共振瑞利散射光谱法测定氯硝柳胺的研究

在PH=1.65的BR缓冲溶液中,十六烷基三甲基溴化铵和氯硝柳胺本身的共振瑞利散射(RRS)均很微弱。但两者相互作用形成离子缔合物时,溶液的RRS显著增强,并产生了新的RRS光谱,其RRS峰位于385nm。在一定条件下氯硝柳胺的浓度与散射强度成正比,据此建立了测定微量氯硝柳胺的瑞利散射光谱分析法。该方法灵敏度高,其检出限为0.0576μmol/L。用于氯硝柳胺的测定,具有操作简便、快速、灵敏、选择性好等特点。     

金纳米探针瑞利共振散射法测定溶菌酶

利用共振散射技术研究了金纳米微粒与溶菌酶的相互作用.金纳米微粒可以通过静电引力及疏水作用与溶菌酶结合,使金纳米微粒粒径变大,从而导致纳米金瑞利共振散射光谱显著增强,并且在一定范围内光散射强度的增加量与溶菌酶浓度成正比.考查了作用时间、溶液酸度、共存离子及有机溶剂等条件的影响.将纳米金作为测定溶菌酶的探针,在最佳反应条件下,对溶菌酶的检出限为0.08 μg/mL.将此方法用于蛋清中溶菌酶的测定,检测结果与文献报道方法一致,结果令人满意.     

基于[Ru(phen)2(bpip)] 2+-DNA相互作用的共振光散射增强建立纳克级DNA的测定方法

通过研究钌多吡啶类配合物［Ru(phen)2(bpip)］2+与DNA相互作用的共振光散射等光谱,我们发现［Ru(phen)2(bpip)］²⁺与DNA相互作用的方式包括插入作用和静电作用模式.同时基于［Ru(phen)2(bpip)］²⁺ DNA体系增强的共振光散射现象,建立了一种简单、快速的测定纳克级核酸的新方法.实验结果表明体系在373 nm处共振光散射强度的增强与DNA的浓度呈线性关系.线性范围为0.025～1.250 mg/L,线性公式为△I_RLS=283.14C+2.26 (mg/L),相关系数为0.9983,DNA的检出限为5.7 ng/mL. 应用到实际样品的分析中,结果令人满意.     

甲胎蛋白和癌胚抗原的胶体金免疫层析同步检测

目的:探讨建立一种同时快速检测甲胎蛋白(AFP)和癌胚抗原(CEA)两种肿瘤标志物的方法.方法:用胶体金分别标记鼠抗AFP单克隆抗体和鼠抗CEA单克隆抗体,喷于胶金垫上.与之配对的抗体喷在硝酸纤维素膜(NC膜)上分别做检测线T2和T1,兔抗鼠二抗喷在NC膜上做质控线,利用免疫层析技术进行检测.结果:采用Frens方法制备的胶体金粒径在20nm,紫外可见吸收峰在521 nm处,金标抗体的最适pH在9.0,最适抗体量为200μl,胶体金标记2.4μg抗体.测试结果表明同时检测AFP和CEA的试纸条灵敏度分别为20 ng.mL-1和5 ng.mL-1,检测时间仅需10min,与PSA、CA125、CA15-3、Ferritin、Human Albumin等没有交叉反应,稳定性好.结论:应用胶体金免疫层析技术,实现了AFP和CEA的同步检测.     

东海原甲藻细胞色素b基因实时荧光定量PCR检测体系优化

为定量检测现场样品中东海原甲藻的细胞色素b(cytochrome b,Cytb)基因,本研究设计了该基因的特异性引物,并对现场样品反转录反应体系中加入的模板数量和定量PCR反应条件进行了优化.结果表明:设计的引物具有较好的特异性,可有效区分不同的藻类;针对现场样品,在20μL的反转录体系中,适宜加入的RNA模板的量为50 ～ 200 ng;PCR模板稀释10倍或向定量PCR反应体系中加入终浓度为0.2μg·μL-1的牛血清蛋白(BSA)均能有效降低现场样品中抑制物的抑制作用,减小干扰.该方法的建立对从分子水平探讨东海原甲藻暴发和消亡的内在机制具有重要意义.     

多用途树种无患子ISSR-PCR体系建立与检测

无患子(Sapindus mukorossi)是我国长江以南地区传统的重要绿化树种,其果皮富含皂苷,种仁富含油脂,为新型木本油料树种之一。为了获得基于KOD FX高保真DNA聚合酶试剂盒的无患子ISSR-PCR的最佳反应体系,该文采用正交优化设计相结合的方法,研究了引物浓度、d NTPs浓度、KOD FX酶、模板DNA浓度和Mg2+浓度对无患子ISSR-PCR反应体系的影响,并在此基础上对退火温度进一步优化,最终确立了适合无患子的ISSR-PCR反应的最佳体系和程序,即20μL PCR反应体系中,引物0.5μmol·L-1、d NTPs 0.05 mmol·L-1、KOD FX酶0.06 U·μL-1、模板DNA浓度1.0 ng·μL-1和Mg2+1.0 mmol·L-1。当以UBC841为引物时,PCR扩增程序为94℃预变性2 min,98℃变性10 s,48.6℃退火30 s,68℃延伸90 s,35个循环,68℃延伸7 min,4℃保存。这一优化的ISSR-PCR反应体系的建立,为无患子种质资源的遗传多样性、亲缘关系及遗传结构研究、种质创新与分子辅助育种等奠定了良好的基础。     

中国李简单重复序列(SSR)反应体系的建立

探讨中国李品种美丽李(Prunus salicina‘Beauty')简单重复序列(simple sequence repeat,SSR)反应体系中5种主要成分浓度对反应产物的影响.结果表明,引物对中国李有通用性,25μLSSR反应体系中,TaqDNA酶、Mg2 、每个引物、模板DNA和dNTPs等5种成分的适宜浓度分别是:1.5 U、2.0 mmol·L-1、0.8μmol·L-1、30～40 ng和0.16～0.24 mmol·L-1.     

甲胎蛋白的原核表达及复性优化

构建甲胎蛋白的原核表达载体p ET32a-AFP,对包涵体形式表达的甲胎蛋白进行复性优化。将构建的重组质粒p ET32a-AFP转化入E.coli,IPTG诱导表达后,经亲和层析纯化获得AFP包涵体,通过对复性过程、p H、添加剂等的研究摸索,获得最佳复性条件。当采用添加0.5 mol/L L-精氨酸的一步法透析复性方法,且透析液p H值为8.5,重组人AFP包涵体蛋白起始浓度为1.0 mg/m L时,复性效率最高。该复性方法获得蛋白质具有较高的回收率且操作简便。     

HPLC-MS/MS法快速测定人血浆中美利曲辛及生物等效性研究

目的:建立测定人血浆中关利曲辛浓度的高效液相串联质谱法(HPLC-MS/MS),用于氟哌噻吨美利曲辛片的生物等效性研究.方法:以Agilent ZORBAX Eclipse Plus C18(4.6mm× 150mm,5μm)为色谱柱,流动相为乙腈(含1%甲酸):0.02mo·L-1甲酸铵水溶液(80∶20,V∶),流速:0.8mL·min-1;柱温:40℃,以醋酸乙酯:二氯甲烷(4∶1,V∶V)为提取剂.样品经电喷雾离子源正离子化后,通过三重四级杆串联质谱仪,采用选择反应监测(SRM)对美利曲辛(m/z 292.2→232.2)和阿米替林(m/z 278.1-91.0)进行测定.结果:美利曲辛的高(50μg·L-1)、中(20μg·L-1)、低(0.5μg·L-1)3个浓度的平均回收率分别为97.53%、104.03%和106.87%,日内(n=5)、日间(n=3)RSD均小于15%;分析方法的最低定量限为0.2μg·L-1.线性范围为:0.2～60μg·L-1,回归方程为:F=1.8691ρ+0.0555,r=0.9986(n=9),权重为1/ρ2.结论:该方法灵敏、准确、简单、快速,可用于临床血浓监测和药动学研究.     

细河河水及其沿岸地下水水质的有机污染特征

调查了沈阳市细河河水及其沿岸地下水水质中石油类和美国环保局(USEPA)"黑名单"上的16种优先控制多环芳烃(PAHs)的有机污染现状,评价细河污染带来的生态风险。结果表明:细河河水中石油类浓度和16种PAHs总浓度范围分别为0.031～1.819mg.L-1和0.026～0.384μg.L-1,平均浓度分别为1.007mg.L-1和0.151μg.L-1;细河沿岸地下水中石油类浓度和16种PAHs总浓度范围分别为0.020～0.987mg.L-1和0.051～0.389μg.L-1,平均浓度分别为0.364mg.L-1和0.133μg.L-1;细河河水和地下水中的石油类污染严重;河水中的PAHs浓度在流经规模较大的城镇或乡镇处出现高值点,苯并(b)荧蒽和苯并(k)荧蒽在河水与地下水中的检出率较高,对水生生态系统健康构成了潜在的威胁。     

Resonance Rayleigh scattering study of interaction of hyaluronic acid with ethyl violet dye and its analytical application

In near weak acid to neutral medium, ethyl violet (EV) can react rapidly with hyaluronic acid (HA) to form a complex, which results in a significant enhancement of resonance Rayleigh scattering (RRS) and an appearance of a new spectrum, and the scattering wavelengths appear at 231, 274, 326, 498 and 640 nm. The maximum scattering wavelength appears at 326 nm. The RRS intensity is directly proportional to the concentration of HA in the range of 0.4-48.0 microg mL(-1). A new method for the determination of trace amounts of HA based on the RRS method has been developed. The method exhibits high sensitivity, and the detection limit for HA is 9.6 x 10(-2) microg mL(-1). This method was applied for determining HA in eyedrops and in sodium hyaluronate injection samples with satisfactory results. Furthermore, the enhancement reasons of RRS and the relationship between RRS spectral characteristics of the HA-EV complex and its absorption spectrum have been discussed.     

LC-MS/MS 法测定人血浆中伊伐布雷定浓度及药动学

目的:建立人血浆中伊伐布雷定的液相色谱-质谱-质谱联用测定方法,研究健康人体药代动力学.方法:以地西泮为内标物,采用液相色谱-质谱-质谱联用法,电喷雾电离源选择性正离子峰检测.测30名健康志愿者单剂量口服盐酸伊伐布雷定片的体内血药浓度,获得药动学参数.结果:伊伐布雷定在0.101-101 ng·mL-1浓度范围内呈良好的线性关系(r=0.998),最低检测浓度为0.101 ng·mL-1.高、中、低浓度的方法提取回收率分别为93.2%、86.6%、87.5%,日内、日间精密度RSD均小于15%.结论:LC-MS/MS方法灵敏度高,专属性强,准确,简便,适用于盐酸伊伐布雷定片的人体药代动力学研究.     

A sensitive and selective immuno‐nanogold resonance‐scattering spectral method for the determination of trace penicillin G

A sensitive and selective immuno‐nanogold resonance scattering spectral assay was developed for the determination of trace hapten penicillin G, based on the resonance scattering (RS) effect of the nanogold at 560 nm, and the nanogold‐labelled immunoreaction took place in pH 5.4 phosphate citric acid buffer solutions and in the presence of polythylene glycol (PEG). The nanogold‐labelled immunocomplex formed more and more with addition of penicillin G. The enhanced RS intensity at 560 nm ΔI_RS was linear to the penicillin G concentration in the range 7.5–1700 ng/mL, with a detection limit of 0.78 ng/mL. The results indicate that the immunonanogold‐labelled RS spectral assay has a high specificity and sensitivity for quantitative determination of penicillin G in raw milk samples. Copyright © 2008 John Wiley & Sons, Ltd.     

Global structure analysis of acid-unfolded myoglobin with consideration to effects of intermolecular coulomb repulsion on solution X-ray scattering

To obtain information on the global structure of protein in the acid-unfolded (AU) state, the structure of apomyoglobin (apoMb) was analyzed by using the solution X-ray scattering (SXS) method. SXS profiles were obtained over a wide range of protein concentrations, 1-18 mg mL-1, under strongly acidic conditions. From analysis of the SXS profile extrapolated to a zero protein concentration, the mean square radius, Rsq, of AU-apoMb at 20 mM HCl was estimated to be 4.81 +/- 0.31 nm. This estimate is more than 1.3 nm larger than those of 3.0-3.5 nm reported thus far. The difference originates from the fact that effects of Coulomb repulsive forces acting between AU-apoMb molecules have not been correctly taken into account in the conventional analysis. In fact, even at a low protein concentration of 1 mg mL-1 close to the limit of measurement in the present SXS method, the solution condition applicable to estimating accurately structural parameters of AU-apoMb is very limited. At HCl concentrations lower than 10 mM, the scattering intensity at a small scattering vector decreases remarkably through the effect of intermolecular repulsive forces and the forward scattering intensity is significantly lower than the estimate from the partial specific volume of protein. On the other hand, at HCl concentrations higher than 50 mM, some compact molten-globule-like structures emerge. As a result, the intermediate concentration of 20 mM HCl is the best choice of the solution condition for determining Rsq of AU-apoMb. The effect of intermolecular Coulomb repulsion on the SXS profile of AU-apoMb is at its maximum for forward scattering and decreases monotonously with an increase in the scattering angle to be virtually negligible at K approximately 0.63 nm(-1). Whereas urea-denatured apoMb shows a SXS profile typical of Gaussian chains, the intrinsic SXS profile of AU-apoMb differs significantly from those of Gaussian chains.     

A rapid and sensitive resonance Rayleigh scattering spectra method for the determination of quinolones in human urine and pharmaceutical preparation

A new method based on resonance Rayleigh scattering (RRS) was proposed for the determination of quinolones (QNS) at the nanogram level. In pH 3.3–4.4 Britton–Robinson buffer medium, quinolones such as ciprofloxacin, pipemidic acid (PIP), lomefloxacin (LOM), norfloxacin (NOR) and sarafloxacin (SAR) were protonated and reacted with methyl orange (MO) to form an ion‐pair complex, which then further formed a six‐membered ring chelate with Pd(II). As a result, new RRS spectra appeared and the RRS intensities were enhanced greatly. RRS spectral characteristics of the MO–QNS–Pd(II) systems, the optimum conditions for the reaction, and the influencing factors were investigated. Under optimum conditions, the scattering intensity (∆I) increments were directly proportional to the concentration of QNS with in certain ranges. The method had high sensitivity, and the detection limits (3σ) ranged from 6.8 to 12.6 ng/mL. The proposed method had been successfully applied for the determination of QNS in pharmaceutical formulations and human urine samples. In addition, the mechanism of the reaction system was discussed based on IR, absorption and fluorescence spectral studies. The reasons for the enhancement of scattering spectra were discussed in terms of fluorescence‐scattering resonance energy transfer, hydrophobicity and molecular size. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of propafenone hydrochloride by flow‐injection analysis coupled with resonance light scattering detection

A simple, sensitive and rapid flow injection analysis (FIA) method with resonance light scattering (RLS) was described for the determination of propafenone (PPF). The method was based on the ion‐association reaction of 12‐tungstophosphoric acid (TP) with propafenone. In pH 1.0 acidic medium, TP reacted with PPF to form an ion‐associate complex, which resulted in a significant enhancement of RLS intensity. The maximum scattering peak was located at 340 nm, the RLS intensity was proportional to the concentration of PPF in the range 0.003–9.0 µg/mL, and the detection limit (3σ) of 1.0 ng/mL was obtained at a sampling rate of 60 samples/h. The feasible reaction conditions and FIA parameters for the system were optimized. The method proposed in this paper shows satisfactory reproducibility with a relative standard deviation (RSD) of 2.1% for 10 successive determinations of 2.0 µg/mL PPF. The present method had been successfully applied to the determination of PPF in serum samples and pharmaceutical samples. The results obtained were in agreement with the method used in the Chinese Pharmacopoeia. Copyright © 2008 John Wiley & Sons, Ltd.     

Determination of bismuth in pharmaceutical products using phosphoric acid as molecular probe by resonance light scattering

A novel method for the sensitive determination of bismuth(III) in pharmaceutical products using phosphoric acid as a molecular probe by resonance light scattering (RLS) is discussed. In 0.5 mol/L phosphoric acid (H₃PO₄) medium, bismuth(III) reacted with PO₄^3? to form an ion association compound, which resulted in the significant enhancement of RLS intensity and the appearance of the corresponding RLS spectral characteristics. The maximum scattering peak of the system existed at 364 nm. Under optimal conditions, there was linear relationship between the relative intensity of RLS and concentration of bismuth(III) in the range of 0.06–10.0 µg/mL for the system. A low detection limit for bismuth(III) of 3.22 ng/mL was achieved. The relative standard deviations (RSD) for the determination of 0.40 and 0.80 µg/mL bismuth(III) were 2.1% and 1.1%, respectively, for five determinations. Based on this fact, a simple, rapid, and sensitive method was developed for the determination of bismuth(III) at nanogram level by RLS technique with a common spectrofluorimeter. This analytical system was successfully applied to determine the trace amounts of bismuth(III) in pharmaceutical products, which was in good agreement with the results obtained by atomic absorption spectrometry (AAS). Copyright © 2011 John Wiley & Sons, Ltd.     

Detection of DNA using cationic polyhedral oligomeric silsesquioxane nanoparticles as the probe by resonance light scattering technique

A novel cationic polyhedral oligomeric silsesquioxane nanoparticle (cationic POSS) was synthesized and successfully used as a new probe for the detection of DNA by resonance light scattering technique (RLS). It was found that the electrostatic interaction of cationic POSS and DNA could obviously enhance the RLS signal, the enhanced RLS intensity at 360 nm was proportional to the concentration of nucleic acids within the range of 0.35-42.82 microg ml-1 for calf thymus DNA, the determination limit (3sigma) was 0.32 ng ml-1. The results showed this method was very sensitive, convenient, rapid and reproducible.     

A simple and sensitive resonance Rayleigh scattering method for determination of As(III) using aptamer‐modified nanogold as a probe

A simple and selective aptamer (ssDNA)‐modified nanogold probe (AussDNA) was prepared for the determination of trace As(III) in HEPES buffer solution (pH 8.2) containing 0.05 mol/L NaCl. The method coupled the aptamer reaction of AussDNA–As(III) and the resonance Rayleigh scattering (RRS) of nanogold aggregations at 278 nm. When the As(III) concentration increased, the RRS intensity at 278 nm increased to form more nanogold aggregation and a stable As(III)–ssDNA complex. Under selected conditions, the increased RRS intensity (ΔI) was linear to the concentration of As(III) in the range 3.8–230.4 ng/mL, with a detection limit of 1.9 ng/mL. This RRS method was applied to detect As(III) in water samples, with simplicity, sensitivity and selectivity. Copyright © 2013 John Wiley & Sons, Ltd.     

Resonance scattering detection of trace microalbumin using immunonanogold probe as the catalyst of Fehling reagent-glucose reaction

A novel and sensitive resonance scattering (RS) spectral immunoassay for the determination of microalbumin (Malb) was developed, based on the catalytic effect of immunonanogold (ING) probe on Fehling reagent-glucose reaction, and resonance scattering effect of Cu(2)O particles. Nanogold particles in size of 10nm were used to label goat anti-human microalbumin (GMalb) to obtain an ING probe (AuGMalb) for Malb. The probe produced unspecific aggregation in pH 5.0 citric acid-Na(2)HPO(4) buffer solutions. Upon addition of Malb, the dispersed ING complex formed. The ING complex in supernatant was obtained by centrifuging and was used as catalyst for the reaction between Fehling reagent and glucose to form the Cu(2)O particles to amplify the resonance scattering signal at 610 nm. With addition of Malb, the ING complex in the supernatant increased and the RS intensity at 610 nm (I(610 nm)) enhanced linearly. The enhanced intensity DeltaI(610 nm) was proportional to the Malb concentration in the range of 0.014-0.43 ng ml(-1), with a detection limit of 7.2 pg ml(-1). The proposed method was applied to detect Malb in human urine sample with satisfactory results.