生物传感器快速测定BOD的研究

生化需氧量(biochemicaloxygendemand,BOD)是一种表征水体有机污染程度的综合指标,广泛应用于水体监测和废水处理厂的运行控制。由于BOD的标准测定方法需时5天,不能及时地反映水质状况和反馈处理信息,因此快速测定BOD的方法和仪器化研究近年来得到广泛的重视。利用生物传感器测定BOD是一种有效地快速测定废水中可生化降解有机物的方法。介绍生物传感器的工作原理及其生物敏感材料,讨论BOD传感器的性能参数以及BOD快速测定值(BODst)与标准BOD5值的一致性问题。对现阶段市场上常见的几种BOD快速测定仪进行简单的介绍,并对它们的性能进行比较 。     

气相色谱法测定液相合成蛋氨酸脑啡肽中杂质残留量

测定液相法合成蛋氨酸脑啡肽中三氟乙酸和醋酸的残留含量。应用气相色谱法,程序升温,离子火焰反应检测器,定量测定。结果可见,该法线性范围为0.001～0.08 mg/L,在此浓度范围内与峰面积呈良好的线性关系,方法的检出限为0.0003～0.012 mg/L,测定的回收率为97.2%～101.7%,相对标准偏差<1.09%。所建立的检测方法简便,快速,灵敏度高,重复性好,可应用于检测蛋氨酸脑啡肽中残留醋酸的含量。     

乙酯型鱼油中二十碳五烯酸乙酯和二十二碳六烯酸乙酯的定量检测

建立了快速、灵敏测定鱼油乙酯中二十碳五烯酸乙酯(EPA-EE)和二十二碳六烯酸乙酯(DHA-EE)的气相色谱方法(GC)。采用HP-INNOWAX毛细管柱,氢火焰离子化检测器(FID),优化了分析检测鱼油乙酯中EPA-EE和DHA-EE的气相色谱条件。结果表明:EPA-EE在0～1.00 mg/mL范围内具有较好线性,相关系数r≥0.9998,检测限为0.0015 mg/mL,相对标准偏差(RSD)为0.60%,回收率在95.27%～103.86%;DHA-EE在0～1.00 mg/mL范围内线性良好,相关系数r≥0.9994,检测限为0.0040 mg/mL,RSD为0.95%,回收率在95.92%～104.38%。其正己烷溶液在-18℃条件下保存较稳定。     

氮、磷营养盐对底栖硅藻的生长及生化组成影响

目的:研究了从福建连江海域分离得到的一株野生底栖硅藻(Nitzschia.constricta)的生长和生理特性.方法:测定了在不同氮磷浓度条件下比增长速率、色素,以及不同时期底栖硅藻蛋白和胞外多糖的积累情况.结果:当硝酸氮浓度为900mg/l时,获得最大比增长速率0.21和叶绿素a含量4.31mg/l、类胡萝卜素含量3.44mg/l、胞外多糖产率90.57μg/ml.当氮浓度为75mg/l时,在第10d得到最大蛋白产率28.90μg/ml.当磷浓度为4.4mg/l时,可得到最大比增长速率0.13、叶绿素a含量3.42mg/l、类胡萝卜素2.82mg/l、蛋白含量23.16μg/ml,无磷培养基中第5d的胞外多糖产率最高13.51μg/ml.结论:氮磷营养盐浓度的增加促进了此种底栖硅藻的生长,但不一定会促进蛋白及胞外多糖的产生.     

气相色谱质谱法测定食品包装材料中24种邻苯二甲酸酯(英文)

目的:建立同时检测食品包装材料中24种邻苯二甲酸酯类化合物的气相色谱质谱法(GC-MS)分析方法。方法:用正己烷提取包装材料,GC-MS选择离子监测模式(SIM)测定,运用气质联用仪测定24种邻苯二甲酸酯类物质。结果:24种邻苯二甲酸酯类物质的线性范围为0.05 mg/L~10 mg/L,除了邻苯二甲酸二异壬酯(DINP)和邻苯二甲酸二异癸酯(DIDP)为0.5 mg/L~10 mg/L,相关系数(r2)除DINP、DIDP外均大于0.99,方法的检出限(信噪比为3)为0.002 mg/kg~0.05 mg/kg,在食品包装材料基质中3个加标水平的平均回收率为85.2%~108%,相对标准偏差(RSD,n=6)为5.9%~10.2%。结论:该方法快速、灵敏、准确可靠,适用于食品包装材料中邻苯二甲酸酯类化合物的分析检测。     

FCLA-PPIX光触发式静态化学发光体系检测人血清白蛋白

本文对基于FCLA-PPIX触发式化学发光的人血清白蛋白检测体系进行了研究。该触发式化学发光体系利用光敏剂和活性氧探针FCLA组成了一个基本的光触发式化学发光检测体系,将待检测样品和体系充分混合后,通过脉冲光触发和延迟发光检测技术测定人血清白蛋白含量。脉冲触发式静态检测体系克服了其他化学发光体系操作复杂、试剂和样品消耗量大以及信号不稳定的缺点,灵敏度高,检测稳定。本文考察了该体系激发光强度、试剂浓度及温度等对人血清白蛋白检测结果的影响。在优化的实验条件下,测定人血清白蛋白的线性范围为2.5×10-9~6.4×10-7mol/L;方法的检出限为1.6×10-10mol/L;本法对5×10-8mol/L的标准样品平行重复测定11次相对标准偏差为4%。     

尼罗红荧光优化法快速检测微拟球藻细胞内油脂含量

【目的】利用尼罗红荧光染色法快速检测微拟球藻细胞内的油脂含量。【方法】系统性地调整激发波长与发射波长,确定最佳二甲基亚砜(DMSO)浓度、尼罗红终浓度、染色时间和细胞密度的范围,比较重量法和Triolein标准品的油脂含量分别与荧光强度之间的关系。【结果】分析得出了尼罗红荧光强度与微拟球藻细胞油脂含量的关系,确定优化后染色条件为:二甲基亚砜浓度为5%,尼罗红终浓度为1 mg/L,染色时间为6 min,且细胞密度为(0.5-3.0)×10~6 cells/m L的范围内,激发波长和发射波长分别为515 nm和570 nm。重量法和Triolein标准品的油脂含量分别与尼罗红染色荧光强度之间的关联性,表明尼罗红荧光染色方法可以用来快速准确地检测细胞内的油脂含量,且油脂含量与荧光强度之间正相关,相关系数R~2为0.997 3。尼罗红染色优化后油脂的检测下限达到2μg,大大减少了测定油脂含量所需细胞量。【结论】针对不同种属系统性地确认了荧光激发波长和发射波长,并优化验证得到了最佳尼罗红荧光染色条件,可以快速准确地检测微量微拟球藻细胞内的油脂含量,便于大规模筛选高产油突变藻株。     

人促卵泡激素体外生物学活性测定方法的建立

目的:建立人促卵泡激素(FSH)体外生物学活性检测方法,并对方法进行验证。方法:以人卵巢颗粒细胞KGN为基质细胞,用不同浓度的FSH与KGN细胞表面的FSH受体结合,刺激细胞产生不同浓度的孕酮,通过测定细胞培养上清中的孕酮含量来评价FSH的生物学活性,结果用国际标准品进行校正;对方法的专属性、线性与范围、回收率、精密度、耐用性进行验证;对市售FSH产品进行检测,并与传统动物体内活性检测结果进行比较。结果:KGN细胞对FSH有很好的剂量反应关系,方法专属性符合要求;线性范围为0.1~200 ng/m L,相关系数R2≥0.99;回收率为80%~120%;经3次重复测定,3批市售样品和3批自制样品的活性分别为(13.4±0.4)~(13.5±0.8)和(12.9±0.7)~(14.3±1.2)IU/μg,变异系数在15%之内;结果显示该方法有很好的耐用性。测定3批市售重组人FSH产品和1批尿源FSH国家标准品,其体外活性测定结果与传统动物体内活性测定结果一致。结论:建立并验证了一种利用KGN细胞体外测定FSH生物学活性的方法,有望代替传统的动物体内活性测定方法,可用于FSH的生物学活性评价和质量控制。     

高效毛细管电泳检测脑膜炎球菌结合疫苗原液中二甲亚砜残余量

目的采用毛细管区带电泳法(capillary zone electrophoresis,CZE)测定脑膜炎球菌结合疫苗原液中二甲亚砜(dimethyl sulfoxide,DMSO)的含量。方法以25 mmol/L四硼酸钠(p H 8.0)为背景电解质,以20 cm×50μm I.D.未涂层的毛细管为色谱分离柱,于200 nm波长检测脑膜炎球菌结合疫苗原液中二甲亚砜的残余量。结果DMSO在不同溶液中质量浓度为1~100 mg/L的线性范围内线性关系良好,R2≥0.998 9。加标回收率为98.07%~103.13%,变异系数为0.38%~2.57%。结论该方法简单、快速、经济、环保,不受混合物组分中其他物质的干扰,适用于快速检测结合疫苗中DMSO的残余量。     

醋酸纤维素膜为基础的葡萄糖生物传感器的研制

用共价法将酶固定在醋酸纤维素膜上,方法简便易行,制造的酶膜稳定,比活力高。同时采用该方法制备了葡萄糖氧化酶酶膜,与氧电极组装成测定葡萄糖的生物传感器,线性范围为50~800mg／dl,仪器工作的最适pH为6．0,最适温度为40℃。将该膜与过氧化氢电极组装得到的传感器具有以下特性：线性范围为10~200mg／dl,最适pH为6．0,测定结果与酶试制盒有良好相关性。     

Novel BOD optical fiber biosensor based on co-immobilized microorganisms in ormosils matrix

A biochemical oxygen demand (BOD) sensor has been developed, which is based on an immobilized mixed culture of microorganisms combined with a dissolved oxygen (DO) optical fiber. The sensing film for BOD measurement consists of an organically-modified silicate (ORMOSIL) film embedded with tri(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) perchlorate and three kinds of seawater microorganisms immobilized on a polyvinyl alcohol sol-gel matrix. The BOD measurements were carried out in the kinetic mode inside a light-proof cell and with constant temperature. Measurements were taken for 3 min followed by 10 min recovery time in 10 mg/L glucose/glutamate (GGA) BOD standard solution, and the range of determination was from 0.2 to 40 mg/L GGA. The effects of temperature, pH and sodium chloride concentration on the BOD sensing films were studied. BOD values estimated by this optical BOD sensing film correlate well with those determined by the conventional BOD5 method for seawater samples.     

Immobilized multi-species based biosensor for rapid biochemical oxygen demand measurement

To improve the practicability of rapid biochemical oxygen demand (BOD) method, we proposed a stable BOD sensor based on immobilizing multi-species BODseed for wastewater monitoring in the flow system. The activation time of the biofilm was greatly shortened for the biofilm prepared by BODseed in the organic-inorganic hybrid material. Some influence factors such as temperature, pH, and concentration of phosphate buffer solution (PBS) were investigated in detail in which high tolerance to environment was validated for the BOD sensor permitted a wide pH and PBS concentration ranges. The minimum detectable BOD was around 0.5 mg/l BOD under the optimized 1.0 mg/ml BODseed immobilized concentration. The as-prepared BOD sensor exhibited excellent stability and reproducibility for different samples. Furthermore, the as-prepared BOD biosensor displayed a notable advantage in indiscriminate biodegradation to different organic compounds and their mixture, similar to the character of conventional BOD(5) results. The results of the BOD sensor method are well agreed with those obtained from conventional BOD(5) method for wastewater samples. The proposed rapid BOD sensor method should be promising in practical application of wastewater monitoring.     

Development and characterization of a novel immobilized microbial membrane for rapid determination of biochemical oxygen demand load in industrial waste-waters

The rapid determination of waste-water quality of waste-water treatment plants in terms of pollutional strength, i.e. biochemical oxygen demand (BOD) is difficult or even impossible using the chemical determination method. The present study reports the determination of BOD within minutes using microbial BOD sensors, as compared to the 5-day determination using the conventional method. Multiple criteria establish the basis for the development of a BOD biosensor useful for rapid and reliable BOD estimation in industrial waste-waters. Of these, preparation of a suitable novel immobilized microbial membrane used in conjunction with an apt transducer is discussed. As a result, a microbial biosensor based on a formulated, synergistic, pre-tested microbial consortium has been developed for the measurement of BOD load of various industrial waste-waters. The sensor showed maximum response in terms of current difference, when a cell concentration of 2.25 x 10(10) CFU, harvested in their log phase of growth were utilized for microbial membrane construction. The sensor showed a stability of 180 days when the prepared membranes were stored at a temperature of 4 degrees C in 50 mM phosphate buffer of pH 6.8. The reusability of the immobilized membranes was up to 200 cycles without appreciable loss of their response characteristics. A linear relationship between the current change and a glucose-glutamic acid (GAA) concentration up to 60 mg l(-1) was observed (r=0.999). The lower detection limit was 1.0 mg l(-1) BOD. The sensor response was reproducible within +/-5% of the mean in a series of ten samples having 44 mg l(-1) BOD using standard a GGA solution. When used for the BOD estimation of industrial waste-waters, a relatively good agreement was found between the two methods, i.e. 5-day BOD and that measured by the developed microbial sensor.     

The use of co-immobilization of Trichosporon cutaneum and Bacillus licheniformis for a BOD sensor

The microorganisms Trichosporon cutaneum and Bacillus licheniformis were used to develop a microbial biochemical oxygen demand (BOD) sensor. It was found that T. cutaneum gave a greater response to glucose, whereas B. licheniformis gave a better response to glutamic acid. Hence, co-immobilized T. cutaneum and B. licheniformis were used to construct a glucose and glutamic acid sensor with improved sensitivity and dynamic range. A membrane loading of T. cutaneum at 1.1x10(8 )cells ml(-1) cm(-2) and B. licheniformis at 2.2x10(8) cells ml(-1) cm(-2) gave the optimum result: a linear range up to 40 mg BOD l(-1) with a sensitivity of 5.84 nA mg(-1) BOD l. The optimized BOD sensor showed operation stability for 58 intermittent batch measurements, with a standard deviation of 0.0362 and a variance of 0.131 nA. The response time of the co-immobilized microbial BOD sensor was within 5-10 min by steady-state measurement and the detection limit was 0.5 mg BOD l(-1). The BOD sensor was insensitive to pH in the range of pH 6.8-7.2.     

Investigation and evaluation of a method for determination of ethanol with the SIRE Biosensor P100, using alcohol dehydrogenase as recognition element

A new method for rapid determination of ethanol was developed, using alcohol dehydrogenase as recognition element for the SIRE (sensors based on injection of the recognition element) Biosensor, which is an amperometric biosensor. The method was simple, fast, accurate, specific and cost-effective. The recognition element solution used was stable at least for 24 h in room temperature, and at least one month when lyophilised. The optimal potential versus the silver wire electrode, the optimal pH of the buffer and the optimal temperature of the water bath was determined to be +950 mV, 8.1 and 308 K, respectively. The optimal concentrations of alcohol dehydrogenase, BSA and NAD(+) were determined to be 200 U/ml, 20 mg/ml and 15 mM, respectively. The total analysis time was between 50 s and 4 min per analysis, depending on the concentration range. The linear range was 0-12.5 mM. The detection limit was less than 0.1 mM. The repeatability (%R.S.D.) was 3-5% (n=10). The reproducibility was 5-8% (n=5). Methanol gave no signal at all, but higher alcohols, such as propanol, pentanol and hexanol, gave significant signals, decreasing with increasing length of the carbon chain. The price for one measurement was calculated to be 0.052 euro. The results from measurements with the biosensor were compared to those from an established analysis kit for ethanol. The results correlated well (R(2)=0.9874). The concentration of ethanol in different alcoholic beverages was investigated and correlated well with the concentrations given by the manufacturers.     

Designing an amperometric thick-film microbial BOD sensor

Thick film oxygen electrodes manufactured by screen print method have been used as a transducer for a biochemical oxygen demand (BOD) sensor. The kinetics of the immobilized yeast, Arxula adeninivorans (Arxula) has been studied. The apparent KM of immobilized Arxula (> 100 microM) is higher than free cells of Arxula (70 microM). The increase in KM caused by the effect of immobilization extends the linear range of the sensor. End-point measurement and quasi-kinetic measurement have been studied comparatively as measurement procedures with a good correlation. The Vmax for end-point measurement is 790.7 microM/s and that for quasi-kinetic measurement is 537.3 microM/s. The limit of detection is calculated 1.24 mg/l BOD. Using the quasi-kinetic measurement, instead of end-point measurements, the measuring time can be reduced from 5-30 min to 100 s. The sensor layer thickness or increase in the layer of covering gel can increase the KM that is accompanied with the extension of the linear range of the sensor. Nevertheless, increase in the layer of covering gel will not increase the saturation signal. Domestic wastewater was checked by the thick film BOD sensor and the results are satisfactory.     

固定化微生物在生物膜式生化需氧量传感器中的应用现状

生化需氧量(Biochemical oxygen demand,BOD)微生物传感器是一种快速检测水样中有机污染物含量的设备,固定化微生物是其核心部件之一,对其稳定性、响应时间、使用寿命及实际应用范围等性能有着重要影响。生物膜式BOD传感器较其他类型的BOD微生物传感器具有结构简单、灵敏度高、响应时间短等优点,受到广泛的研究和应用。本文主要针对固定化微生物在生物膜式BOD传感器中的应用情况,概述较典型的微生物固定化方式的原理、特点及应用;总结几类应用较多或具有较好前景的载体材料,并讨论载体特性与传感器性能之间的关系;综述微生物在该领域的应用现状;简要介绍生物膜式BOD传感器的实际应用及商业化现状,比较其与另外几种BOD微生物传感器的优缺点;分析生物膜式BOD传感器中固定化微生物现存的一些问题及其发展趋势。     

Degradation of 4-aminobenzenesulfonate by alginate encapsulated cells of Agrobacterium sp. PNS-1

Studies were carried out on 4-aminobenzenesulfonate (4-ABS) degradation by free and alginate entrapped cells of Agrobacterium sp. PNS-1. Degradation rate in batch reactors with free cells was marginally higher than Ca-encapsulated cells. Comparison of Ca2+ and Ba2+ as gelling agents showed that 4-ABS removal rate was significantly less with Ba-alginate entrapped cells. Specific degradation rates, using linear regression analysis and based on the initial biomass in the beads, varied from 49.7 mg/mg biomass/h to 92.0 mg/mg biomass/h for Ca-alginate encapsulated cells for different initial 4-ABS concentrations ranging from 200 to 800 mg/L. UV spectra of the aliquots drawn at different time intervals from batch reactors did not show accumulation of any intermediate during degradation. Ca-alginate immobilized cells could be repeatedly reused upto five cycles without any loss of activity. Studies with packed bed reactors, operated in a semi-continuous mode, showed that this could be used for 4-ABS degradation.     

Continuous determination of biochemical oxygen demand using microbial fuel cell type biosensor

A mediator-less microbial fuel cell (MFC) was used as a biochemical oxygen demand (BOD) sensor in an amperometric mode for real-time wastewater monitoring. At a hydraulic retention time of 1.05 h, BOD values of up to 100 mg/l were measured based on a linear relationship, while higher BOD values were measured using a lower feeding rate. About 60 min was required to reach a new steady-state current after the MFCs had been fed with different strength artificial wastewaters (Aws). The current generated from the MFCs fed with AW with a BOD of 100 mg/l was compared to determine the repeatability, and the difference was less than 10%. When the MFC was starved, the original current value was regained with a varying recovery time depending on the length of the starvation. During starvation, the MFC generated a background level current, probably due to an endogenous metabolism.     

Submersible microbial fuel cell sensor for monitoring microbial activity and BOD in groundwater: Focusing on impact of anodic biofilm on sensor applicability

A sensor, based on a submersible microbial fuel cell (SUMFC), was developed for in situ monitoring of microbial activity and biochemical oxygen demand (BOD) in groundwater. Presence or absence of a biofilm on the anode was a decisive factor for the applicability of the sensor. Fresh anode was required for application of the sensor for microbial activity measurement, while biofilm‐colonized anode was needed for utilizing the sensor for BOD content measurement. The current density of SUMFC sensor equipped with a biofilm‐colonized anode showed linear relationship with BOD content, to up to 250 mg/L (～233 ± 1 mA/m²), with a response time of <0.67 h. This sensor could, however, not measure microbial activity, as indicated by the indifferent current produced at varying active microorganisms concentration, which was expressed as microbial adenosine‐triphosphate (ATP) concentration. On the contrary, the current density (0.6 ± 0.1 to 12.4 ± 0.1 mA/m²) of the SUMFC sensor equipped with a fresh anode showed linear relationship, with active microorganism concentrations from 0 to 6.52 nmol‐ATP/L, while no correlation between the current and BOD was observed. It was found that temperature, pH, conductivity, and inorganic solid content were significantly affecting the sensitivity of the sensor. Lastly, the sensor was tested with real contaminated groundwater, where the microbial activity and BOD content could be detected in <3.1 h. The microbial activity and BOD concentration measured by SUMFC sensor fitted well with the one measured by the standard methods, with deviations ranging from 15% to 22% and 6% to 16%, respectively. The SUMFC sensor provides a new way for in situ and quantitative monitoring contaminants content and biological activity during bioremediation process in variety of anoxic aquifers. Biotechnol. Bioeng. 2011;108: 2339–2347. © 2011 Wiley Periodicals, Inc.