谷氨酸棒状杆菌尿素传感器的研究

基于腺酶催化尿素分解产生氨,以氨气敏电极为基础电极,用含脲酶丰富的谷氨酸棒状杆菌研制成测定尿素的微生物传感器.在30℃、pH8.0、0.1mol/L磷酸盐缓冲液中,该传感器的线性范围为1.1×10^-4～1.4×10^-2mol/L,斜率为51.2mV/decade,检测下限为1.0×10^-5mol/L,寿命可达45d.考察了传感器响应初速和底物浓度之间的关系,测定了微生物膜中脲酶的表观米氏常数K_m及最大响应初速v_m.     

谷氨酸发酵中二价阳离子对噬菌体抑制作用初步观察

本文报道适当浓度的Ca²⁺、Fe²⁺在发酵液中能抑制噬菌体530对谷氨酸生产菌株T₆^-13、B₄的裂解,谷氨酸发酵产量不低于对照菌株。     

O -·2增强谷氨酸与其受体的结合力及EBSELEN的保护作用

用放射配体测定受体法研究了黄嘌呤(X)/黄嘌呤氧化酶(XO)体系产生的超氧阴离子自由基(O -·2)对［3H］DL-谷氨酸与大鼠大脑皮层突触膜谷氨酸受体结合的影响,结果表明O -·2明显增强谷氨酸与其受体的结合力,此作用能被2-苯基-1,2-苯并异硒唑-3(2H)酮(EBSELEN)(1 μmol/L)所抑制.     

鸟氨酸脱羧酶活性微量测定法

本文采用一种简单,微量反应系统,根据 ¹⁴C-鸟氨酸释放的 ¹⁴CO₂量测定鸟氨酸脱羧酶(ODC)的活性,酶反应在置于液闪计数瓶内的玻璃小管中进行,释放的 ¹⁴CO₂被瓶内滤纸片上的海胺吸收。实验结果表明,加酸释放 ¹⁴CO₂后30分钟 ¹⁴CO₂吸收已达最大值,且吸收量与释放量成正比,酶反应测定证明 ¹⁴CO₂释放速度在40分钟内保持恒定。ODC活性与酶浓度呈线性关系,此方法不仅用于ODC活性测定,而且亦可用于其他脱羧酶活性的测定。     

亲和层析纯化肌质网Ca2+－ATP酶

建立了一种亲和层析纯化肌质网Ca²⁺-ATP酶的方法．用非离子型去污剂C₁₂E₈ 溶解肌质网,再通过反应红－120琼脂糖亲和层析柱使肌质网Ca²⁺-ATP酶纯度从粗品中的65％提高到99％,并具有较高ATP水解活性．经SDS－聚丙烯酰胺凝胶电泳检测,为电泳纯．     

谷氨酸脱羧酶放射测量法的改良及应用

用NaOH代替苯乙胺作为¹⁴CO₂的吸附剂,改进谷氨酸脱羧酶（GAD）活性的放射测定方法,结果发现NaOH为吸附剂组内变异系数为9.6%, 以苯乙胺为吸附剂组内变异系数为31.9%;以NaOH为吸附剂72 h后测量其放射活性仍稳定不变,以苯乙胺为吸附剂者1 h后放射性活性即下降47%,6 h后已降低至本底水平;¹⁴CO₂重吸收实验亦证明以苯乙胺为吸附剂吸附的¹⁴CO₂ 6 h内已有80%以上重新被NaOH吸附;以NaOH作为吸附剂测定GAD的活性,在0.39～17.8 mg脑组织样品范围内GAD量与¹⁴CO₂生成量之间有线性关系.NaOH代替苯乙胺作为¹⁴CO₂的吸附剂测定GAD的活性其灵敏度提高1.66倍.用此方法测定组织和细胞内GAD活性证明其具有良好的重复性和稳定性,值得推广应用.     

快速测定BOD的微生物传感器

异常汉逊氏酵母菌(Hansenula anomala var schneggil)细胞经海藻胶固定,与氧电极组装成BOD传感器。采用恒容动态法,可在5分钟内完成一个样品BOD的测定。用于某些污水测定时,与BOD₅²⁰法有较好的相关性。传感器可使用90天和测定650次以上。     

H2O2－Fe3+所致人淋巴细胞DNA双链断裂损伤

采用脉冲电场凝胶电泳法检测H₂O₂－Fe³⁺体系产生的OH^·对人淋巴细胞DNA的双链断裂损伤．H₂O₂－Fe³⁺浓度与DNA双链断裂呈明显量效关系;随OH^·作用时间延长,细胞DNA双链断裂加重;过氧化氢酶对OH^·损伤有明显抑制作用．脉冲电场凝胶电泳法可检测到的H₂O₂和FeCl₃引起细胞DNA双链断裂的最低浓度为0.3 mmol/L和6 μmol/L．     

代谢型谷氨酸受体激动引起星形神经胶质细胞肿胀

用[~3H]-3-氧-甲基-D-葡萄糖摄取的方法测定细胞水含量,观察谷氨酸受体激动剂、拮抗剂对培养的星形神经胶质细胞水含量的影响,并观察细胞内、外钙的作用.结果发现:0.5,1,10 mmol/L的谷氨酸和1mmol/L的trans-ACPD(代谢型谷氨酸受体激动剂)1h均可以引起细胞的水含量增加,1mmol/L的 AMPA(离子型谷氨酸受体激动剂)不影响细胞的水含量,1mmol/L的L-AP3(代谢型谷氨酸受体拮抗剂)可以拮抗1mmol/L谷氨酸和trans-ACPD的作用;撤除细胞外液的钙,谷氨酸不再引起细胞的水含量增加, 20 μmol/ L 的 CdCl₂不能减轻谷氨酸的作用,而300μmol/L的CdCl₂及30μmol/L的胆罗啉(Dantrolene)均可以减轻谷氨酸的作用,提示代谢型谷氨酸受体激动引起星形细胞肿胀,细胞内、外Ca²⁺在谷氨酸引起的星形细胞肿胀中起一定的作用.     

活性氧清除剂和钙离子通道阻断剂对人淋巴细胞化学发光的效应

本文报道了活性氧(ROS)清除剂——苯甲酸钠、维生素C、甘露醇、L-组氨酸、过氧化氢酶和超氧化物歧化酶对Con A诱导的人外周血淋巴细胞化学发光(Ly-CL)均有抑制效应,提示人Ly-CL与ROS的生成有关,参与人Ly-CL的ROS类型有·OH、¹O₂、H₂O₂和O₂^-_·。钙通道阻断剂——Verapamil对人Ly-CL也有抑制效应,表明人Ly-CL依赖于人淋巴细胞内钙离子浓度的增加。     

Microbial corrosion monitoring by an amperometric microbial biosensor developed using whole cell of Pseudomonas sp.

A microbial biosensor was developed for monitoring microbiologically influenced corrosion (MIC) of metallic materials in industrial systems. The Pseudomonas sp. isolated from corroded metal surface was immobilized on acetylcellulose membrane and its respiratory activity was estimated by measuring oxygen consumption. The microbial biosensor was used for the measurement of sulfuric acid in a batch culture medium contaminated by microorganisms. A linear relationship between the microbial sensor response and the concentration of sulfuric acid was observed. The response time of biosensor was 5 min and was dependent on the immobilized cell loading of Pseudomonas sp., pH, temperature and corrosive environments. The microbial biosensor response was stable, reproducible and specific for sensing of sulfur oxidizing bacterial activity.     

Microbial corrosion monitoring by an amperometric microbial biosensor developed using whole cell of Pseudomonas sp.

A microbial biosensor was developed for monitoring microbiologically influenced corrosion (MIC) of metallic materials in industrial systems. The Pseudomonas sp. isolated from corroded metal surface was immobilized on acetylcellulose membrane and its respiratory activity was estimated by measuring oxygen consumption. The microbial biosensor was used for the measurement of sulfuric acid in a batch culture medium contaminated by microorganisms. A linear relationship between the microbial sensor response and the concentration of sulfuric acid was observed. The response time of biosensor was 5 min and was dependent on the immobilized cell loading of Pseudomonas sp., pH, temperature and corrosive environments. The microbial biosensor response was stable, reproducible and specific for sensing of sulfur oxidizing bacterial activity.     

Microbial electrode sensor for alcohols

A microbial electrode consisting of immobilized microorganisms, a gas permeable Teflon membrane, and an oxygen electrode was prepared for the continuous determination of methyl and ethyl alcohols. Immobilized Trichosporon brassicae was employed for a microbial electrode sensor for ethyl alcohol. When a sample solution containing ethyl alcohol was injected into a microbial electrode system, the current of the electrode decreased markedly with time until a steady state was reached. The response time was within 10 min by the steady state method and within 6 min by the pulse method. A linear relationship was observed between the current decrease and the concentration of ethyl alcohol below 22.5 mg/liter. The current was reproducible within ± 6% of the relative error when a sample solution containing 16.5 mg/liter ethyl alcohol. The standard deviation was 0.5 mg/liter in 40 experiments. The selectivity of the microbial electrode sensor for ethyl alcohol was satisfactory. The microbial electrode sensor was applied to a fermentation broth of yeasts and satisfactory comparative results were obtained (correlation coefficient 0.98). The current output of the microbial electrode sensor was almost constant for more than three weeks and 2100 assays. A microbial electrode sensor using immobilized bacteria for methyl alcohol was also described.     

Microbial sensor system for nondestructive evaluation of fish meat quality

A microbial sensor system consisting of the bacterium (Alteromonas putrefaciens) immobilized within membranes, a flow cell, an oxygen electrode, peristaltic pumps, a buffer tank, a thermostatically controlled bath and a recorder, was constructed for the nondestructive quality evaluation of bluefin tuna. The chemical compounds on fish meat surfaces which are the indicators of fish meat quality were rapidly determined by using the proposed sensor system. Fish meat quality was determined from the rate of current decrease of the sensor. Good correlations were obtained between fish meat quality and sensor response. One assay could be completed within one minute.     

Microbial sensor for selective determination of sulphide

A microbial sensor consisting of immobilized Thiobacillus thiooxidans, a gas-permeable membrane, and an O₂ electrode was prepared for the determination of sulphide. When a sample solution containing sulphide was passed into the flow cell, the output of the microbial sensor decreased markedly with time until a steady state was reached. The total time required for an assay was 20–30 min by the steady-state method. In the pulse method, the total time required for an assay was about 5 min. A linear relationship was obtained between the sensor output and the concentration of sodium sulphide below 0.40 mm. The minimum detectable concentration of sodium sulphide was 0.02 mm. Selectivity of the sensor was satisfactory. The microbial sensor was applied to the determination of sulphide in spring water. A good agreement was obtained between the microbial sensor and the methylene blue method. The regression coefficient was 0.97 for five experiments. The activity of the microbial membrane was stable for more than 25 days. The response was reproducible with 2.5% of the relative standard deviation when a sample solution containing 0.2 mm sodium sulphide was employed. *** DIRECT SUPPORT *** AG903053 00005     

Microbial sensor for aspartame

Summary A microbial amperometric sensor using immobilized Bacillus subtilis cells was developed for the determination of the dipeptide sweetener aspartame (l-aspartyl-l-phenylalaninemethylester). From 0.07 to 0.6 mmol/l aspartame, a linear dependence of the initial current change (i.e., change in respiration rate) was obtained. The sensitivity for aspartame was one order of magnitude higher than for its amino acid constituents. The microbial sensor was stable for 8 weeks.     

Application of a flow-type antibody sensor to the detection of Escherichia coli in various foods

A flow-type biosensor system which uses a broad-spectrum anti-Escherichia coli antibody and quartz crystal microbalance as biological component and transducer was developed. Biosensor responses were initiated by injecting viable E. coli suspensions through a flow cell and the sensor system was optimized for response time according to flow rate and injection time, followed by the measurement of responses for various E. coli strains. As expected, the sensor system showed a characteristic broad binding feature against E. coli strains. A linear sensor response in double-logarithmic scale was observed for the microbial suspensions ranging from 1.7 x 10(5) to 8.7 x 10(7) CFU/ml. Sample measurements could be done within 20-30 min after Stomacher treatment followed by spiking or enrichment.     

A chemiluminescence fiber-optic biosensor system for the determination of glutamine in mammalian cell cultures.

A chemiluminescence fiber-optic biosensor system has been developed for determining glutamine in hybridoma cell cultures producing monoclonal antibodies against viral surface antigens. Glutaminase and glutamate oxidase (GLO) were immobilized onto aminopropyl glass beads via glutaraldehyde activation separately and packed in a column. Two separate columns containing immobilized GLO and catalase were placed upstream to eliminate endogenous glutamate. In the presence of ferricyanide, luminol reacted with hydrogen peroxide released from the enzymatic reactions to produce a chemiluminescence (CL) light signal which was detected and quantitated with a fiber-optic system. In combination with flow injection analysis it was possible to process samples virtually identically, thus avoiding difficulties in reproducing the CL signal. There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 10(-6) to 10(-3) M. A complete analysis could be performed in 2 min including sampling and washing. Each immobilized enzyme column was stable for at least 300 repeated analyses without any loss of activity. When the biosensor system was used for the determination of glutamine in spent mammalian cell cultures, the values obtained compared well with those of high-performance liquid chromatography, thus validating the applicability of the CL fiber-optic system.     

Use of a microbial sensor: a new approach to the measurement of inhibitory effects on the microbial activity of activated sludge

This paper presents a new method for the measurement of inhibitory effects in wastewater treatment plants on the basis of a continuous measurement of the microbial respiration product (CO(2)). The microbial sensor developed for this purpose consists of a small conical fluidized bed reactor connected to a cylindrical chamber that comprises part of the sample recirculation system. Activated sludge microbes are immobilized on spherical (diameter=1-2 mm) reticulated sinter glass carriers. Pure oxygen is supplied via the cylindrical chamber in order to sustain a highly dense population of microbial mass. The mean hydraulic retention time in the microbial sensor ranges between 30 and 40 min, while temperature is maintained at 30 degrees C, and pH 6.4. Carbon dioxide in the off-gas, which reflects the microbial activity, is continuously analyzed by means of an infrared analyzer. Inhibition of microbial activity (toxicity) can be determined as the mean percent reduction in carbon dioxide concentration. Several substances were tested and proved toxic to the microbes. With this microbial sensor, early detection of toxic substances becomes feasible, preventing them from entering an activated sludge unit operation.     

Measurement of biodegradable substances using the salt-tolerant yeast Arxula adeninivorans for a microbial sensor immobilized with poly(carbamoyl) sulfonate (PCS) Part I: Construction and characterization of the microbial sensor

A microbial biosensor based on the yeast Arxula adeninivorans LS3 has been developed for measurement of biodegradable substances. Arxula is immobilized in the hydrogel poly(carbamoyl) sulfonate (PCS). The immobilized yeast membrane is placed in front of an oxygen electrode with -600 mV versus Ag/AgCl. Arxula is salt tolerant; it can give a stable signal up to 2.5 M NaCl in sample (120 mM in measuring cell). The sensor's measurements are highly correlated to BOD5 measurements. It has a very high stability which can last for 40 day without any decrease in signal. The linear range of the sensor is up to a corresponding BOD value of 550 mg/l.