Luminescent Bioassay Research Articles

Preparation and Time-Resolved Luminescence Bioassay Application of Multicolor Luminescent Lanthanide Nanoparticles

Because highly luminescent lanthanide compounds are limited to Eu(3+) and Tb(3+) compounds with red (Eu, approximately 615 nm) and green (Tb, approximately 545 nm) emission colors, the development and application of time-resolved luminescence bioassay technique using lanthanide-based multicolor luminescent biolabels have rarely been investigated. In this work, a series of lanthanide complexes covalently bound silica nanoparticles with an excitation maximum wavelength at 335 nm and red, orange, yellow and green emission colors has been prepared by co-binding different molar ratios of luminescent Eu(3+)-Tb(3+) complexes with a ligand N,N,N(1),N(1)-(4'-phenyl-2,2':6',2''-terpyridine-6,6''-diyl)bis(methylenenitrilo) tetrakis (acetic acid) inside the silica nanoparticles. The nanoparticles characterized by transmission electron microscopy and luminescence spectroscopy methods were used for streptavidin labeling, and time-resolved fluoroimmunoassay (TR-FIA) of human prostate-specific antigen (PSA) as well as time-resolved luminescence imaging detection of an environmental pathogen, Giardia lamblia. The results demonstrated the utility of the new multicolor luminescent lanthanide nanoparticles for time-resolved luminescence bioassays.

Hormesis response of marine and freshwater luminescent bacteria to metal exposure.

The stimulatory effect of low concentrations of toxic chemicals on organismal metabolism, referred to as hormesis, has been found to be common in the widely used luminescence bioassay. This paper aims to study the hormesis phenomenon in both marine and freshwater luminescent bacteria, named Photobacterium phosphorem and Vibrio qinghaiensis. The effects of Cu (II), Zn (II), Cd (II) and Cr (VI) on luminescence of these two bacteria were studied for 0 to 75 minutes exposure by establishing dose- and time-response curves. A clear hormesis phenomenon was observed in all four testing metals at low concentrations under the condition of luminescence assays.

Hormesis response of marine and freshwater luminescent bacteria to metal exposure

The stimulatory effect of low concentrations of toxic chemicals on organismal metabolism, referred to as hormesis, has been found to be common in the widely used luminescence bioassay. This paper aims to study the hormesis phenomenon in both marine and freshwater luminescent bacteria, named Photobacterium phosphorem and Vibrio qinghaiensis. The effects of Cu (II), Zn (II), Cd (II) and Cr (VI) on luminescence of these two bacteria were studied for 0 to 75 minutes exposure by establishing dose- and time-response curves. A clear hormesis phenomenon was observed in all four testing metals at low concentrations under the condition of luminescence assays.

Optimization of a rapid luminescence bioassay for the assessment of toxicity of contaminants in XTC's pills

Evaluation of biological effects of synthesized molecules are generally based on cell cultures and animals in-vitro and in-vivo assays. These tests are expensive and time-consuming. In case of toxicity screening, recent studies have emphasized the benefits of rapid, reproducible and cost effective bacterial assays [1]. In aim to evaluate the possible toxicity of contaminants found in illegally purchased XTC's pills, we have optimized a bioassay coupling directly luminescent bacteria, Vibrio fischeri, with thin-layer chromatography [2]. For the chemical characterization of investigated tablets, we have developed an extraction method of XTC's components and chromatographic conditions for GC/MS analysis. In all investigated pills, the major constituent, 3,4-methylenedioxymethamphetamine (MDMA or „Ecstasy“) was jointly present with a series of by-products, precursors (natural products like safrole) and adjuvants. Although the toxicity of MDMA has been thoroughly investigated, few authors have considered the possible importance of such contaminants. The CCM-bioluminescence assay allows the separation of these complex mixtures and points out biologically active compounds by spot inhibition of bioluminescence visualized with the use of Bioluminizer (Camag, Switzerland). In order to identify the active compounds, the inhibition zones were scrapped and eluted with methanol, concentrated and analyzed by GC/MS. No relevant molecules have yet been identified, but further studies by LC/MS/MS could help to identify molecules not-detectable in GC/MS.

Ecotoxicological characterization of photoelectrocatalytic process for degradation of pentachlorophenol on titania nanotubes electrode

Luminescence bioassay using Vibrio fischeri and in vitro ethoxyresorufin- O-deethylase (EROD) induction bioassay using H4-IIE rat hepatoma cell were employed to test toxicity changes during the photoelectrocatalysis process for the degradation of pentachlorophenol (PCP) on TiO 2 nanotubes electrode. Acute toxicity of the reaction solution reduced by 83% after 120 min of irradiation. Bioassay-derived 2,3,7,8-TCDD equivalents (TEQ bio) increased from 0.72 to 6.72 ng l −1 during the former reaction stage, decreasing rapidly to 1.25 ng l −1 during the latter reaction stage. HRGC/HRMS analysis detected polychlorinated dibenzo- p-dioxins/dibenzofurans (PCDD/Fs) congeners in the reaction mixtures. TEQ bio and chemical-derived 2,3,7,8-TCDD equivalents (TEQ cal) were about of the same order of magnitude. Both results illustrated the formation of dioxins and their removal by photoelectrocatalysis. Moreover, photoelectrocatalysis showed a significant priority over direct photolysis and photocatalysis depending on the reaction conditions, not only for PCP removal but also for reduction in toxicity.

Lanthanide-based luminescence probes and time-resolved luminescence bioassays

Time-resolved luminescence bioassays based on lanthanide-luminescence probes have become common, useful tools in clinical diagnostics and bioanalytical chemistry. The unique spectral and temporal properties of lanthanide-luminescence probes, such as very long luminescence lifetime, large Stokes Shift and sharp emission profile, make them an ideal choice for use in time-resolved luminescence bioassays to discriminate background noises effectively. This review gives an overview of recent developments in lanthanide-luminescence probes and their applications in time-resolved luminescence bioassays.

Preparation and Time-Resolved Fluorometric Application of Luminescent Europium Nanoparticles

Novel silica-coated luminescent europium (SCLE) nanoparticles have been prepared by copolymerization of tetraethyl orthosilicate and 3-aminopropyl(triethoxy)silane with ammonium hydroxide in a water-in-oil microemulsion containing a luminescent Eu3+ chelate, Triton X-100, n-octanol, and cyclohexane. The nanoparticles were characterized by transmission electron microscopy, X-ray diffraction, and UV−vis and luminescence spectra. The results show that the nanoparticles are spherical and uniform in size, 50 ± 5 nm in diameter, and have high photostability and long luminescence lifetime (770 μs). Because primary amino groups have been directly introduced to the nanoparticle's surface by using 3-aminopropyl(triethoxyl)silane in nanoparticle preparation, the surface modification and bioconjugation of the nanoparticles are easier and reproducible. The nanoparticles were used for streptavidin (SA) labeling, and the nanoparticle-labeled streptavidin was successfully used in time-resolved fluoroimmunoassay of human α-fetoprotein. The results reveal that the new nanoparticles are favorable for use as a new type of luminescence probe for highly sensitive time-resolved luminescence bioassay.

Detection of a luxS-signaling molecule in Bacillus anthracis.

Quorum-sensing regulation of density-dependent genes has been described for numerous bacterial species. The partially annotated genome sequence of Bacillus anthracis contains an open reading frame (BA5047) predicted to encode an ortholog of luxS, required for synthesis of the quorum-sensing signaling molecule autoinducer-2 (AI-2). To determine whether B. anthracis produces AI-2, the Vibrio harveyi luminescence bioassay was used. Cell-free conditioned media from vaccine (Sterne) strain 34F(2) induced luminescence in V. harveyi reporter strain BB170, indicating its production of AI-2. Cloned BA5047, expressed in Escherichia coli DH5 alpha cells, restored AI-2 activity to these cells. To evaluate whether BA5047 is essential for AI-2 synthesis, it was deleted through allelic exchange with marker rescue; the resulting mutant had no functional luxS activity and had reduced growth in vitro. In the wild-type strain, AI-2 activity was greatest during the exponential phase of growth. In total, these data indicate that BA5047 is a functional luxS ortholog in B. anthracis necessary for growth-phase-specific AI-2 expression. Thus, B. anthracis may utilize extracellular signaling molecules to regulate density-dependent gene expression.

Early Changes in Thyroid-Stimulating Antibody Activity following Radioiodine Therapy

Objective: The aim of this study was to determine whether or not the titre of thyroid-stimulating hormone receptor antibody with stimulating (TRAb-S) activity changes in patients with Graves’ disease (GD) or toxic multinodular goitres (TMNG) 3 months after treatment with sodium iodide (¹³¹I). Subjects and Methods: Serum specimens were obtained from 21 hyperthyroid patients (15 with GD and 6 with TMNG) at a median 0.5 months before and 3 months after ¹³¹I treatment using a standard ablative dose of 555 MBq. TRAb-S activity was measured in a sensitive and specific luminescent bioassay employing the lulu cell line and expressed as a stimulation index (SI; normal ≤1.5). Results: The mean TRAb-S in the GD patients was 2.72 SI (95% CI: 1.51–4.03) 0.5 months before administration of ¹³¹I and 3.98 SI (95% CI: 1.20–6.76) 3 months after administration of ¹³¹I. The difference was not statistically significant at p < 0.8. It was not elevated in the TMNG patients before (0.57 SI; 95% CI: 0.41– 0.73) and after (1.00 SI; 95% CI: 0.74–1.26) treatment either. Conclusions: Radioiodine therapy for GD or TMNG did not induce a significant change in TRAb-S activity at 3 months after treatment with ¹³¹I, probably due to effective antithyroid therapy or the timing of samples.

Influence of organic solvents on the sensitivity of a bioluminescence toxicity test with Vibrio harveyi.

The possibility of using organic solvents (OSs) to increase the susceptibility of bioluminescent microorganisms in a bioassay for assessing the toxicity of chemicals dissolved in water was investigated. To conduct the tests acetonitrile, dimethyl sulfoxide, ethanol, methanol, and isopropanol were used as OSs and Cd, Hg, and Zn as reference toxicants. The addition of OSs modified the toxicity of the three metals to Vibrio harveyi, according on the bioluminescence assay used. The sensitivity of the luminescence bioassay for Hg increased in the presence of the five OSs, thus indicating a greater toxic effect. However, the sensitivity of the assay for the other two metals, Cd and Zn, increased or decreased (lesser toxic effect) depending on the concentration at which the OSs were used. No correlation was observed between the concentration of the five OSs and the toxicity of the three reference toxicants. From this it can be deduced that none of these OSs could be recommended for increasing generically the sensitivity of toxicity biotests using V. harveyi.

Correlation of Two Bioluminescence and One Fluorogenic Bioassay for the Detection of Toxic Chemicals

The linear correlation between the EC 50 values of 50 substances obtained in luminescence bioassays using Vibrio fischeri and Vibrio harveyi and in a fluorogenic bioassay using Escherichia coli was investigated. As a result, a significant correlation was found between the said values in all three toxicity tests. The bioassay using V.harveyi had a sensitivity similar to that of the fluorogenic bioassay, and the bioassay using V. fischeri was the least sensitive of all. The sensitivity of the three bioassays for each of the tested substances, chiefly heavy metals, organic solvents, orgnochlorated compounds, and pesticides, differed in the majority of the cases. The three bioassays were quantified using the same laboratory apparatus and the data were processed in the same way. The possibility of designing a battery of toxicity tests that can be performed using the same apparatus but different organisms and parameters is discussed.

Hormesis Responses of Free and Immobilized Light-Emitting Bacteria

The stimulatory effect of sublethal or low concentrations of toxic chemicals on organismal metabolism, referred to as hormesis, has been found to be common in the widely used Vibrio fischeri luminescence bioassay. In addition to the “normal” type α, we have demonstrated type β and, possibly, type γ, dose–response curves in free and immobilized V. fischeri bioassays developed. Understanding and utilizing data from hormesis responses are necessary in determining the toxicity of chemicals, singly or in complex mixtures, to natural biota without imposing excessive penalties to dischargers. At the same time, care must be taken not to relax environmental standards. This can only arise by fully investigating and understanding the role of hormesis in toxicity data used for risk assessment.

Comparative evaluation of the effects of pesticides in acute toxicity luminescence bioassays

Acute toxicity of pesticides in water was assessed singly and in mixtures using the responses of the luminescent bacterium Vibrio fischeri (BioTox™), the aquatic invertebrate Daphnia magna (Daphtoxkit™), and the MitoScan™ assay. The latter utilized fragmented mitochondria to enzymatically convert β-nicotinamide adenine dinucleotide (NADH) to its oxidized form, NAD +. The rate of the conversion being sensitive to type and concentration of toxicants. The pesticides tested were Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate), Cyromazine ( N-cyclopropyl-1,3,5-triazine-2,4,6-triamine), Fenamiphos (ethyl 4-methylthio-m-tolyl isopropylphosphoramidate), and Formetanate (3-dimethylaminomethyleneiminophenyl methylcarbamate). The toxicity bioassays were characterized in terms of relative sensitivity, reproducibility, range of the linear response, and the ability to reveal synergistic/antagonistic interactions among toxicants. The D. magna assay was the most sensitive and best able to detect toxic interactions of mixtures. Also, unlike the other assays used, the response of the daphnid system was linear over a 10-fold change in pesticide concentration. Relative to the BioTox™, the MitoScan™ was 2- to 11-fold more sensitive for the compounds and mixtures tested. The EC 50 reproducibility of all tests was within ±20% coefficient of variation; however, the lowest observable effect concentration (LOEC) were only reproducible to ±35% on average. Cyromazine was the least toxic of the pesticides tested. To test the predictive value of the concept of concentration addition, toxicities of binary and quaternary mixtures of four different pesticides were analyzed. Synergistic/antagonistic responses were most frequently observed in testing with D. magna. Synergistic/antagonistic effects were seen only in 25 and 50% of the cases with the BioTox™ and the MitoScan™ assays, respectively.

Assessing the quality of marine coastal environments: comparison of scope for growth and Microtox® bioassay results of pollution gradient areas in eastern Mediterranean (Greece)

Methods for assessing the quality of marine coastal environments are numerous and rapidly evolving. The integration of physiological parameters termed as Scope for Growth (SfG) and the luminescent bioassay Microtox® were applied in several sites of Saronikos and Amvrakikos Gulfs (Greece) to assess the environmental quality and compare the results of the two methods. Each site in the two areas undergoes different types and levels of pollution. Both methods could identify a pollution gradient reflecting the quality of each site. A very good linear correlation was noticed between the two methods ( r=0.98). The stress order, which was similar in both methods, exhibited the expected pollution gradient according to the pressure that each site undergoes. Results suggest that for screening and fast isolation of polluted sites before further analysis, the rapid and simple Microtox® assay could operate as an alternative to the more complex SfG method.

Determination of Acute Zn Toxicity in Pore Water from Soils Previously Treated with Sewage Sludge Using Bioluminescence Assays

The effects of increasing concentrations of Zn and Cu in soil pore water from soils of a long-term sewage sludge field experiment on microbial bioluminescence were investigated. Concentrations of total soluble Zn, free Zn2+, and soluble Cu increased sharply in soil pore water with increasing total soil metal concentrations above 140 mg of Zn kg-1 or 100 mg of Cu kg-1. Two luminescence bioassays were tested, based on two bacteria (Escherichia coli and Pseudomonas fluorescens) with the lux genes encoding bacterial luminescence inserted into them. The bioluminescence response of the two microorganisms declined as total soil Zn, soil pore water soluble Zn, and soil pore water free Zn2+ concentrations increased. The EC25 values for E. coli and P. fluorescens were 1.3 ± 0.2 and 4.3 ± 0.5 mg L-1 on a free Zn2+ basis, respectively. The EC50 values were 2.5 ± 0.2 and 9.6 ± 0.9 mg of free Zn2+ L-1, respectively. Copper had no significant effect on bioluminescence in the two assays, even at the largest soil pore water concentration of about 620 μg L-1, corresponding to a total Cu concentration in bulk soil of about 350 mg kg-1. Thus, the decline in bioluminescence of the two assays was ascribed to increasing soil pore water free Zn2+ and not soluble Cu.

Development of a Luminescent Bioassay for Thyroid Stimulating Antibodies

The hyperthyroidism of Graves Disease (GD) is due to thyroid stimulating antibodies (TSAb) which are thyrotropin (TSH) agonists. They are detected routinely by measuring their ability to inhibit TSH binding to the receptor (TBII), which does not reflect their true biological activity. Current bioassays which measure cAMP by RIA, are not suitable for routine use. We have developed a luminescent bioassay for TSAb, by introducing a cAMP responsive luciferase construct into CHO cells stably expressing the human TSH receptor (TSHR). Clone lulu1 displays dose dependent TSH response detectable from 10 μU/ml and maximal at 10 mU/ml when a >25 fold increase in light output is obtained. 34 euthyroid sera were tested to determine a reference range, with values >1.5 relative light units (R.L.U.) being considered positive. An international TSAb standard responded in a dose dependent manner with 10 mIU/ml giving an R.L.U. of >10. The assay was adapted to a 96 well format for automatic readout and 100 treated GD samples (50 TBII negative and 50 TBII positive) were tested, 73% being positive. In contrast only 4% of 79 control sera from individuals with Hashimoto’s, non-thyroid autoimmunity or multinodular goitre produced R.L.U. >1.5. When 44 of the GD sera were compared in a traditional salt-free bioassay, 61% were positive compared with 75% in the new luminescent assay. In conclusion, we have developed a luminescent bioassay for TSAb, using unfractionated serum which is capable of high throughput suitable for routine use.

Development of a luminescent bioassay for thyroid stimulating antibodies.

The hyperthyroidism of Graves Disease (GD) is due to thyroid stimulating antibodies (TSAb) which are thyrotropin (TSH) agonists. They are detected routinely by measuring their ability to inhibit TSH binding to the receptor (TBII), which does not reflect their true biological activity. Current bioassays which measure cAMP by RIA, are not suitable for routine use. We have developed a luminescent bioassay for TSAb, by introducing a cAMP responsive luciferase construct into CHO cells stably expressing the human TSH receptor (TSHR). Clone lulul displays dose dependent TSH response detectable from 10 microU/ml and maximal at 10 mU/ml when a >25 fold increase in light output is obtained. 34 euthyroid sera were tested to determine a reference range, with values >1.5 relative light units (R.L.U.) being considered positive. An international TSAb standard responded in a dose dependent manner with 10 mIU/ml giving an R.L.U. of >10. The assay was adapted to a 96 well format for automatic readout and 100 treated GD samples (50 TBII negative and 50 TBII positive) were tested, 73% being positive. In contrast only 4% of 79 control sera from individuals with Hashimoto's, non-thyroid autoimmunity or multinodular goitre produced R.L.U. >1.5. When 44 of the GD sera were compared in a traditional salt-free bioassay, 61% were positive compared with 75% in the new luminescent assay. In conclusion, we have developed a luminescent bioassay for TSAb, using unfractionated serum which is capable of high throughput suitable for routine use.

Increased aquatic toxicity following photolytic conversion of an organochlorine pollutant

In studies of the photolytic degradation of chlorophenolic substances in aqueous media, 2,4,6-trichlorophenol was found to be converted into products with increased toxic effects on the Microtox® luminescence bioassay system. The chlorophenol was mainly photohydrolysed at the 2- or 6- positions and 3,5-dichlorocatechol was formed. The increased toxic effects were confined to this photolytic product. Although there were indications that the catechol was further modified, it accumulated in the aquatic medium at the test conditions, and most probably will appear in the aquatic environment upon illumination of the polluted parent compound. The 2,4,6-trichlorophenol/3,5-dichlorocatechol system here described therefore increases the environmental impact of an abiotic transformation of a polluted organic compound.

38] Luminescent bioassays based on macrophage cell lines

Publisher Summary Cells of the monocyte/macrophage lineage serve essential functions for immune responses. Considering the central role of macrophage cells in the immune system, assay systems had to be developed to probe accurately the functional activity of macrophage cells and their regulation. An extreme sensitive method to monitor macrophage activity, at least when studying the process of antigen catabolism is provided by chemiluminescent measurements. This activity, termed the respiratory burst, appears to represent the major mechanism by which macrophage cells exert their antimicrobial and antitumor activity and tissue injury that may accompany inflammatory reactions. The cellular bioassay described here is based on the assumption that provided the quantity and functional capacity of the macrophage cell population are held constant, using a homogeneous macrophage cell line, the degree of stimulated macrophage oxygenation activity and resulting CL emission will reflect the stimulating capacity of a triggering agent. As such, the triggering capacity of a particular agent is proportional to the macrophage oxygenation activity and therefore to the measured CL. If the triggering agent is held constant, one is able to measure the activity of other agents, which modulate the activity of either the responding macrophage cells or the inherent stimulating activity of different triggering agents. Hence, the modulating activity of a particular agent is proportional to the macrophage oxygenation activity, and therefore, to the measured CL.