精吡氟禾草灵酶联免疫吸附分析方法研究

建立了定量测定精吡氟禾草灵的间接竞争酶联免疫吸附分析方法(ic-ELISA)。将精吡氟禾草灵在碱性条件下水解制得半抗原,再将半抗原与蛋白质偶联形成抗原后免疫新西兰大白兔,获得多克隆抗体。对其进行条件优化后,得到了精吡氟禾草灵的ic-ELISA方法的标准曲线。该方法的Ic50为0.553mg/L。检出限为0.0062mg/L。方法对其他芳氧苯氧基丙酸酯类除草剂没有明显交叉反应。精吡氟禾草灵在样品中的回收率为86.80%～103.41%,变异系数为3.96%～12.32%。表明本研究建立的精吡氟禾草灵的ELISA方法符合农药残留分析的要求。     

基于纳米金比色检测NOS1AP基因单碱基突变

基于单、双链DNA与纳米金颗粒间的不同静电作用, 建立了一种基于颜色反应检测NOS1AP基因单碱基突变的方法. 根据NOS1AP基因的单碱基多态位点设计检测探针、互补靶序列及带有单碱基突变序列寡核苷酸DNA. 室温下, 检测探针分别与互补序列、单碱基突变序列在缓冲液中进行杂交, 再分别加入纳米金溶液以及NaCl溶液. 用肉眼可以观察到纳米金溶液在两种不同杂交溶液中产生明显不同的颜色变化. 这种变化可通过紫外-可见分光光度计测定纳米金溶液的紫外吸收峰值的变化来证实. 实验结果表明, 纳米金溶液在一定浓度NaCl存在的条件下, 对互补双链NOS1AP DNA及单碱基突变NOS1AP DNA呈现出不同的颜色反应及紫外吸收光谱的改变. 此方法可望用于相关疾病的医学诊断及单碱基突变的检测.     

Kinetics of comet formation in single‐cell gel electrophoresis: Loops and fragments

We investigated the mechanisms of DNA exit during single‐cell gel electrophoresis (the comet assay) by measuring the kinetics of the comet tail formation. In the neutral comet assay, the rate of DNA exit was found to be dependent on the topological state of DNA, which was influenced by either ethidium bromide or a low radiation dose. The results clearly show that the comet tail is formed by extended DNA loops: the loop extension, being reversible when the DNA torsional constraint remains in the loops, is favored when the constraint is relaxed. The kinetics of the comet formation in the case of a high radiation dose points out that accumulation of the single‐strand breaks causes DNA fragmentation. In contrast to the neutral comet assay, the alkaline comet assay is not related to the chromatin loops. Our results imply that the alkaline treatment induces detachment of the loops from the nuclear matrix, and the comet tail is formed by ssDNA fragments, the ends of which are pulled out from the comet head by electric force. We suggest that the kinetic approach can be considered as an important improvement of the comet assay.     

Analysis of microcystins by capillary zone electrophoresis coupling with electrospray ionization mass spectrometry

In this paper, a rapid and effective method based on capillary zone electrophoresis (CZE) coupled with electrospray ionization mass spectrometry (ESI-MS) was established for the trace analysis of microcystin (MC) isomers in crude algae sample. The experimental conditions including the composition, acidity and concentration of buffer, separation voltage, injection time, and MS detection parameters were investigated in detail. A capillary separation system was as follows: a uncoated fused-silica capillary tube (50 μm i.d. × 90 cm), 40 mmol L⁻¹ ammonium acetate solution (pH 9.86) as running buffer, 25 kV as separation voltage, 20 kV × 3 s water first and 20 kV × 20 s for sample injection. Mass analysis was performed in ESI source, with sheath gas temperature 150 °C, sheath gas pressure 10 psi, and sheath gas flow 6 L min⁻¹. And sheath liquid was 7.5 mmol L⁻¹ acetic acid in 50% isopropanol-water (3 μL min⁻¹). Protonation and ammonium adduct molecular ions m/z 506.9 (MC-LR) and 532.0 (MC-YR) were used for the quantification of MCs. Under these conditions, two MCs were baseline separated within 9 min, the calibration curves were obtained in the range of 0.11-10.0 μg mL⁻¹ and 0.16-10.5 μg mL⁻¹ for MC-LR and MC-YR, respectively. Meanwhile, limits of detection were 0.05 and 0.08 μg mL⁻¹ for MC-LR and MC-YR, respectively. The recoveries for the two MCs were in the range of 95.8-108%. The developed approach had been successfully applied to the analysis of MCs in crude algae samples.     

Development of an immunoassay for rapid screening of vardenafil and its potential analogues in herbal products based on a group specific monoclonal antibody

Vardenafil is a phosphodiesterase-5 (PDE-5) inhibitor for the treatment of erectile dysfunction (ED). Undeclared vardenafil and related analogues adulterated in herbal products are a threat to public health. To screen vardenafil and its analogues in herbal matrix rapidly, an immunoassay based on a group specific monoclonal antibody (McAb) was developed.Glutaraldehyde was used to link vardenafil to immunogen and coating-antigen, respectively. Through the assessment of the structural specificity of eight anti-vardenafil McAbs, the McAb of 4B9 was characterized as being specific to the common structure of vardenafil and its analogues. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established based on this McAb, the limit of detection of vardenafil was 5.0 ng mL⁻¹, the calibration curve was linear from 5.0 to 40 ng mL⁻¹ (R² = 0.952) with an IC₅₀ value of 18.2 ng mL⁻¹. In the extracts of 20 Chinese traditional drugs, the detection capability (CCβ) of vardenafil was 0.08 mg g⁻¹, the recoveries were 76-116% and the coefficients of variation (CV%) were 9.7%-16.2%. The ic-ELISA was in good agreement with LC-UV when detected herbal products containing vardenafil and its analogue.The method is a suitable tool for screening vardenafil and its analogues as illegal additives in herbal products.     

Beyond labels: A review of the application of quantum dots as integrated components of assays, bioprobes, and biosensors utilizing optical transduction

A comprehensive review of the development of assays, bioprobes, and biosensors using quantum dots (QDs) as integrated components is presented. In contrast to a QD that is selectively introduced as a label, an integrated QD is one that is present in a system throughout a bioanalysis, and simultaneously has a role in transduction and as a scaffold for biorecognition. Through a diverse array of coatings and bioconjugation strategies, it is possible to use QDs as a scaffold for biorecognition events. The modulation of QD luminescence provides the opportunity for the transduction of these events via fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), charge transfer quenching, and electrochemiluminescence (ECL). An overview of the basic concepts and principles underlying the use of QDs with each of these transduction methods is provided, along with many examples of their application in biological sensing. The latter include: the detection of small molecules using enzyme-linked methods, or using aptamers as affinity probes; the detection of proteins via immunoassays or aptamers; nucleic acid hybridization assays; and assays for protease or nuclease activity. Strategies for multiplexed detection are highlighted among these examples. Although the majority of developments to date have been in vitro, QD-based methods for ex vivo biological sensing are emerging. Some special attention is given to the development of solid-phase assays, which offer certain advantages over their solution-phase counterparts.     

Microtiterplate phosphate assay based on luminescence quenching of a terbium complex amenable to decay time detection

We describe a terbium-ligand complex (TbL) for a microtiterplate assay for phosphate (P) in the 0.3-100 μmol L⁻¹ range based on luminescence quenching. As the pH optimum is at neutral pH (7.4) the probe is quenched by both, primary (H₂PO₄⁻) and secondary phosphate (HPO₄²⁻). The LOD is 110 nmol L⁻¹. A Stern-Volmer study revealed that quenching is mostly static. Due to the ms-decay time of TbL, the first luminescence lifetime assay for phosphate could also be developed. The lifetime-based calibration plot is linear between 0.5 and 5 μmol L⁻¹ of P. The effect of various surfactants on assay performance and a study on interferents are presented. The probe was successfully applied to determination of P in commercial plant fertilizers and validated against the molybdenum blue test. The probe is the most sensitive lanthanide-based probe for phosphate.     

Nanocatalytic resonance scattering spectral analysis

The resonance scattering spectral technique has been established using the synchronous scanning technique on spectrofluorometry.Because of its advantages of simplicity,rapidity and sensitivity,it has been widely applied to analyses of proteins,nucleic acids and inorganic ions.This paper summarizes the application of immunonanogold and aptamer modified nanogold(AptAu) catalytic resonance scattering spectral technique in combination with the work of our group,citing 53 references.     

中药致肾毒性成分马兜铃酸A单抗制备及酶联免疫分析方法的建立

采用活化酯法,将马兜铃酸A分别与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联,得到免疫抗原马兜铃酸A-BSA和包被抗原马兜铃酸A-OVA.利用马兜铃酸A-BSA免疫Bal b/c小鼠,制得鼠单克隆抗体1A11,单抗效价为2×104;单抗为IgG1类,轻链为κ型;与其结构类似物马兜铃酸B、C和D的交叉反应率分别为2.8%,3.5%和31.2%.基于抗马兜铃酸A单克隆抗体的间接竞争酶联免疫分析方法(icELISA)的IC50为1.9 μg/L,检测范围为0.5～7.5 μg/L.icELISA添加回收率为86%～97%,相对标准偏差在5.2%～11.1%之间.利用所建立的icELISA测定了6个中药材和5个中成药中马兜铃酸A的含量,并用高效液相色谱法(HPLC)进行了验证,其中关木通、广防己、天仙藤、马兜铃和青木香中均检测出马兜铃酸A,而川木通和5个中成药中未检测到马兜铃酸A.结果表明: 本方法可用于中药中马兜铃酸A的快速检测.     

表面等离子共振技术测定生长抑素含量

应用表面等离子共振(SPR)技术建立了一种快速检测生长抑素(SST)含量的方法, 在pH=5.0, SST浓度为75 μg/mL的最佳偶联条件下, SST在CM5芯片上的偶联值为1231.7 Response unit(RU). 选择117.7 nmol/L作为固定的大分子量抗体浓度, 用抗原-抗体-抗原结合法检测SST纯品, 在20~2000 pg范围内, SST纯品含量和RU值之间可建立直线相关的标准曲线, R=-0.824, p<0.05, 变异系数CV=1.3%. 所建立的方法测定SST含量的范围为20~2000 pg, 重复性好且可随时检测, 有望用于SST的临床即时检测.