发光细菌传感器在纳米氧化物紫外屏蔽性能评估分析中的应用研究

本文发现发光细菌在紫外光照射下能够被杀死，其荧光强度相应地发生降低，而在纳米氧化物的保护下，细菌荧光强度的降低得到了抑制，因此发光细菌可以被用来分析和评价纳米氧化物的紫外屏蔽性能。青海弧菌Q67是发光细菌的一种，本文研究了不同浓度及不同种类的纳米氧化物对本发光细菌在分别受到UVA、UVB、UVC紫外光照射下的发光强度的影响，根据细菌发光强度降低的相对值建立了一种分析和评价纳米氧化物的紫外屏蔽性能的方法。该方法可以对纳米氧化物在UVA、UVB、UVC区的紫外屏蔽性能进行评价，同时也为化妆品等行业提供了一种对防晒剂紫外屏蔽性能评估的有效方法。     

Bioactivity screening by chromatography-bioluminescence coupling

Summary Because luminescent microorganisms change their light emission in the presence of bioactive substances, they provide an effective way of monitoring toxicity. Here we report the direct coupling of bioluminescence to chromatography which is capable of detecting single toxic components in complex mixtures. Two approaches were investigated, utilizing thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The TLC-bioluminescence imaging technique was developed into a versatile and rugged method and proved to be superior to the HPLC-based design. Employing genetically engineered luminescent bacteria gives customized selectivity of bioactivity detection. The combination of analytical separation technology with the biospecific sensing ability of living cells provides a novel screening tool for targeting bioactive components in complex samples.     

Toxicity Assessment of Cyanide and Tetramethylene Disulfotetramine （Tetramine） Using Luminescent Bacteria Vibrio-qinghaiensis and PbO= Electrochemical Sensor

The toxicities of cyanide and tetramethylene disulfotetramine （tetramine） were evaluated by two methods of luminescent bacteria and PbO2 electrochemical sensor. Vibrio-qinghaiensis, a kind of luminescent bacteria, can produce bioluminescence and the bioluminescence was decreased with the addition of toxicants. The toxicities of cyanide and tetrarnine were expressed as 10 min-EC50 value, which was the concentration of chemical that reduces the light output by 50% after contact for 10 min. Nano PbO2 modified electrode, a rapid toxicity determination method was also described in this work. By the PbO2 modified electrode, the current responses of Escherichia coli （E. coli） were changed with the addition of toxicants. The value of 10 min-EC50 was also provided with the PbO2 electrochemical sensor. Compared with the 10 min-EC50 and detection limits （38.38 and 0.60 μg/mL for cyanide, 0.24 and 0.02 μg/mL for tetramine） with luminescent bacteria, the PbO2 sensor provided a simple and convenient method with lower 10 min-EC50 and detection limits （26.37 and 0.52 μg/mL for cyanide, 0.21 and 0.01 μg/mL for tetramine） and fast response time.     

Theoretical Study of Dinoflagellate Bioluminescence

Dinoflagellates are the most ubiquitous luminescent protists in the marine environment and have drawn much attention for their crucial roles in marine ecosystems. Dinoflagellate bioluminescence has been applied in underwater target detection. The luminescent system of dinoflagellates is a typical luciferin–luciferase one. However, the excited‐state oxyluciferin is not the light emitter of dinoflagellate bioluminescence as in most luciferin–luciferase bioluminescent organisms. The oxyluciferin of bioluminescent dinoflagellates is not fluorescent, whereas its luciferin emits bright fluorescence with similar wavelength of the bioluminescence. What is the light emitter of dinoflagellate bioluminescence and what is the chemical process of the light emission like? These questions have not been answered by the limited experimental evidence so far. In this study, for the first time, the density functional calculation is employed to investigate the geometries and properties of luciferin and oxyluciferin of bioluminescent dinoflagellate. The calculated results agree with the experimental observations and indicate the luciferin or its analogue, rather than oxyluciferin, is the bioluminophore of dinoflagellate bioluminescence. A rough mechanism involving energy transfer is proposed for dinoflagellate bioluminescence.     

BIOLUMINESCENCE: BIOCHEMICAL AND PHYSIOLOGICAL ADVANCES

Bioluminescent organisms are to be found among the bacteria, fungi, unicellular algae, and most of the major animal phyla, some of which contain hundreds of luminescent species. Early biochemical studies reflected this taxonomic diversity and often a primary focus of attention was the uniqueness of each newly isolated system. Relatively few in vitro cross-reactions were discovered among unrelated species and five of the first six luciferins to be characterized proved to be structurally unrelated compounds. The functions of bioluminescence in many organisms were unknown and, while several theories arose, these theories seldom addressed the part that light production might play in the ecology of an organism or a population. Major advances on each of these fronts have been made during the past decade. Biochemical research has centered on the chemical mechanism(s) of luminescence and a single type of chemical species, a dioxetanone, has emerged as a common intermediate in several (but not all) bioluminescence and chemiluminescence systems. Likewise, in the last 10 years, numerous interphyletic cross-reactions have been discovered and the ecological functions of bioluminescence for a large number of species have been established. Intensive studies are continuing on reaction mechanisms, especially those involving the bioluminescent bacteria, and much remains to be learned about the protein biochemistry of all luminescence systems. Amino acid sequence determinations and X-ray crystallographic studies have been initiated in several laboratories and, in others, attempts are being made to clone the genes that code for bioluminescence proteins. Sensitized bioluminescence has been implicated in representatives of both prokaryotic and eukaryotic organisms. Low level biological chemiluminescence has been investigated in a variety of systems including liver microsomes and phagocytic leucocytes using sensitive photon counting devices. Perhaps the area of greatest growth, however, has been the application of bioluminescence and chemiluminescence techniques as tools of clinical research. The need for safe, sensitive and specific assay methods has, for example, stimulated the development of immobilized luciferases and luciferase-linked enzyme systems. In addition, luminescence immunoassay has emerged as a reasonable alternative to the commonly used, but more troublesome, method of radioimmunoassay. This trend toward applications development has shifted the emphasis of some of the university laboratories and in general has improved the lines of communication between basic and applied research groups working in the area of bioluminescence.     

Bacterial bioluminescence, bioelectromagnetics and function

The biological functions of light emission in bacterial bioluminescence are not always obvious, especially if the bacteria are in a free-living mode. Experimental evidence suggests that light emission confers benefit to the bacteria themselves such as through photoreactivation and involves as much as 20% of cell energy metabolism. A theoretical model shows if the effect is mediated solely by light then cells should be luminescent at both high and low cell densities, therefore raising doubt over the photoreactivation hypothesis and suggesting that another cofactor is involved. It has been postulated that bioelectromagnetics may be involved in biological processes and be involved with coordinated activity in quorate cells. The cell densities associated with autoinduction coincide with a large change in coupling efficiency in the millimeter and submillimeter spectral region. In this paper it is suggested that one function of bioluminescence is as a pump, involving millimeter and submillimeter wave coupling that is of benefit to the quorum. This may be related to the observation that millimeter wave radiation exposure has been reported to induce changes in DNA conformation and possibly gene expression. Agents that change DNA conformation in bioluminescent bacteria can cause increases in light emission. This work may have implications for electromagnetic fields as quorum-quenching agents.     

Chemical Analysis of the Luminous Slime Secreted by the Marine Worm Chaetopterus (Annelida,Polychaeta)

The marine annelid Chaetopterus variopedatus produces bioluminescence by an unknown and potentially novel mechanism. We have advanced the study of this fascinating phenomenon, which has not been investigated for nearly 60 years after initial studies were first reported for this species. Here, we show that the luminous slime produced by the worm exhibits blue fluorescence that matches the bioluminescence emission. This result suggests that the oxyluciferin emitter is present. However, while the blue fluorescence decays over time green fluorescence is increasingly revealed that is likely associated with products of the luminescence reaction. LC/MS and fluorescence analysis of harvested luminescent material revealed riboflavin as the major green fluorescent component. Riboflavin is usually associated with the mechanism of light production in bacteria, yet luminous bacteria were not found in the worm mucus, and accordingly were not reported to be directly responsible for the light emission, which is under nervous control in the worm. We therefore propose a hypothesis in which riboflavin or a structurally related derivative serves as the emitter in the worm's light producing reaction.     

Analytik bakterientoxischer Effekte mit Hilfe genetisch konstruierter Leuchtbakterien

Zusammenfassung Leuchtbakterienteste stellen eine elegante Methode zur schnellen, reproduzierbaren Bestimmung bakterientoxischer Effekte dar. Einer allgemeinen Anwendung steht allerdings entgegen, daß die Luminescenzeigenschaften auf relativ wenige Bakterienfamilien mit zum Teil extremen Standortansprüchen beschränkt sind. Die Übertragbarkeit der Meßergebnisse auf interessierende Anwendungen ist deshalb in vielen Fällen nicht gegeben. In der vorliegenden Arbeit wird am Beispiel eines gentechnologisch konstruierten luminescenten Abwasserstammes gezeigt, daß durch Verwendung von anwendungsspezifischen Testorganismen das Anwendungsspektrum von Leuchtbakterientesten erweitert werden kann.

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Analysis of microbial toxicity using gentechnologically constructed luminescent bacteria

Summary Luminescent bacteria bioassays represent an elegant method for fast and reproducible determination of microbial toxicity. A general application of this method is however prevented by the fact that the capability for bioluminescence is restricted to a relatively small group of bacteria with extreme environmental demands. Therefore it seems questionable whether results obtained with those organisms can be used for all interesting applications. It is shown in this paper, that the field of applications for bacterial luminescent assays can be widened using gentechnologically constructed luminescent bacteria.

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Herrn Prof. Dr. H. Hartkamp zum 60. Geburtstag gewidmet     

Efficiency of bioelectrocatalytic oxidation of ethanol by whole cells and membrane fractions of <Emphasis Type="Italic">Gluconobacter Oxydans</Emphasis> bacteria in the presence of mediators of ferrocene series

Bioelectrocatalytic oxidation of ethanol by whole cells and membrane fraction of Gluconobacter oxydans bacteria is studied on modified graphite-paste electrodes in mediator biosensors. Ferrocene derivatives are used as electron transport mediators for effective coupling of enzymatic and electrochemical processes on graphite electrodes. Electrochemical kinetics of the processes are studied; the obtained data are interpreted in the terms of the mechanism of two-substrate enzymatic reaction. It is shown that mediators of ferrocene series are promising compounds for development of mediator biosensors based both on whole cells of Gluconobacter oxydans bacteria and on membrane fractions of these bacteria. Bioelectrocatalytic processes of ethanol oxidation on graphite paste electrodes occur more efficiently when the bacterial membrane fraction is used as a biocatalyst and ferrocenemonocarboxylic acid is used as a mediator.     

Effect of halogenated fluorescent compounds on bioluminescent reactions

The paper investigates an application of luminescent bioassays to monitor the toxicity of organic halides. Effects of xanthene dyes (fluorescein, eosin Y, and erythrosin B), used as model compounds, on bioluminescent reactions of firefly Luciola mingrelica, marine bacteria Photobacterium leiognathi, and hydroid polyp Obelia longissima were studied. Dependence of bioluminescence quenching constants on the atomic weight of halogen substituents in dye molecules was demonstrated. Bacterial bioluminescence was shown to be most sensitive to heavy halogen atoms involved in molecular structure; hence, it is suitable for construction of sensors to monitor toxicity of halogenated compounds. Mechanisms of bioluminescence quenching—energy transfer processes, collisional interactions, and enzyme–dye binding—were considered. Changes of bioluminescence (BL) spectra in the presence of the dyes were analyzed. Interactions of the dyes with enzymes were studied using fluorescence characteristics of the dyes in steady-state and time-resolved experiments. The dependences of fluorescence anisotropy of enzyme-bound dyes, the average fluorescence lifetime, and the number of exponential components in fluorescence decay on the atomic weight of halogen substituents were demonstrated. The results are discussed in terms of “dark effect of heavy halogen atom” in the process of enzyme–dye binding; hydrophobic interactions were assumed to be responsible for the effect.     

Current Status of Research on Fungal Bioluminescence: Biochemistry and Prospects for Ecotoxicological Application

Over the last half decade the study of fungal bioluminescence has regained momentum since the involvement of enzymes has been confirmed after over 40 years of controversy. Since then our laboratory has worked mainly on further characterizing the substances involved in fungal bioluminescence and its mechanism, as well as the development of an ecotoxicological bioluminescent assay with fungi. Previously, we proved the involvement of a NAD(P)H‐dependent reductase and a membrane‐bound luciferase in a two‐step reaction triggered by addition of NAD(P)H and molecular oxygen to generate green light. The fungal luminescent system is also likely shared across all lineages of bioluminescent fungi based on cross‐reaction studies. Moreover, fungal bioluminescence is inhibited by the mycelium exposure to toxicants. The change in light emission under optimal and controlled conditions has been used as endpoint in the development of toxicological bioassays. These bioassays are useful to better understand the interactions and effects of hazardous compounds to terrestrial species and to assist the assessment of soil contaminations by biotic or abiotic sources. In this work, we present an overview of the current state of the study of fungal luminescence and the application of bioluminescent fungi as versatile tool in ecotoxicology.     

基于金属有机骨架材料的生物传感器在食源性致病菌或毒素检测中的应用

食源性致病菌或其产生的毒素污染的食物会给人体健康带来严重威胁,并造成巨大的经济损失。近年来,金属有机骨架材料(MOFs)作为一种新型的多孔晶体材料,因其具有大的表面积、高的孔隙率等特点,受到人们的广泛关注。将MOFs与生物传感器结合用于食源性致病菌或毒素的检测引起了研究者的兴趣。基于此,本文介绍了MOFs用于生物传感器的优势,概述了MOFs在不同的电化学和光学生物传感器的应用,综述了基于MOFs的生物传感器在致病菌或毒素的研究进展,讨论了基于MOFs的生物传感器在致病菌或毒素所面临的挑战和展望。     

超氧负离子在生物发光中的作用

活性氧物种和超氧负离子是生物体内的重要物质,本文通过超氧负离子在生物发光反应中的作用,针对几种不同的典型生物发光体系,综述了超氧负离子参与发光反应的相关理论和实验研究进展.     

Electrochemical Biosensors for Detection of Biological Warfare Agents

This review discusses current development in electrochemical biosensors for detection of biological warfare agents. This could include bacteria, viruses and toxins that are aerosoled deliberately in air, food or water to spread terrorism and cause disease or death to humans, animals or plants. The rapid and unequivocal detection and identification of biological warfare agents is a major challenge for any government including military, health and other government agents. Reliable, specific characterization and identification of the microorganism from sampling location, either air, water, soil or others is required. This review will survey different types of electrochemical biosensors has been developed based on the following: i) Immunosensors ii) PCR (DNA base Sensor) iii) Bacteria or whole cell sensor and iv) Enzyme sensor. This article gives an overview of electrochemical biosensor for detection of biological warfare agents. Electrochemical biosensors have the advantages of sensitivity, selectivity, to operate in turbid media, and amenable to miniaturization. Recent developments in immunofiltration, flow injection, and flow‐through electrochemical biosensors for bacteria, viruses, and toxin detection are reviewed. The current research and development in biosensors for biological warfare agents detection is of interest to the public as well as to the defense is also discussed.     

Recent development in chitosan nanocomposites for surface‐based biosensor applications

Recent years have witnessed ever expanding use of biosensors in the fields of environmental monitoring, homeland security, pharmaceutical, food and bioprocessing, and agricultural industries. To produce effective and reliable biosensors, good quality immobilization of biological recognition elements is critical. Chitosan and its nanocomposites emerge as an excellent immobilization matrix on biosensor surface. As a natural polysaccharide, chitosan has many useful characteristics, such as high permeability and mechanical strength, biocompatibility and non‐toxicity, availability, and low cost. Due to the presence of amino and hydroxyl groups on chitosan, chitosan can easily crosslink with a variety of nanomaterials. This investigation of chitosan nanocomposite‐based biosensors presents recent development and innovations in the preparation of chitosan nanocomposites in coordination with biosensors for various bio‐detection applications, including chitosan nanocomposites formed with carbon nanomaterials, various inorganic and biological complexes. These chitosan nanocomposite based biosensors have demonstrated good sensitivity selectivity and stability for the detection of different types of targets ranging from glucose, proteins, DNAs, small biomolecules to bacteria. It is in our hope that this review will offer guidance for the development of novel biosensors and open up opportunities in the field of biosensor research.     

INDUCTION OF BIOLUMINESCENCE IN THE MARINE FISH, Porichthys, BY Vargula (CRUSTACEAN) LUCIFERIN. EVIDENCE FOR de novo SYNTHESIS OR RECYCLING OF LUCIFERIN

Abstract— The marine fish, Porichthys notatus , emits light by a classical luciferin-luciferase reaction whose components are similar, if not identical, to those found in the luminescent crustacean, Vargula. Porichthys is divided geographically into a southern luminescent and a northern nonluminescent population. Specimens of nonluminescent Porichthys can be induced to become luminescent by injection or ingestion of Vargula luciferin. After feeding a known quantity of Vargula luciferin, light emitted by Porichthys was monitored for a 2-yr period. Summation of light produced during each bioluminescence episode demonstrated that the total quanta emitted over 2 yr exceeded the theoretical yield from the administered luciferin. These results indicate that the administered luciferin either recycles or induces de novo synthesis of additional luciferin in Porichthys .     

Larval and adult emission spectra of bioluminescence in three European firefly species

We studied the spectral characteristics of the larvae of three sympatric Belgian species of fireflies, Lampyris noctiluca, Phosphaenus hemipterus and Lamprohiza splendidula. An in vivo spectral study was performed to compare bioluminescence spectra. The emission spectrum of a laboratory reared female L. noctiluca was recorded by a different, more exact method. The mean peak wavelength (lambdamax = 546 nm) and shapes of the unimodal emission spectra are visually similar for the larvae of all three species. The emission spectrum of the adult female L. noctiluca peaked in the same range as the larval bioluminescence between 546 and 551 nm. The bandwidth at half-maximum intensity was slightly greater for larval L. noctiluca (77 +/- 4 nm) compared with P. hemipterus (70 +/- 10 nm). The bandwidth of larval L. splendidula (77 +/- 8 nm) was not different compared with the other larvae, whereas the females' bandwidth was somewhat narrower (68 nm). The ecological significance of the color of bioluminescence and conservancy of green emission in larval fireflies and other luminescent beetle larvae is discussed.     

Two bioluminescent diptera: the North American Orfelia fultoni and the Australian Arachnocampa flava. Similar niche, different bioluminescence systems

Orfelia fultoni is the only bioluminescent dipteran (Mycetophilidae) found in North America. Its larvae live on stream banks in the Appalachian Mountains. Like their Australasian relative Arachnocampa spp., they build sticky webs to which their bioluminescence attracts flying prey. They bear two translucent lanterns at the extremities of the body, histologically distinct from the single caudal lantern of Arachnocampa spp., and emit the bluest bioluminescence recorded for luminescent insects (lambda(max) = 460 nm versus 484 nm from Arachnocampa). A preliminary characterization of these two bioluminescent systems indicates that they are markedly different. In Orfelia a luciferin-luciferase reaction was demonstrated by mixing a hot extract prepared with dithiothreitol (DTT) under argon with a crude cold extract. Bioluminescence is not activated by adenosine triphosphate (ATP) but is strongly stimulated by DTT and ascorbic acid. Using gel filtration, we isolated a luciferase fraction of approximately 140 kDa and an additional high molecular weight fraction (possibly a luciferin-binding protein) that activated bioluminescence in the presence of luciferase and DTT. The Arachnocampa luciferin-luciferase system involves a 36 kDa luciferase and a luciferin soluble in ethyl acetate under acidic conditions; the bioluminescence is activated by ATP but not by DTT. The present findings indicate that the bioluminescence of O. fultoni constitutes a novel bioluminescent system unrelated to that of Arachnocampa.     

Reporter gene bioassays in environmental analysis

In parallel to the continuous development of increasingly more sophisticated physical and chemical analytical technologies for the detection of environmental pollutants, there is a progressively more urgent need also for bioassays which report not only on the presence of a chemical but also on its bioavailability and its biological effects. As a partial fulfillment of that need, there has been a rapid development of biosensors based on genetically engineered bacteria. Such microorganisms typically combine a promoter-operator, which acts as the sensing element, with reporter gene(s) coding for easily detectable proteins. These sensors have the ability to detect global parameters such as stress conditions, toxicity or DNA-damaging agents as well as specific organic and inorganic compounds. The systems described in this review, designed to detect different groups of target chemicals, vary greatly in their detection limits, specificity, response times and more. These variations reflect on their potential applicability which, for most of the constructs described, is presently rather limited. Nevertheless, present trends promise that additional improvements will make microbial biosensors an important tool for future environmental analysis.