用于污染黄曲霉毒素花生分选的荧光信号研究

为在加工前将黄曲霉毒素超限的带衣花生米从原料中剔除,参照已有的色选系统,提出一种依据黄曲霉毒素含量超限带衣花生米的专属荧光信号进行逐粒分选的技术构想。采用Cary Eclipse荧光分光光度计测定100粒外观具有代表性的带衣花生米表面的紫外-荧光规律,通过与免疫亲和层析净化荧光光度法(GB/T18979-2003)检测结果对比,判定了黄曲霉毒素超限带衣花生米的荧光光谱特征;通过绘制450/490、460/490荧光强度比值的箱线图,评估了表面荧光法判断黄曲霉毒素超限带衣花生米的准确率;在搭建的荧光成像系统上,对黄曲霉毒素超限带衣花生米进行了荧光成像。检测发现,在365 nm波长激发下,黄曲霉毒素超限带衣花生米在420~460 nm处有荧光峰;以450/490荧光强度比值为依据剔除超限值带衣花生米的判断准确率为81%;a.u.40的带衣花生米可在图像中呈现亮蓝荧光光斑。表明表面荧光信号可作为带衣花生米在线、无损、逐粒分选的专属光学信号,用于黄曲霉毒素超限带衣花生米的剔除。     

Quantitative determination of diuron in ground and surface water by time-resolved fluoroimmunoassay: seasonal variations of diuron, carbofuran, and paraquat in an agricultural area

The aim of this research is to develop an ultrasensitive time-resolved fluorescence immunoassay (TR-FIA) for herbicide diuron in water samples. This method appears to be a promising approach, instead of conventional analytical techniques, in the screening procedure of organic pollutants because it is simple, rapid, and specific, and it does not require sample preconcentration or cleanup. Lanthanide chelate used as label allows to achieve sensitivity even 10 times higher than most of the other techniques. It has been applied to monitoring diuron contamination in specimens collected along a year in an agricultural area. The water specimens were collected monthly from lake, well, and irrigation ditch in the agricultural area south of Milan. Assay was performed using diuron-specific polyclonal antibody raised in sheep; as fluorescent marker, we used rabbit antisheep IgG conjugated with a chelating molecule complexed with Eu3+. The compound 4-(3-(3,4-dichloro-phenyl)-1-methyl-ureido)-butyric acid (CPD) was synthesized and conjugated with bovine serum albumin (BSA) to prepare a solid phase. Sensitivity achieved was 20 ng L-1 below the European Community limits. Paraquat (PQ) and carbofuran (CF) presence in the same samples has been also evaluated in a similar way, using immunoassays with time-resolved revelation systems. Diuron concentration shows a peak coinciding with a peak of carbofuran during summer periods. The peak of diuron was 65 pg/mL in June and 180 pg/mL in September in ditch and lake water samples, respectively; carbofuran concentration was higher than diuron in all samples: a carbofuran peak was revealed in September and October resulting in 87 ng/mL. Herbicide paraquat was not detectable in any assayed sample.     

Fluorescence-based method, exploiting lipofuscin, for real-time detection of central nervous system tissues on bovine carcasses

The removal of central nervous system (CNS) tissues as part of bovine spongiform encephalopathy (BSE) risk material is one of the highest priority tasks to avoid contamination of the human food chain with BSE. No currently available method enables the real-time detection of possible CNS tissue contamination on carcasses during slaughter. The fluorescent pigment lipofuscin is a heterogeneous, high-molecular weight material that has been shown to be enriched in high concentrations in neuronal tissues. In this study, lipofuscin fluorescence was investigated as a marker for real-time detection of CNS contamination. Front-faced fluorescence spectra of brain and spinal cord samples from 11 cattle gave identical, reproducible fluorescence signal patterns with high intensities. The specificity of these spectra was assessed by investigating 13 different non-CNS tissues enabling the differentiation of brain and spinal cord by signal intensity and structure of the spectra, respectively. Small quantities of bovine spinal cord were reliably detected in the presence of raw bovine skeletal muscle, fat, and vertebrae. The presented data are a fundamental basis for the development of a prototype device allowing real-time monitoring of CNS tissue contamination on bovine carcasses and meat cuts.     

Identification of an Assemblage of Indicator Organisms to Assess Timing and Source of Bacterial Contamination in Groundwater

Bacterial contamination of drinking water wells is a commonproblem in many rural areas. Some of this contamination may berelated to manure spreading or housing of livestock; another source is on-site septic systems. Current indicator organisms are able to detect the presence of fecal contamination, but where there may be more than one potential source of fecal material, the current indicators are unable to ascertain the origin. This laboratory investigation was undertaken to determine the longevity and reliability of a selected suite of indicator organisms. Total coliform, fecal coliform, fecal streptococci and Clostridium perfringens were monitoredin a simulated contaminated groundwater environment for 6 months. All four indicator organisms were present at the end of6 months. The number of fecal streptococi bacteria decreased most noticeably, allowing assessment of relative age of contamination. C. perfringens was found to be a reliableindicator of contamination from animal manure. Fecal material from 28 different animals and three septic systems were assessed for the presence of the indicator organisms. Totalcoliform, fecal coliform and fecal streptococci were present in the fecal material of all animals tested including reptiles.C. perfringens was detected in feces from all but two of the animals assessed. Using an assemblage of indicator organisms provides more information regarding source and timingof contamination than just testing for total coliform and fecalcoliform bacteria.     

Fluorescence Tracing of Diffuse Landfill Leachate Contamination in Rivers

Landfill leachates are composed of a complex mixture of degradation products which include a wide range of potentially fluorescent organic molecules and compounds. Here we investigate the use of fluorescence excitation–emission matrix (EEM) analysis in detecting diffuse landfill leachate contamination in rivers. Landfill leachates from three unlined landfill sites adjacent to our study river are characterised by intense fluorescence at excitation wavelength 220–230 nm, and emission wavelength 340–370 nm, which derives from fluorescent components of the xenobiotic organic matter fraction. Seven surface water sample sites on an adjacent polluted river system were analysed for fluorescence and water quality properties. The 220–230 nm excitation wavelength, 340–370 nm emission wavelength fluorescent centre was also detected in this river system at the sample locations downstream of the landfills, but not at upstream control sites, demonstrating its use as a tracer of landfill leachate contamination. Negative correlations are observed between this fluorescence centre and dissolved oxygen in the river water samples, demonstrating the water quality implications of leachate contamination at this study site. The fluorescence intensity at the 220–230 nm excitation wavelength, 340–370 nm emission wavelength fluorescent centre in landfill leachates is such that it remains detectable at dilutions of 10²–10³, and the fluorescence EEM technique is rapid and cost-effective for use by river managers and water quality regulators.     

Enterocyte and M-cell transport of native and heat-denatured bovine beta-lactoglobulin: significance of heat denaturation

The three-dimensional structure, digestibility, and immunological properties of bovine beta-lactoglobulin (beta-lg) are modified by heat treatments used in processing of liquid milk products. Because it is not known if such treatments also modify the intestinal transport properties of beta-lg, the transport of native and heat-denatured bovine beta-lg was investigated in experimental cell models using Caco-2 cells and M cells. Transport of beta-lg labeled with a fluorescent marker was followed with fluorometric measurements, electrophoretic analyses, and fluorescence microscopy. The data show that both cell types transported native beta-lg more efficiently than they did heat-denatured beta-lg. In addition, M cells transported native beta-lg more than Caco-2 cells. Transport of native and heat-denatured beta-lg was transcellular. The electrophoretic data also suggest that heat-denatured beta-lg may have degraded more than native beta-lg during the transport.     

以黄连素/羟丙基-β-环糊精为荧光探针检测金刚烷胺

为了建立一种金刚烷胺(AMD)检测的新方法,以盐酸黄连素/羟丙基-P-环糊精(BRH/HP-β-CD)为荧光探针,将HP-β-CD分别与BRH、AMD相互作用,测定AMD、BRH/HP-β-CD及两者结合的荧光强度变化规律,并验证分析该荧光分光光度法的适用性。结果表明,HP-β-CD与BRH生成1∶1的包合物并显著增强黄连素水溶液的荧光强度,当加入AMD后,BRH/HP-β-CD包合物的荧光强度逐渐减弱,据此成功建立一种以BRH/HP-β-CD为荧光探针的金刚烷胺检测新方法。当AMD溶液浓度在0.05~4.5 mg·L^-1范围内时与荧光猝灭值△F之间呈线性关系,相关系数为0.989 3,检测限(S/N=3)为0.03 mg·L^-1。溶液pH值和常见的药物赋形剂均不会对测量结果造成干扰,将本方法用于盐酸金刚烷胺药片的分析,其回收率在92%~101%范围内,相对标准偏差小于1%,说明该方法能够成功用于金刚烷胺的测定。本研究为以超分子包合物为荧光探针用于金刚烷胺类药物的检测提供了理论依据。     

Functional assembly of a microbial consortium with autofluorescent and mineralizing activity for the biodegradation of organophosphates

Organophosphorus pesticides (OPs) cause serious environmental problems, and bioremediation using bacterial enzymes may provide an efficient and economical method for their detoxification. Green fluorescent protein (GFP) is a stable and easily detectable marker in monitoring genetically engineered microorganisms (GEMs) in the environment. In our research, the methyl parathion hydrolase gene (mpd) and enhanced green fluorescent protein gene (egfp) were successfully coexpressed using pETDuet vector in E. coli BL21 (DE3). The coexpression of methyl parathion hydrolase (MPH) and enhanced green fluorescent protein (EGFP) were confirmed by determining MPH activity and fluorescence intensity. The recombinant protein MPH showed high enzymatic degradative activity of several widely used OP residues on vegetables determined by GC analysis. Subsequently, a dual-species consortium comprising engineered E. coli and a natural p-nitrophenol (PNP) degrader Ochrobactrum sp. strain LL-1 for complete mineralization of dimethyl OPs was studied. It could completely mineralize methyl parathion (MP) via MP through PNP and hydroquinone and eventually through the TCA cycle as determined by HPLC analysis. The accumulation of PNP in suspended culture was prevented. The consortium could be further utilized for complete mineralization of PNP-substituted OPs in a laboratory-scale bioreactor and easily monitored by fluorescence of EGFP for its activity and fate.     

Evaluation of microbial indicators for the determination of bacterial groundwater contamination sources

Several different Microbial source tracking methods (MSTs) can be used to distinguish human from animal fecal contamination in water; In this study, experiments were carried out to test the effectiveness and reliability of three bacteria based approaches (the fecal coliforms to fecal streptococci ratio, antibiotic-resistant Clostridium perfringens, and human bifidobacteria) in a simulated groundwater micro-environment. The methods were evaluated in three phases: initially, the specificity of each indicator was validated on groundwater samples affected by known pollution source; then the variation of performance with time of each method was determined, and finally, the die-off coefficients for pure species of Clostridium perfringens and Bifidobacterium adolescentis were measured. The results indicate that only the determination of human bifidobacteria concentration can be considered reliable in distinguishing human from animal pollution in groundwater at the conditions applied. Nevertheless, human bifidobacteria were detectable only for two weeks after the contamination event. This study also shows that antibiotic resistant Clostridium perfringens detected using the Shahidi-Ferguson medium is not source specific, whereas it confirms that this species can be useful for timing general fecal contamination events.     

Simple Measurement of 4,4’-bis(2-sulfostyryl)-biphenyl in River Water by Fluorescence Analysis and Its Application as an Indicator of Domestic Wastewater Contamination

A characteristic peak of fluorescent whitening agents (FWAs) was detected by fluorescence excitation spectrum (FES) measurement of river water samples. The main causative chemical was 4,4’-bis(2-sulfostyryl)-biphenyl (DSBP), which is commonly added to household detergents in Japan. As the fluorescence of DSBP overlaps with that of fulvic-like organic matter in the spectral fluorescent signatures, DSBP concentration was determined by the newly proposed calculation method, which uses fluorescence intensity at three excitation wavelengths of 320, 345 and 360 nm at emission wavelength of 430 nm for baseline correction. The concentration of DSBP calculated using this method showed strong correlation (correlation coefficient: r = 0.992) with that obtained by high-performance liquid chromatography analysis. The concentrations of DSBP detected in river water samples were 0.28 to 1.84 μg l^?1, with high concentrations observed at the stations with relatively high flow rates of upstream sources of treated domestic wastewater and untreated gray water (domestic wastewater excluding flush toilet wastewater). It was proved that the concentration of DSBP in river water is useful for giving rough estimation of the magnitude of domestic wastewater contamination in river water.     

Coliform detection from river waters: Comparison between MPN and MF techniques

This paper compares three different methods used to determine fecal pollution in river water. Two of the methods are based on direct estimation using standard aerobic incubation of membrane filters (S-MF) and anaerobic incubation of membrane filters (A-MF); the third is based on statistical evaluation by the most probable number method (MPN). It was seen from regression analysis of the results obtained that the MPN, S-MF and A-MF techniques are equally valid as regards the enumeration of fecal coliforms, whereas the S-MF and A-MF techniques are more sensitive than the MPN procedure for the enumeration of total coliforms.     

接种物耐酸驯化对菌糠厌氧干发酵产气的影响

为了提高厌氧干发酵体系运行稳定性,使中间产物酸及时转化为甲烷,避免出现酸抑制现象,该研究采用逐步提高乙酸浓度降低pH值的方式对接种物进行驯化,得到了在乙酸浓度10200mg/kg、pH值6.0条件下仍能快速产气的耐酸接种物,并以易水解酸化的菌糠为原料进行了厌氧干发酵试验。结果表明,驯化过程中产甲烷古菌类群的多样性下降,乙酸营养型甲烷八叠球菌丰度大幅提高,乙酸转化率和甲烷浓度逐渐提高;耐酸接种物的脱氢酶活性下降,辅酶F420和纤维素酶活性升高;添加耐酸接种物可以加快菌糠厌氧干发酵启动速度,避免酸抑制现象的发生;接种物含有75%耐酸接种物的试验组甲烷产量提高了56.1%。该研究成果能够为有效解决厌氧干发酵过程酸抑制现象提供一定的理论指导。     

基于液滴微流控的壳寡糖植物免疫诱导剂荧光检测

为了建立基于液滴微流控芯片平台的植物免疫诱导剂的荧光信号检测技术,该文设计制作了具有液滴生成结构的芯片,制备包裹烟草(BY-2)细胞的液滴,并对包裹了细胞的液滴数目进行统计分析。将包裹细胞的液滴孵育一氧化氮探针后使用质量浓度为50μg/m L壳寡糖处理,在荧光显微镜下观测其产生的荧光,利用荧光酶标仪对比采用96孔板和液滴承载的细胞的荧光变化趋势。结果显示,水相流速为100μL/h,油相流速为300μL/h时,微流控芯片产生的液滴尺寸适合包裹细胞,其中液滴包裹单细胞的比例达22.9%。经壳寡糖处理后的细胞在生成的液滴中产生了明显的荧光,且用96孔板和液滴承载的细胞的荧光变化趋势一致。研究结果为开发基于液滴微流控技术的植物免疫诱导剂的高通量筛选平台提供参考。     

Limitations of acid digestion techniques for the determination of fluoride in plant material

Abstract

Procedures for determination of fluoride (F) in plant material employing acid digestion and solution analysis by ion specific electrode (ISE) were compared to alkali fusion using a range of plant materials. The efficiency of the methods were assessed using standard reference plant material (SRM) not previously available for plant F analysis. All acid digestion procedures tested failed to obtain the certified value for F in the SRM. This was due to failure of the acids to liberate F bound strongly within silicate minerals found in the plant materials. Acid digestion is therefore not recommended for determination of total F, but could be used to determine labile F in plant materials. During investigation of the acid digestion procedures, it was also found that F concentrations determined in solution using the ISE are sensitive to solution pH, even at solution pH values where complexation of F with hydrogen ions (H⁺) can be discounted. It is therefore recommended that both ionic strength and pH of sample and standard solutions be matched when determining F concentrations in solution using the F‐ISE.     

Fluorescence of raw cane sugars evaluated by chemometrics

In a fluorescence study of raw cane sugar samples, two-way and three-way chemometric methods have been used to extract information about the individual fluorophores in the sugar from fluorescence excitation-emission landscapes. A sample set of 47 raw sugar samples representing a varied selection was analyzed, and three individual fluorophores with (275, 350) nm, (340, 420) nm, and (390, 460) nm as their approximate excitation and emission maxima were found. The spectral profiles of the fluorophores were estimated with the three-way decomposition model PARAFAC. Two-way principal component analysis (PCA) of unfolded fluorescence landscapes confirmed the PARAFAC results and showed patterns of samples related to time of storage. Partial least squares (PLS) calibration models of color at 420 nm had a high model error due to the very high color range of the raw sugars, but variable selection performed on the fluorescence data revealed that all three fluorophores were correlated to color. The (275, 350) nm fluorophore is considered as a color precursor to the color developed on storage and the (340, 420) nm and (390, 460) nm fluorophores show colorant polymer characteristics.     

Fluorescent properties of low-molecular-weight fractions from chernozem humic acids

The polyacrylamide gel electrophoresis of chernozem humic acids (HAs) followed by ultraviolet detection (λ = 312 nm) has revealed a new highly fluorescent fraction that has the highest electrophoretic mobility and the lowest nominal molecular weight (NMW). The preparative isolation of the fraction has been performed using the multiple microfiltration of the same HA sample in a 7 M carbamide solution on a membrane with a nominal pore size of 5 kDa. Thirty ultrafiltrates with NMW < 5 kDa and different fluorescence maximums in the region of 475–505 nm have been prepared, as well as a nonfluorescent concentrate with NMW > 5 kDa. Fluorescence maximums at and below 490 nm have been noted only in the first four ultrafiltrates. All the ultrafiltrates have been combined into the fraction with NMW < 5 kDa, which has been successively passed through membranes of 3 and 1 kDa. Solutions of subfractions F 3–5 kDa, F 1–3 kDa, and F < 1 kDa with fluorescence maximums at 505, 488, and 465 nm, respectively, have been prepared. The F < 1 kDa subfraction with the lowest NMW had the highest fluorescence intensity. The distribution of the fluorescence maximums in the ultrafiltrates has indicated the presence of at least two groups of fluorophores and has confirmed the supramolecular organization of the extracted soil HAs.     

Decline in leaf growth under salt stress is due to an inhibition of H+‐pumping activity and increase in apoplastic pH of maize leaves

In this study, salt‐induced changes in the growth rate of maize (Zea mays L.) were investigated during the first phase of salt stress. Leaf growth was reduced in the presence of 100 mM NaCl, and effects were more pronounced for the salt‐sensitive cv. Pioneer 3906 in comparison to the hybrid SR03. While hydrolytic activity of plasma membrane remained unaffected, H⁺‐pumping activity was reduced by 47% in Pioneer 3906, but was unchanged in SR03. Changes in apoplastic pH were detected by ratiometric fluorescence microscopy using the fluorescent dye fluorescein isothiocyanate‐dextran (50 mM). Pioneer 3906 responded with an increase of 0.2 pH units in contrast to SR03 for which no apoplastic alkalization was found. With respect to the hypothesis that the apoplastic pH is influenced by salinity, it is suggested that salt resistance is partly achieved due to efficient H⁺‐ATPase proton pumping, which results in cell‐wall acidification and loosening.     

Inactive fluorescently labeled xylanase as a novel probe for microscopic analysis of arabinoxylan containing cereal cell walls

A new technique to visualize cereal cell walls by fluorescence microscopy was developed. The novel staining technique is based on an inactive fluorescently labeled xylanase binding to arabinoxylan (AX), an important polysaccharide in grain cell walls in terms of the technological and physiological functionalities of grain. The xylanase probe could stain AX in the seed coat, nucellar epidermis, aleurone layer, and starchy endosperm, but not the highly substituted AX of the pericarp layer. The advantage of this new staining technique over the existing immunolabeling techniques is that the staining procedure is clearly faster and less laborious, and uses a smaller probe that can easily be produced by marking a well characterized enzyme with a fluorescent label. In the future, the here proposed technology can be used to develop probes having specificity also for cell wall components other than AX and thus to study plant cell walls further through fluorescence microscopy.     

Synchronous fluorescence as a rapid method for the simultaneous determination of folic acid and riboflavin in nutritional beverages

A rapid synchronous spectrofluorimetric method was first developed for the simultaneous determination of folic acid and riboflavin in nutrimental beverages. Folic acid could be detected by using H(2)O(2) plus Cu(II) as oxidation system to produce pterine-6-carboxylic acid, which had strong fluorescence in aqueous solution, and riboflavin itself was obviously fluorescent. Various operational parameters were thoroughly discussed in terms of their effects on the fluorescence signals, including instrumental parameters, concentration of the oxidation system, and pH. Under optimum conditions, the calibration curves were linear in the ranges of 100-250 μg/L for folic acid and 1-250 μg/L for riboflavin, and the detection limits were 2.0 and 0.014 μg/L, respectively. In addition, this method was applied to the determination of folic acid and riboflavin in nutrimental beverages with satisfactory results.