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      Colorimetric detection of Shewanella oneidensis based on immunomagnetic capture and bacterial intrinsic peroxidase activity

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      Scientific Reports
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          Abstract

          Rapid detection and enumeration of target microorganisms is considered as a powerful tool for monitoring bioremediation process that typically involves cleaning up polluted environments with functional microbes. A novel colorimetric assay is presented based on immunomagnetic capture and bacterial intrinsic peroxidase activity for rapidly detecting Shewanella oneidensis, an important model organism for environmental bioremediation because of its remarkably diverse respiratory abilities. Analyte bacteria captured on the immunomagnetic beads provided a bacterial out-membrane peroxidase-amplified colorimetric readout of the immunorecognition event by oxidizing 3, 3′, 5, 5′-tetramethylbenzidine (TMB) in the present of hydrogen peroxide. The high-efficiency of immunomagnetic capture and signal amplification of peroxidase activity offers an excellent detection performance with a wide dynamic range between 5.0 × 10 3 and 5.0 × 10 6 CFU/mL toward target cells. Furthermore, this method was demonstrated to be feasible in detecting S. oneidensis cells spiked in environmental samples. The proposed colorimetric assay shows promising environmental applications for rapid detection of target microorganisms.

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          Most cited references28

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          Fe3O4 magnetic nanoparticles as peroxidase mimetics and their applications in H2O2 and glucose detection.

          Artificial enzyme mimetics are a current research interest because natural enzymes bear some serious disadvantages, such as their catalytic activity can be easily inhibited and they can be digested by proteases. A very recently study reported by Yan et al. has proven that Fe(3)O(4) magnetic nanoparticles (MNPs) exhibit an intrinsic enzyme mimetic activity similar to that found in natural peroxidases, though MNPs are usually thought to be biological and chemical inert (Gao, L. Z.; Zhuang, J.; Nie, L.; Zhang, J. B.; Zhang, Y.; Gu, N.; Wang, T. H.; Feng, J.; Yang, D. L.; Perrett, S.; Yan, X. Y. Nat. Nanotechnol. 2007, 2, 577-583). In the present work, we just make use of the novel properties of Fe(3)O(4) MNPs as peroxidase mimetics reported by Yan et al. to detect H(2)O(2). The Fe(3)O(4) MNPs were prepared via a coprecipitation method. The as-prepared Fe(3)O(4) MNPs were then used to catalyze the oxidation of a peroxidase substrate 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) by H(2)O(2) to the oxidized colored product (see eq 1) which provides a colorimetric detection of H(2)O(2). As low as 3 x 10(-6) mol/L H(2)O(2) could be detected with a linear range from 5 x 10(-6) to 1 x 10(-4) mol/L via our method. More importantly, a sensitive and selective method for glucose detection was developed using glucose oxidase (GOx) and the as-prepared Fe(3)O(4) MNPs. The detection platforms for H(2)O(2) and glucose developed in the present work not only further confirmed that the Fe(3)O(4) MNPs possess intrinsic peroxidase-like activity but also showed great potential applications in varieties of simple, robust, and easy-to-make analytical approaches in the future.
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            Horseradish peroxidase: a modern view of a classic enzyme.

            C Veitch (2004)
            Horseradish peroxidase is an important heme-containing enzyme that has been studied for more than a century. In recent years new information has become available on the three-dimensional structure of the enzyme and its catalytic intermediates, mechanisms of catalysis and the function of specific amino acid residues. Site-directed mutagenesis and directed evolution techniques are now used routinely to investigate the structure and function of horseradish peroxidase and offer the opportunity to develop engineered enzymes for practical applications in natural product and fine chemicals synthesis, medical diagnostics and bioremediation. A combination of horseradish peroxidase and indole-3-acetic acid or its derivatives is currently being evaluated as an agent for use in targeted cancer therapies. Physiological roles traditionally associated with the enzyme that include indole-3-acetic acid metabolism, cross-linking of biological polymers and lignification are becoming better understood at the molecular level, but the involvement of specific horseradish peroxidase isoenzymes in these processes is not yet clearly defined. Progress in this area should result from the identification of the entire peroxidase gene family of Arabidopsis thaliana, which has now been completed.
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              Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes

              Dissimilatory reduction of metal (e.g. Fe, Mn) (hydr)oxides represents a challenge for microorganisms, as their cell envelopes are impermeable to metal (hydr)oxides that are poorly soluble in water. To overcome this physical barrier, the Gram-negative bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens have developed electron transfer (ET) strategies that require multihaem c-type cytochromes (c-Cyts). In S. oneidensis MR-1, multihaem c-Cyts CymA and MtrA are believed to transfer electrons from the inner membrane quinone/quinol pool through the periplasm to the outer membrane. The type II secretion system of S. oneidensis MR-1 has been implicated in the reduction of metal (hydr)oxides, most likely by translocating decahaem c-Cyts MtrC and OmcA across outer membrane to the surface of bacterial cells where they form a protein complex. The extracellular MtrC and OmcA can directly reduce solid metal (hydr)oxides. Likewise, outer membrane multihaem c-Cyts OmcE and OmcS of G. sulfurreducens are suggested to transfer electrons from outer membrane to type IV pili that are hypothesized to relay the electrons to solid metal (hydr)oxides. Thus, multihaem c-Cyts play critical roles in S. oneidensis MR-1- and G. sulfurreducens-mediated dissimilatory reduction of solid metal (hydr)oxides by facilitating ET across the bacterial cell envelope.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                05 June 2014
                2014
                : 4
                : 5191
                Affiliations
                [1 ]Guangdong Institute of Eco-environmental and Soil Sciences , Guangzhou 510650, P. R. China
                Author notes
                Article
                srep05191
                10.1038/srep05191
                4046127
                24898751
                cdbfe035-fd81-43a3-9f46-cfdd541d4276
                Copyright © 2014, Macmillan Publishers Limited. All rights reserved

                This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. The images in this article are included in the article's Creative Commons license, unless indicated otherwise in the image credit; if the image is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the image. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

                History
                : 03 February 2014
                : 16 May 2014
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