4 of the benzene ring; and 3. a mixture of branched NP isomers, conjugated to the carrier protein via a benzene ring by the Mannich reaction, and expected to be the closest mimic of NP structure by preserving its natural alkyl moiety.Fluorescence polarization immunoassays based on different combinations of antibody and labeled antigen for screening detection of NP were developed and structural aspects of assay sensitivity and specificity were investigated. The assays based on the antisera raised against omega-(4-hydroxyphenyl)nonanoic acid and NP conjugate via Mannich reaction are capable of express detection of NP with detection limit of 7 microg mL(-1 )and assay dynamic range of 18-300 microg mL(-1).