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      Beetroot-Pigment-Derived Colorimetric Sensor for Detection of Calcium Dipicolinate in Bacterial Spores

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          Abstract

          In this proof-of-concept study, we describe the use of the main red beet pigment betanin for the quantification of calcium dipicolinate in bacterial spores, including Bacillus anthracis. In the presence of europium(III) ions, betanin is converted to a water-soluble, non-luminescent orange 1∶1 complex with a stability constant of 1.4×10 5 L mol –1. The addition of calcium dipicolinate, largely found in bacterial spores, changes the color of the aqueous solution of [Eu(Bn) +] from orange to magenta. The limit of detection (LOD) of calcium dipicolinate is around 2.0×10 –6 mol L –1 and the LOD determined for both spores, B. cereus and B. anthracis, is (1.1±0.3) ×10 6 spores mL –1. This simple, green, fast and low cost colorimetric assay was selective for calcium dipicolinate when compared to several analogous compounds. The importance of this work relies on the potential use of betalains, raw natural pigments, as colorimetric sensors for biological applications.

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          Most cited references22

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          Rapid detection of an anthrax biomarker by surface-enhanced Raman spectroscopy.

          A rapid detection protocol suitable for use by first-responders to detect anthrax spores using a low-cost, battery-powered, portable Raman spectrometer has been developed. Bacillus subtilis spores, harmless simulants for Bacillus anthracis, were studied using surface-enhanced Raman spectroscopy (SERS) on silver film over nanosphere (AgFON) substrates. Calcium dipicolinate (CaDPA), a biomarker for bacillus spores, was efficiently extracted by sonication in nitric acid and rapidly detected by SERS. AgFON surfaces optimized for 750 nm laser excitation have been fabricated and characterized by UV-vis diffuse reflectance spectroscopy and SERS. The SERS signal from extracted CaDPA was measured over the spore concentration range of 10(-14)-10(-12) M to determine the saturation binding capacity of the AgFON surface and to calculate the adsorption constant (Kspore=1.7 x 10(13) M(-1)). At present, an 11 min procedure is capable of achieving a limit of detection (LOD) of approximately 2.6 x 10(3) spores, below the anthrax infectious dose of 10(4) spores. The data presented herein also demonstrate that the shelf life of prefabricated AgFON substrates can be as long as 40 days prior to use. Finally, these sensing capabilities have been successfully transitioned from a laboratory spectrometer to a field-portable instrument. Using this technology, 10(4) bacillus spores were detected with a 5 s data acquisition period on a 1 month old AgFON substrate. The speed and sensitivity of this SERS sensor indicate that this technology can be used as a viable option for the field analysis of potentially harmful environmental samples.
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            Spore germination.

            The germination of dormant spores of Bacillus species is the first crucial step in the return of spores to vegetative growth, and is induced by nutrients and a variety of non-nutrient agents. Nutrient germinants bind to receptors in the spore's inner membrane and this interaction triggers the release of the spore core's huge depot of dipicolinic acid and cations, and replacement of these components by water. These latter events trigger the hydrolysis of the spore's peptidoglycan cortex by either of two redundant enzymes in B. subtilis, and completion of cortex hydrolysis and subsequent germ cell wall expansion allows full spore core hydration and resumption of spore metabolism and macromolecular synthesis.
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              The ecology of anthrax spores: tough but not invincible.

              Bacillus anthracis is the causative agent of anthrax, a serious and often fatal disease of wild and domestic animals. Central to the persistence of anthrax in an area is the ability of B. anthracis to form long-lasting, highly resistant spores. Understanding the ecology of anthrax spores is essential if one hopes to control epidemics. Studies on the ecology of anthrax have found a correlation between the disease and specific soil factors, such as alkaline pH, high moisture, and high organic content. Researchers initially suggested that these factors influenced vegetative anthrax bacilli. However, subsequent research has shown that vegetative cells of B. anthracis have very specific nutrient and physiological requirements and are unlikely to survive outside a host. Review of the properties of spores of B. anthracis and other Bacillus species suggests that the specific soil factors linked to epidemic areas reflect important environmental conditions that aid the anthrax spores in causing epidemics. Specifically, high levels of calcium in the soil may help to maintain spore vitality for prolonged periods, thereby increasing the chance of spores encountering and infecting a new host. Cycles of runoff and evaporation may collect spores dispersed from previous epidemics into storage areas, thereby concentrating them. Uptake of large doses of viable spores from storage areas by susceptible animals, via altered feeding or breeding behavior, may then allow the bacterium to establish infection and cause a new epidemic. Literature search for this review was done by scanning the Life Sciences Collection 1982-1994 using the keywords "anthrax" and "calcium and spore."
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                3 September 2013
                : 8
                : 9
                : e73701
                Affiliations
                [1 ]Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, SP, Brazil
                [2 ]Biosystems and Biomaterials Division, Chemical Science Technology Laboratory, National Institute of Standards and Technology. Gaithersburg, Maryland, United States of America
                [3 ]Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil
                Loyola University Medical Center, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: ELB LCPG SMS RAA. Performed the experiments: LCPG SMS RAA. Analyzed the data: ELB LCPG SMS RAA PCD. Wrote the paper: ELB LCPG SMS RAA PCD.

                Article
                PONE-D-13-20577
                10.1371/journal.pone.0073701
                3760816
                24019934
                fc8d4871-0d9e-43a0-8dd7-0ae137e48eb3
                Copyright @ 2013

                This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 18 May 2013
                : 20 July 2013
                Page count
                Pages: 6
                Funding
                Funding was provided by the São Paulo Research Foundation (FAPESP, 07/00684-6 and 11/23036-5). LCPG was supported by a FAPESP fellowship (07/59407-1) and the Coordination for the Improvement of Higher Education Personnel (CAPES, PNPD 427-10/2009) fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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