Multiplexed assessment of engineered bacterial constructs for intracellular ß-galactosidase expression by redox amplification on catechol-chitosan modified nanoporous gold.

Synthetic biology approaches for rewiring of bacterial constructs to express particular intracellular factors upon induction with the target analyte are emerging as sensing paradigms for applications in environmental and in vivo monitoring. To aid in the design and optimization of bacterial constructs for sensing analytes, there is a need for lysis-free intracellular detection modalities that monitor the signal level and kinetics of expressed factors within different modified bacteria in a multiplexed manner, without requiring cumbersome surface immobilization. Herein, an electrochemical detection system on nanoporous gold that is electrofabricated with a biomaterial redox capacitor is presented for quantifying ß-galactosidase expressed inside modified Escherichia coli constructs upon induction with dopamine. This nanostructure-mediated redox amplification approach on a microfluidic platform allows for multiplexed assessment of the expressed intracellular factors from different bacterial constructs suspended in distinct microchannels, with no need for cell lysis or immobilization. Since redox mediators present over the entire depth of the microchannel can interact with the electrode and with the E. coli construct in each channel, the platform exhibits high sensitivity and enables multiplexing. We envision its application in assessing synthetic biology-based approaches for comparing specificity, sensitivity, and signal response time upon induction with target analytes of interest.

Detection of Bacteria in Water with ß-Galactosidase-Coated Magnetic Nanoparticles.

Pathogenic contamination and resistant bacterial infections remain critical concerns in both developed and developing countries. Rapid and sensitive detection of pathogens is still a key requirement for both environmental and clinical settings. This article introduces a simple, colorimetric, cost-effective, and high-throughput system based on a positively charged iron oxide/enzyme complex for the detection of both gram-positive and gram-negative bacteria in water between 103 and 108 cfu/mL. This study provides an effective strategy for the identification and purification of pathogen contamination in drinking water.

Overview and assessment of the histochemical methods and reagents for the detection of ß-galactosidase activity in transgenic animals.

Bacterial ß-galactosidase is one of the most widely used reporter genes in experiments involving transgenic and knockout animals. In this review we discuss the current histochemical methods and available reagents to detect ß-galactosidase activity. Different substrates are available, but the most commonly used is X-gal in combination with potassium ferri- and ferro-cyanide. The reaction produces a characteristic blue precipitate in the cells expressing ß-galactosidase, and despite its efficiency in staining whole embryos, its detection on thin tissue sections is difficult. Salmon-gal is another substrate, which in combination with ferric and ferrous ions gives a reddish-pink precipitate. Its sensitivity for staining tissue sections is similar to that of X-gal. Combining X-gal or Salmon-gal with tetrazolium salts provides a faster and more sensitive reaction than traditional ß-galactosidase histochemistry. Here, we compare the traditional ß-galactosidase assay and the combination of X-gal or Salmon-gal with three tetrazolium salts: nitroblue tetrazolium, tetranitroblue tetrazolium and iodonitrotetrazolium. Based on an assessment of the sensitivity and specificity of the different combinations of substrates, we are proposing an optimized and enhanced method for ß-galactosidase detection in histological sections of the transgenic mouse brain. Optimal staining was obtained with X-gal in combination with nitroblue tetrazolium, which provides a faster and more specific staining than the traditional X-gal combination with potassium ferri- and ferro-cyanide. We recommend the X-gal/nitroblue tetrazolium staining mixture as the first choice for the detection of ß-galactosidase activity on histological sections. When faster results are needed, Salmon-gal/nitroblue tetrazolium should be considered as an alternative, while maintaining acceptable levels of noise.

Printing silicone-based hydrophobic barriers on paper for microfluidic assays using low-cost ink jet printers.

Paper-based microfluidic devices exhibit many advantages for biological assays. Normally, the assays are restricted to certain areas of the paper by hydrophobic barriers comprised of wax or alkyl ketene dimers (AKD). Neither hydrophobic barrier is able to constrain aqueous solutions of surfactants, which are frequently used in biological assays. We demonstrate that rapidly curing silicone resins can be inkjet printed onto pure cellulose paper using inexpensive thermal ink-jet printers. The Piers-Rubinsztajn (PR) reaction dominates the cure chemistry leading to cellulose fibers that are surface coated with a silicone resin. The resulting barriers are able to resist penetration by surfactant solutions and even by the lower surface energy solvents DMF and DMSO. The utility of the barrier was demonstrated using a coliform assay based on detection of ß-galactosidase.

Assessment of a novel, capsid-modified adenovirus with an improved vascular gene transfer profile.

BACKGROUND: Cardiovascular disorders, including coronary artery bypass graft failure and in-stent restenosis remain significant opportunities for the advancement of novel therapeutics that target neointimal hyperplasia, a characteristic of both pathologies. Gene therapy may provide a successful approach to improve the clinical outcome of these conditions, but would benefit from the development of more efficient vectors for vascular gene delivery. The aim of this study was to assess whether a novel genetically engineered Adenovirus could be utilised to produce enhanced levels of vascular gene expression. METHODS: Vascular transduction capacity was assessed in primary human saphenous vein smooth muscle and endothelial cells using vectors expressing the LacZ reporter gene. The therapeutic capacity of the vectors was compared by measuring smooth muscle cell metabolic activity and migration following infection with vectors that over-express the candidate therapeutic gene tissue inhibitor of matrix metalloproteinase-3 (TIMP-3). RESULTS: Compared to Adenovirus serotype 5 (Ad5), the novel vector Ad5T*F35++ demonstrated improved binding and transduction of human vascular cells. Ad5T*F35++ mediated expression of TIMP-3 reduced smooth muscle cell metabolic activity and migration in vitro. We also demonstrated that in human serum samples pre-existing neutralising antibodies to Ad5T*F35++ were less prevalent than Ad5 neutralising antibodies. CONCLUSIONS: We have developed a novel vector with improved vascular transduction and improved resistance to human serum neutralisation. This may provide a novel vector platform for human vascular gene transfer.

Spectrophotometric assessment of salivary ß-galactosidases in halitosis.

The aim of this work was to develop a spectrophotometric method for the measurement of salivary ß-galactosidase for the evaluation of oral malodor. A comparison between different methods for estimating oral malodor has been conducted on 94 healthy adult volunteers. For organoleptic measurements, the subjects were instructed to exhale briefly through the mouth at a distance of approximately 10 cm from the noses of two trained judges. The evaluation of ß-galactosidase activity was accomplished in unstimulated whole saliva of all participants through the colorimetric method and spectrophotometry. A significant association among spectrophotometric evaluation of ß-galactosidase and organoleptic measurements was assessed by Spearman correlations. Although colorimetric and spectrophotometric assessments of ß-galactosidases were estimated to have the same sensitivity, the last method is characterized by a higher specificity. The results suggest that the use of the UV-vis spectrophotometer increases the specificity of the evaluation of salivary ß-galactosidases.

Assessment of anti-oxidant activity of plant extracts using microbial test systems.

AIMS: To evaluate the anti-oxidant properties of extracts from 20 medicinal herbs growing in western Siberia using microbial test systems and different in vitro methods. METHODS AND RESULTS: In vivo anti-oxidant activity of extracts was evaluated for their capacity to protect bacteria, Escherichia coli, against bacteriostatic and bactericidal effects of H(2)O(2) and menadione, and action on anti-oxidant gene expression. In vitro anti-oxidant activity has been examined by a number of methods including: the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH(*))-scavenging assay, chelating activity and capacity to protect plasmid DNA against oxidative damage. In addition, total polyphenol content was determined. The extracts of Fragaria vesca, Rosa majalis, Pentaphylloides fruticosa, Alchemilla vulgaris and Pulmonaria mollis possessed the highest levels of anti-oxidant activity in vivo and in vitro. The protective properties were more closely related to the DPPH(*) radical-scavenging activity, tannin content and action on anti-oxidant gene expression than to other parameters. CONCLUSION: The extracts of medicinal plants may have anti-oxidant effects on bacteria simultaneously through several different pathways, including direct inhibition of reactive oxygen species, iron chelation and anti-oxidant genes induction. SIGNIFICANCE AND IMPACT OF THE STUDY: Using microbial test systems, we revealed herbs that may be used as potential sources of natural anti-oxidants.

Quantitative nanopipettor for miniaturized chemical and biochemical reaction sampling.

A novel and readily available pipettor capable of nanoliter-sized volume manipulation was developed to improve and increase the flexibility of small-scale reaction processing. The volume delivery was found to be reproducible, with typical relative standard deviations of 1-5%, and easily tunable over a range of nanoliter-sized aliquots. The nanopipettor was combined with capillary electrophoresis using laser-induced fluorescence detection to monitor a small-scale enzyme reaction (beta-galactosidase) using a tetramethylrhodamine-labeled substrate. The results were in good agreement with a standard enzyme assay using a micropipet, thus demonstrating the nanopipettor's potential in developing new nanoscale utrasensitive enzyme assays.

Assessment of oestrogenic potency of chemicals used as growth promoter by in-vitro methods.

Three in-vitro bioassays were used to compare the oestrogenic potency of chemicals used as growth promoter in beef cattle in certain non-European Union countries (17beta-oestradiol, alpha-zearalanol, testosterone, trenbolone, trenbolone acetate, melengestrol acetate) or found as food contaminant such as the mycotoxin zearalenone and some of their metabolites (17alpha-oestradiol, oestrone, 17alpha-epitestosterone, 19-nortestosterone, androstendione, zearalanone, alpha-zearalanol, beta-zearalanol, alpha-zearalenol, beta-zearalenol). The strong oestrogens 17alpha-ethinyl oestradiol and diethylstilboestrol were used as standards. The first bioassay was based on the activation of a reporter gene by oestrogens in recombinant yeast expressing human or rainbow trout oestrogen receptor. In the second bioassay, the vitellogenin gene induction of rainbow trout hepatocyte cultures was used as a biomarker for the exposure to oestrogens. The third bioassay was based on the alkaline phosphatase gene induction by oestrogens in the human endometrial Ishikawa cell line. The assessment of oestrogenic potency of these chemicals clearly demonstrates the strong oestrogenicity of the mycotoxin zearalenone and its metabolites and particularly alpha-zearalenol which was as potent as ethinyl oestradiol and diethylstilboestrol in the human endometrial Ishikawa cell line.

Rapid and cost-effective multiparameter toxicity tests for soil microorganisms.

Three biochemical parameters, DNA quantification in soil samples and two enzymatic activities, beta-galactosidase and dehydrogenase have been assessed as potential end-points for the use in cost-effective toxicity tests on soil microorganisms. The assessment included the development of a classical dose-response 24-h assay and the incorporation of measurements of the effects on microbial activities in soil column leaching studies and multispecies miniaturised terrestrial systems (MTS). Four different chemicals, copper, a new herbicide, thiabendazole and fenthion were studied. A rapid fluorescence DNA quantification technique did not produce adequate responses. The efforts to quantify DNA after extraction and clean-up procedures failed due to the presence of humic acids. From the protocol of the technique one could see that the technical procedure is time-consuming and expensive and, for this reason, not suitable for use as a parameter in rapid and cost-effective tests. However, the enzymatic activities showed their potential as toxicity end-points. Copper produced a concentration/response inhibition of beta-galactosidase and dehydrogenase with EC50 values of 78.39 and 24.77 mg Cu/kg soil, respectively. In the soil column study, these endpoints allowed the measurement of the microbial activities through the column. The effects of the new herbicide on beta-galactosidase and dehydrogenase activities were statistically significant for the highest application dose (40 g/ha). Thiabendazole affected the microbial activity when mixed within the soil, but no effects were observed when this fungicide was applied on the soil surface. Fenthion produced effects when applied either in the soil or on the soil surface. These results can be explained by the low mobility of thiabendazole. The results show the capabilities of these biochemical parameters to be included as endpoints in cost-effective bioassays.

A method for the quantification of a colored or fluorescent signal in enzyme immunoassays by photodensitometry.

A colored or fluorescent signal is generally evaluated with the naked eye, or by means of different more or less sophisticated and costly instruments. Photodensitometry is an additional technique which is both inexpensive and simple to perform. This technique can satisfactorily quantify a signal without the use of either a spectrophotometer or a fluorometer. In this study we compared readings obtained by spectrophotometry, fluorometry and photodensitometry in 96-well ELISA plates and in Terasaki plates. In ELISA plates, it is possible to detect 1220-300,000 femtograms (fg) of peroxidase by spectrophotometry and 4800-125,000 fg by photodensitometry. In Terasaki plates, we were able to measure between 3.8 and 8000 fg of beta-galactosidase per sample by spectrofluorometry, and from 30 to 8000 fg by photodensitometry. Using a sandwich procedure in Terasaki plates we were able to measure between 100 and 10,000 fg of IgE per sample by spectrofluorometry and from 500 to 10,000 fg by photodensitometry. Photodensitometry is the least expensive technique for the reliable detection of enzyme or enzymatic marker in small sample volumes treated with a fluorogenic substrate.