化学发光底物分辨双组分免疫分析法测定胶质瘤标志物NSE和CA15-3

本文建立了一种基于辣根过氧化物酶(HRP)和碱性磷酸酯酶(ALP)化学发光底物分辨的双组分免疫分析新技术,用以检测人胶质瘤血清标志物神经元特异性烯醇化酶(NSE)和糖链抗原15-3(CA15-3).实验详细考察了捕获抗体、检测抗体、HRP和ALP酶标记物的用量,结果发现NSE和CA15-3的线性范围分别为0.5～20ng/mL(R20.99)和0.5～20U/mL(R20.99),最低检出限分别为0.2ng/mL和0.2U/mL;高、中和低三个浓度的血清加样回收率良好;天内和天间相对标准偏差均小于10%;且一份血清,两组分同时检测无交叉干扰.综合而言:本法能够一次实验,高灵敏、高特异地同时检测两种疾病标志物,具有血清用量少、检测时间短、操作简单、结果可靠的优点,有望为胶质瘤的临床早期诊断提供坚实的支持.     

Study of the separation and determination of monosaccharides in soluble coffee by capillary zone electrophoresis with electrochemical detection

A simple, fast and reliable method, based on capillary electrophoresis with electrochemical detection, for the separation and determination of six monosaccharides, namely glucose, galactose, arabinose, fructose, xylose and ribose, in soluble coffees was developed. A copper disk electrode was used as the working electrode. The optimum conditions for separation and detection were 50 mmol L-1 sodium hydroxide buffer (pH 12.7), separation voltage 5 kV and detection potential 0.65 V (vs. Ag/AgCl). The linear ranges were from 5.0 x 10(-3) to 0.5 mmol L-1 for all six sugars. All regression coefficients were > 0.99. The detection limits for all the sugars were 1.0 x 10(-3) mmol L-1. The RSD of the peak current was < 4.2% (n = 5). The proposed method was applied directly to the separation and determination of the six sugars without prior derivatization, and the assay results were satisfactory.     

A colorimetric fructose assay

A new spectrophotometric method for measuring fructose is presented. The method uses Tryptamine in HCl acid, is carried out at 60°C, and is complete within 60 min. The assay is read at 518 nm and shows very low interference from other sugars. The method can be used for fructose, fructosans, and inulin.     

酶解小麦蛋白产物-还原糖美拉德反应的光谱研究

采用紫外-可见吸收光谱和荧光光谱研究了酶解小麦蛋白产物与还原糖不同加热条件下的美拉德反应及其产物.美拉德反应在紫外区240和294 nm产生两个特征峰,荧光的最大激发和发射波长为347和450 nm;随反应进行,紫外光吸收和荧光强度迅速增加,表明美拉德反应进入高级阶段,产生的糠醛类、呋喃酮类、吡喃酮类、噻吩类及噻唑类等小分子物质表现较大的积累速率.温度升高,强度增加速率增大.在较高温度时,紫外光吸收出现最大平稳值;荧光强度则到达最大值后降低,表明小分子物质间或与肽聚合生成大分子黑素类物质,小分子物质的积累表现消除速率,反应进入终级阶段.     

A Simplified Filter Paper Assay Method of Cellulase Enzymes Based on HPLC Analysis

A simplified filter paper assay (FPA) method of cellulase enzymes was proposed based on high-performance liquid chromatography (HPLC) measurement. The method was according to the sum of glucose and cellobiose concentrations measured by HPLC that was able to be correlated with filter paper units (FPU) of the cellulase enzymes assayed by the traditional FPA method, regardless of the differences in the sources, activities, and components of the cellulases. This simple and quick assay method for the cellulase enzymes provided another parameter of the ratio of glucose to cellobiose (G/C ratio) representing the capacity of cellulase enzymes degrading cellulose into fermentable monomeric sugars.     

Fast microwave-assisted free sugars washing and hydrolysis pre-treatment for the flow injection determination of starch in food

The approach used consists of a flow injection (FI) manifold assisted by a focused microwave digestor for both fast washing of free sugars and acceleration of the hydrolysis step prior to the determination of starch in food. The action of microwaves reduces both the times for removal of free sugars to a 5 min single washing cycle with ethanol/water and that of the subsequent starch hydrolysis to a 10 min step. The sugars formed in the starch hydrolysis are in-line derivatised and photometrically monitored at λ=460 nm. In this way, automation of pre-treatment and determination is achieved with the minimum of both cost and time. The precision of the overall method, expressed as relative standard deviation, is 3.75% and the total analysis time is 38 min. Comparison of the results, obtained in applying the method to flour and bread, is in agreement with those provided by the manual method.     

Automated flow system on-line to LC with postcolumn derivatisation for determination of sugars in carbohydrate-rich foods

Summary A method for the direct determination of carbohydrates in foods using an automated-flow system coupled on-line to a high-performance liquid chromatograph is proposed. The method is based on postcolumn derivatisation of reducing sugars with p-aminobenzoic acid hydrazide in an alkaline medium following elution from the chromatographic column with an acetonitrile: water gradient as mobile phase. The manifold allows non-reducing sugars to be hydrolysed prior to insertion into the chromatograph, thus making them compatible with the derivatisation reaction and with continuous decolorisation of the sample on an activated carbon column. The method allows determination of six sugars in contents between 0.005 and 4% (w/v) with high precision (3.8–5.0%RSD). For analytical validation, it was applied to determination of fructose, sucrose, maltose, lactose and maltotriose in two reference materials (milk powder and sugar). Finally, the method was used to determine sugars in a variety of carbohydrate-rich foods.     

Detection and quantification of sucrose as dietary biomarker using gas chromatography and liquid chromatography with mass spectrometry

Several epidemiological studies suggest a link between the intake of refined sugars and an increased risk for colorectal, breast, pancreatic and endometrial cancer. However, other studies failed to confirm these conclusions and the reason for this may be the ambiguity of dietary assessment methods - mainly self-reporting - employed. Sucrose is an established biomarker for sugars intake, allowing the objective assessment of dietary sucrose. So far, urinary excretion of sucrose was mainly determined using an enzyme assay. However, this method is time-consuming and labour-intensive. In this study, we present a mass spectrometric method for the determination of sucrose in urine using liquid chromatography with mass spectrometry (LC/MS) which can be used for large-scale epidemiological studies.     

Determination of lactulose and mannitol in urine by HRGC

Alteration of intestinal permeability towards compounds like sugars is an indication of abnormalities observed in some diseases. Measurement of the dual sugar absorption of lactulose and mannitol is a non-invasive test of intestinal permeability. The use of this test is however limited by difficulties in the analysis of sugars in urine at low concentrations. In this study we have developed an analytical procedure for quantitative assay of the lactulose–mannitol pair in urine by high resolution gas chromatography. The minimum detectable amounts are 7.5 pg for lactulose and 4.4 pg for mannitol. The calibration curves are linear in the range of 100–6400 mg L^?1. Recoveries are within the range 86–102 % for mannitol and 84–105 % for lactulose.     

Preparation of starch and soluble sugars of plant material for the analysis of carbon isotope composition: a comparison of methods

Starch and soluble sugars are the major photosynthetic products, and their carbon isotope signatures reflect external versus internal limitations of CO₂ fixation. There has been recent renewed interest in the isotope composition of carbohydrates, mainly for use in CO₂ flux partitioning studies at the ecosystem level. The major obstacle to the use of carbohydrates in such studies has been the lack of an acknowledged method to isolate starch and soluble sugars for isotopic measurements. We here report on the comparison and evaluation of existing methods (acid and enzymatic hydrolysis for starch; ion‐exchange purification and compound‐specific analysis for sugars). The selectivity and reproducibility of the methods were tested using three approaches: (i) an artificial leaf composed of a mixture of isotopically defined compounds, (ii) a C₄ leaf spiked with C₃ starch, and (iii) two natural plant samples (root, leaf). Starch preparation methods based on enzymatic or acid hydrolysis did not yield similar results and exhibited contaminations by non‐starch compounds. The specificity of the acidic hydrolysis method was especially low, and we therefore suggest terming these preparations as HCl‐hydrolysable carbon, rather than starch. Despite being more specific, enzyme‐based methods to isolate starch also need to be further optimized to increase specificity. The analysis of sugars by ion‐exchange methods (bulk preparations) was fast but produced more variable isotope compositions than compound‐specific methods. Compound‐specific approaches did not in all cases correctly reproduce the target values, mainly due to unsatisfactory separation of sugars and background contamination. Our study demonstrates that, despite their wide application, methods for the preparation of starch and soluble sugars for the analysis of carbon isotope composition are not (yet) reliable enough to be routinely applied and further research is urgently needed to resolve the identified problems. Copyright © 2009 John Wiley & Sons, Ltd.     

食品中降解糖的高灵敏、高选择性的检测方法

本文使用岛津公司开发的,以精氨酸为衍生剂的降解糖柱后衍生荧光检测系统,并选择配位体交换色谱法为分离手段,获得了6种降解糖的线性关系和检出限。本法重现性较好。利用该法测定了酱菜汁中的各种糖组分的含量,分析结果满意。     

Typical conversion of lignocellulosic biomass into reducing sugars using dilute acid hydrolysis and alkaline pretreatment

The development and production of fossil fuel alternatives have become one of the main focal points in recent investigations. Lignocellulosic biomass is a renewable source of fermentable sugars for second-generation biofuels and chemicals via biotechnological pathways. However, the presence of lignin and hemicellulose in lignocellulosic biomass makes it difficult for the biomass to be hydrolyzed or digested during fermentation. Thus, effective biomass pretreatment is vital. The present review shows that chemical pretreatment is the current preferred method to obtain high sugar yields at low cost, with dilute acid and alkaline hydrolysis as the two most reported technologies. Dilute acid favours hydrolysis of the hemicelluloses whereas alkaline hydrolysis targets the lignin fraction. Both methods have merits and demerits, and have been combined with other treatments such as hydrothermal and enzymatic hydrolysis. Further investigation is required to improve the pretreatment processes and to ensure the economic viability of bioconversion.     

OZONE/OXONE-MEDIATED OXIDATIONS OF AMINO AND O-BENZYLHYDROXYLAMINO SUGARS

A method for the preparation of C-methyl branched nitro sugars from the corresponding amino sugars by oxidation with mixed oxidants is described. Ozonolysis of the amino sugars in the presence of OXONE (potassium monopersulfate) and acetone (Bashir-Hashemi's procedure) gave nitro sugars in good yields. Oxidation of an O-benzylhydroxylamino sugar by this method resulted in deprotection and oxidation in a single step, giving the nitro sugar directly. The mixed oxidant method is experimentally simpler and more convenient to use than DMDO, which had been previously reported for the oxidation of amino sugars.     

Fabrication of the Enzyme‐less Voltammetric Bilirubin Sensor Based on Sol‐gel Imprinted Polymer

Measuring the serum bilirubin (BR) level is part of the common test for newborns, since high concentrations of bilirubin in the blood can cause brain damage and even death for them. Therefore, achieving a fast, accurate and cost effective method for bilirubin assay in medical diagnosis and Medical management of patients with jaundice, especially neonates, is very important. In this study, an electrochemical sensor was developed to detect low amounts of bilirubin in serum samples. The basis of the work is the BR imprinted polymer synthesis and the use of this polymer as a recognition element in the production of the bilirubin voltammetric sensor. For this purpose, bilirubin imprinted polymer was synthesized using the simple sol gel method and the prepared polymer is characterized by SEM and FT‐IR. In the next step, this polymer was applied to modify the carbon paste electrode and provide a sensitive and selective sensor for the BR molecule. The proposed sensor was able to measure the bilirubin in the concentration range of 1 to 100 μM after optimizing the operating conditions. The limit of detection (LOD) and limit of quantification (LOQ) was calculated 0.75 μM and 2.5 μM respectively. It is also applicable to bilirubin assay in serum samples.     

Synthesis and Preliminary Anticancer Activity Assessment of N-Glycosides of 2-Amino-1,3,4-thiadiazoles

The addition of 2-amino-1,3,4-thiadiazole derivatives with parallel iodination of differently protected glycals has been achieved using a double molar excess of molecular iodine under mild conditions. The corresponding thiadiazole derivatives of N-glycosides were obtained in good yields and anomeric selectivity. The usage of iodine as a catalyst makes this method easy, inexpensive, and successfully useable in reactions with sugars. Thiadiazole derivatives were tested in a panel of three tumor cell lines, MCF-7, HCT116, and HeLa. These compounds initiated biological response in investigated tumor models in a different rate. The MCF-7 is resistant to the tested compounds, and the cytometry assay indicated low increase in cell numbers in the sub- G1 phase. The most sensitive are HCT-116 and HeLa cells. The thiadiazole derivatives have a pro-apoptotic effect on HCT-116 cells. In the case of the HeLa cells, an increase in the number of cells in the sub-G1- phase and the induction of apoptosis was observed.     

A modification of the phenol/sulfuric acid assay for total carbohydrates giving more comparable absorbances

The conditions and acid strength of the phenol/sulfuric acid assay were investigated to improve agreement between absorbances obtained from different sugars. It was found that by increasing acid strength and by cooling the tubes in water after a short reaction time, the values obtained for several sugars, including fructose and xylose, agreed, on an equimolar value, with that for glucose.     

Ethanol from lignocellulosic wastes with utilization of recombinant bacteria

This article presents the advanced technology that has been developed by BioEnergy International of Gainesville, Florida, utilizing novel recombinant strains of bacteria developed by Lonnie Ingram of the University of Florida. The first commercial applications of these unique fermenting organisms convert 5-carbon sugars, as well as 6-carbon sugars, and oligomers of cellulose (e.g., cellobiose and cellotriose) directly to ethanol. The proposed systems that will be utilized for conversion of agricultural wastes, mixed waste papers, and pulp and paper mill waste in forthcoming commercial installations are now under design. This involves the extensive experience of Raphael Katzen Associates International, Inc. in acid hydrolysis, enzyme production, enzymatic hydrolysis, large-scale fermentation engineering, and distillation/dehydration. Specific examples of this advanced technology will be presented in different applications, namely:

New downstream processing strategy for the purification of monoclonal antibodies from transgenic tobacco plants

Affinity chromatography on immobilized Protein A is the current method of choice for the purification of monoclonal antibodies (mAbs). Despite its widespread use it presents certain drawbacks, such as ligand instability, leaching, toxicity and high cost. In the present work, we report a new procedure for the purification of two human monoclonal anti-HIV (human immunodeficiency virus) antibodies (mAbs 2G12 and 4E10) from transgenic tobacco plants using stable and low cost chromatographic materials. The first step of the mAb 2G12 purification procedure is comprised of an aqueous two-phase partition system (ATPS) for the removal of polyphenols while providing an essential initial purification boost (2.01-fold purification). In the second step, mAb 2G12 was purified using cation-exchange chromatography (CEX) on S-Sepharose FF, by elution with 20mM sodium phosphate buffer pH 7.5, containing 0.1M NaCl. The eluted mAb was directly loaded onto an immobilized metal affinity chromatography column (IMAC, Zn(2+)-iminodiacetic acid-Sepharose 6B) and eluted by stepwise pH gradient. The proposed method offered 162-fold purification with 97.2% purity and 63% yield. Analysis of the antibody preparation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme immunosorbent assay (ELISA) and western blot showed that the mAb 2G12 was fully active and free of degraded variants, polyphenols and alkaloids. The effectiveness of the present purification protocol was evaluated by using a second transgenic human monoclonal anti-HIV mAb 4E10. The results showed that the same procedure can be successfully used for the purification of mAb 4E10. In the case of mAb 4E10, the proposed method offered 148-fold purification with 96.2% purity and 36% yield. Therefore, the proposed protocol may be of generic use for the purification of mAbs from transgenic tobacco plants.     

Rapid determination of paeoniflorin and three sugars in Radix Paeoniae Alba by capillary electrophoresis.

A method based on capillary electrophoresis with electrochemical detection has been developed for the separation and determination of paeoniflorin, sucrose, glucose, and fructose in traditional Chinese medicine, Radix Paeoniae Alba. The effects of several important factors, such as the concentration of NaOH, the separation voltage, the injection time, and the detection potential, were investigated to determine the optimum conditions. The detection electrode was a 300-microm diameter copper disc electrode at a working potential of +0.60 V (versus SCE). The four analytes can be well separated within 8 min in a 40 cm length fused-silica capillary at a separation voltage of 12 kV in a 75 mM NaOH aqueous solution. The relation between the peak current and the analyte concentration was linear over about 3 orders of magnitude with detection limits (S/N = 3) ranging from 1 to 2 microM for all analytes. The proposed method has been successfully applied for the determination of the paeoniflorin and sugars in real plant samples with satisfactory assay results.     

Immobilization of glutamate dehydrogenase on glass derivatives. A method for the assay of glutamates in real samples with simplex optimized automated FIA-system

An enzymatic method for the determination of glutamic acid in food samples and pharmaceuticals is described. l-Glutamate dehydrogenase (GLDH) from beef liver was immobilized on isothiocyanate modified Controlled Pore Glass for the construction of a packed bed reactor. The NADH produced from the enzymic reaction was monitored fluorimetrically. The working curve is linear up to 200 mumol/l glutamate for an injection volume of 58 mul. The detection limit of the method is 0.3 mumol/l. The composite modified simplex was employed for the selection of the proper experimental conditions using an in-house flow injection manifold. Many interfering species and several amino acids were tested to verify the specificity of the enzyme reactor. The system works selectively for glutamic acid. The method is ideally suited to the assay of glutamic acid in a large number of samples because of its simplicity, stability and low cost. Forty-five samples per hour can be analyzed with a relative standard deviation better than 2%. The reactor is stable for a period of more than four months under specified storage conditions. The accuracy of the proposed method is tested by comparison of the results with those obtained by the official methods and the manufacturer's specifications for the analyzed samples. Good correlation was attained. Recovery experiments showed results between 97 and 104%.