Fluorometric assay for the determination of glutathione reductase activity

The determination of free sulfhydryl groups is important in many aspects of biotechnology, such as measuring the coupling efficiency of thiol-reactive probes, assaying cysteine-containing haptens, and assaying reductase/thiol transferase activity, as well as in various aspects of the food industry. This is generally achieved colorimetrically using Ellman's reagent, although the assay is relatively insensitive and Ellman's reagent is unstable. In this paper, we describe a highly sensitive fluorometric assay for free sulfhydryl groups based on FRET, which we have used to develop a sensitive assay for glutathione reductase activity. The assay exploits the specific increase in fluorescence intensity that occurs at 520 nm when a probe containing two molecules of fluorescein linked via a disulfide group is cleaved by glutathione. The assay is 2 orders of magnitude more sensitive than the commonly used colorimetric glutathione reductase assay and has a greater dynamic range.     

催化电位法测定环境水样中痕量亚硝酸根的研究

研究了以亚硝酸根对溴酸钾氧化碘化钾的催化作用,用碘离子选择电极跟踪测定催化体系的电位。实验表明,在适宜的条件下,该指示反应为拟一级反应;电位差与亚硝酸讯浓度在测定条件下呈线性关系,其检出限为3.0×10~(-5)mg/mL。该法用于环境水样的测定,其结果良好。     

滴定法测定食品中亚硝酸盐含量的研究

亚硝酸盐含量的测定是食品检测中重要的检测指标之一,现行的检测方法很多。文中采用了滴定法测定食品中亚硝酸盐含量,利用亚硝酸根在酸性条件下跟碘离子反应,生成碘单质,再用硫代硫酸钠滴定法碘单质,间接测定食品中亚硝酸盐的含量。实验中还研究了淀粉用量、不同酸性物质和不同pH等反应条件对回收率的影响。最后研究了此方法测定亚硝酸盐含量的时,食品中其他物质对结果的影响。该方法测亚硝酸盐含量相对偏差为2.4%,限检量为0.30mg/kg。     

Fluorometric broad-range screening of compounds with affinity for nucleic acids

The potential of a nucleic acid-based optical bioprobe for environmental measurements and drug monitoring is described. The sensor employs the long-wavelength intercalating fluorophore TO-PRO-3 (TP3). Compounds that interact with the TP3-DNA complex are indirectly detected by a decrease in the fluorescence intensity. We found that the configuration and length of the DNA dramatically affected the intensity of the fluorescence emitted from the TP3-DNA complex. We compared nucleic acids from different sources and optimized the system for pBR322 plasmid DNA (4363 bp) digested by HindIII restriction endonuclease. This endonuclease has a single recognition site in plasmid pBR322. In the proposed method, we attempt to combine broad-range detection with rapid and simple operation. A fiber-optic capillary fluorescence system was used to analyze toxic aromatic amines, antibiotics, and several kinds of antitumor drugs, using small amounts of sample, down to 10 muL, with a sensitivity comparable to that of current electrochemical methods. The detection limit can be as low as a few ppb or submicromolar. This approach is useful for routine screening in environmental monitoring or for controlling cytotoxic drug administration. The ease of operation and the rapid response allow high-throughput screening.     

Aqueous nitrite ion determination by selective reduction and gas phase nitric oxide chemiluminescence

An improved method of flow injection analysis for aqueous nitrite ion exploits the sensitivity and selectivity of the nitric oxide (NO) chemilluminescence detector. Trace analysis of nitrite ion in a small sample (5-160 microL) is accomplished by conversion of nitrite ion to NO by aqueous iodide in acid. The resulting NO is transported to the gas phase through a semipermeable membrane and subsequently detected by monitoring the photoemission of the reaction between NO and ozone (O3). Chemiluminescence detection is selective for measurement of NO, and, since the detection occurs in the gas-phase, neither sample coloration nor turbidity interfere. The detection limit for a 100-microL sample is 0.04 ppb of nitrite ion. The precision at the 10 ppb level is 2% relative standard deviation, and 60-180 samples can be analyzed per hour. Samples of human saliva and food extracts were analyzed; the results from a standard colorimetric measurement are compared with those from the new chemiluminescence method in order to further validate the latter method. A high degree of selectivity is obtained due to the three discriminating steps in the process: (1) the nitrite ion to NO conversion conditions are virtually specific for nitrite ion, (2) only volatile products of the conversion will be swept to the gas phase (avoiding turbidity or color in spectrophotometric methods), and (3) the NO chemiluminescence detector selectively detects the emission from the NO + O3 reaction. The method is free of interferences, offers detection limits of low parts per billion of nitrite ion, and allows the analysis of up to 180 microL-sized samples per hour, with little sample preparation and no chromatographic separation. Much smaller samples can be analyzed by this method than in previously reported batch analysis methods, which typically require 5 mL or more of sample and often need chromatographic separations as well.     

A new approach for nitrite determination based on a HRP/catalase biosensor

In this paper, a new concept of enzyme inhibition-based biosensor involving a two enzyme system was developed. The latter displays a signal increase instead of a decrease in the presence of an inhibitor. HRP and catalase were thus separately entrapped into Layered Double Hydroxides (LDH), an anionic clay, as a host matrix. The inner layer was constituted of HRP electrically wired by [Zn₂CrABTS] LDH and the outer layer contained catalase immobilized in [Zn₃AlCl] LDH. Both enzymes catalyzed the decomposition of H₂O₂, HRP its reduction and catalase its breakdown into oxygen and water. Nitrite was selected as a specific inhibitor of catalase. In the presence of H₂O₂, the nitrite addition blocked the H₂O₂ consumption by catalase, inducing thus an increase in the amperometric signal of the H₂O₂ reduction at 0 V by the wired HRP. The optimum configuration of the bi-enzyme biosensor displayed in aerated aqueous solutions, a nitrite sensitivity of 102 μA M^− 1· cm^− 2 with a fast response time, the detective limit being 4 μM.     

A new HRP/catalase biosensor based on microconductometric transduction for nitrite determination

An interdigitated conductimetric electrode system using a combination of peroxidase/catalase has been developed to determine nitrite in water samples. A peroxidase (HRP) was located in the inner layer while the outer contained catalase. Catalase catalyzed the breakdown of H₂O₂ into H₂O and O₂ thus consuming totally H₂O₂, the substrate of HRP. The latter, in presence of H₂O₂, generates a conductometric signal due to the reduction of H₂O₂. Nitrite was selected as an inhibitor of catalase. In the presence of H₂O₂, the nitrite addition blocked the H₂O₂ consumption by catalase. Since nitrite had no effect on HRP activity, its inhibitive effect on catalase leads to an increase in the conductometric signal. The bienzyme sensor exhibits an increase in conductometric response for nitrite concentration, leading to high values of conductivity. In both case, the detection limit of nitrite is 0.3 µM and for bienzyme sensor the dynamic range is from 0.3 µM to 446 µM.     

酸性高锰酸钾流动注射分析测定化学需氧量的方法开发

在高温加热的条件下,酸性高锰酸钾将废水中的有机物氧化;根据比色分析高锰酸钾浓度的降低,从而测定样品的化学需氧量。在0～150mg／L的范围内,标准曲线的一次拟合的相关性系数达到0．999;对63mg／L的标准样品的进行12次测定,其相对标注偏差为3．2％。