Article pubs.acs.org/est
CYANOCHIP: An Antibody Microarray for High-TaxonomicalResolution Cyanobacterial Monitoring Yolanda Blanco,† Antonio Quesada,†,‡ Ignacio Gallardo-Carreño,† Jacobo Aguirre,§ and Victor Parro*,† †
Department of Molecular Evolution, Centro de Astrobiología (INTA-CSIC), Carretera de Ajalvir km 4, Torrejón de Ardoz, 28850 Madrid, Spain ‡ Department of Biology, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain § Centro Nacional de Biotecnología (CSIC), c/Darwin 3, 28049 Madrid, Spain S Supporting Information *
ABSTRACT: Cyanobacteria are Gram-negative photosynthetic prokaryotes that are widespread on Earth. Eutrophication and global warming make some aquatic ecosystems behave as bioreactors that trigger rapid and massive cyanobacterial growth with remarkable economic and health consequences. Rapid and efficient early warning systems are required to support decisions by water body authorities. We have produced 17 specific antibodies to the most frequent cyanobacterial strains blooming in freshwater ecosystems, some of which are toxin producers. A sandwich-type antibody microarray immunoassay (CYANOCHIP) was developed for the simultaneous testing of any of the 17 strains, or other closely related strains, in field samples from different habitats (water, rocks, and sediments). We titrated and tested all of the antibodies in succession using a fluorescent sandwich microarray immunoassay. Although most showed high specificity, we applied a deconvolution method based on graph theory to disentangle the few existing crossreactions. The CYANOCHIP sensitivity ranged from 102 to 104 cells mL−1, with most antibodies detecting approximately 102 cells mL−1. We validated the system by testing multiple isolates and crude natural samples from freshwater reservoirs and rocks, both in the laboratory and by in situ testing in the field. The results demonstrated that CYANOCHIP is a valuable tool for the sensitive and reliable detection of cyanobacteria for early warning and research purposes.
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INTRODUCTION Cyanobacteria are Gram-negative photosynthetic prokaryotes that are common members of the plankton found in marine, brackish, and freshwater ecosystems. They also occur on rocks and soils and in symbioses with plants and fungi. Worldwide, aquatic pollution is driving aquatic ecosystems toward eutrophication, and as a consequence, massive cyanobacterial growth is common. Climate change may trigger changes in cyanobacterial blooming dynamics, increasing its frequency and persistence.1 Massive growths of cyanobacteria have been found in both benthic and planktonic systems,1,2,3 and a primary concern is their potential production of toxic substances that may represent hazards to both organisms in these ecosystems and ecosystem services.4 The World Health Organization has published some guidelines for protecting human health from exposure to some of these toxic compounds produced by cyanobacteria.5 Some countries have adopted these guidelines as part of their regulations for both drinking and bathing water.6 National regulations in many countries require the frequent monitoring of cyanobacteria in water masses, by taxonomical and/or toxicological methodologies to ensure that the health risk to users of these water bodies is minimized. This monitoring effort represents a considerable investment of time and resources. Taxonomical cyanobacterial identification and © 2015 American Chemical Society
counting are currently based on light microscopy techniques and require extensive specialized training. This process is laborintensive, and in some cases, distinguishing specific species morphologically, when specialized cells are not present [e.g., Aphanizomenon ovalisporum (Chrysosporum ovalisporum) and Aphanizomenon aphanizomenoides (Spherospermopsis aphanizomenoides)], can be extremely difficult. The in vivo fluorescent detection of cyanobacterial pigments such as phycocyanin or chlorophyll a has been used both in situ7,8 and through remote sensing,1,9 but these methodologies do not discriminate between different cyanobacterial genera and have their own limitations.10,11 Among molecular methodologies, DNA/RNA microarrays have been recently used to discriminate between close microbial species, including cyanobacteria, with the performance depending on probe quality.12 These nucleic acid microarrays require the extraction, cleanup, and labeling of the nucleic acids before the hybridization process in the laboratory.13,14 Although very useful for taxonomical conReceived: Revised: Accepted: Published: 1611
October 21, 2014 January 3, 2015 January 7, 2015 January 7, 2015 DOI: 10.1021/es5051106 Environ. Sci. Technol. 2015, 49, 1611−1620
Article
Environmental Science & Technology Table 1. Antibodies and Antigens Used in This Work antibody code
immunogen (strain)
order
habitat
K1-AnabN K2-Anab K3-Mflo K4-Mnov K5-Maer K6-Aova K7-Phor K8-Rivu K9-Cham K10-Lbor K11-Tdis K12-Aaph K13-Nant K14-Anaa K15-Lant K16-Toly K17-Plan
Anabaena sp. PCC7120 Anabaena sp. PCC7120 M. f los-aquae UAM242 M. novacekii UAM259 M. aeruginosa UAM265 A. ovalisporum UAM290 Phormidium sp. UAM361 Rivularia sp. UAM369 Chamaesiphon sp. UAM386 Leptolyngbya boryana UAM391 Tolypothrix distorta UAM392 A. aphanizomenoides UAM508 Nostoc sp. (Antarctic) Anabaena sp. UAM545 Leptolyngbya sp. UAM550 Tolypothrix sp. UAM546 Planktothrix rubescens
Nostocales Nostocales Chroococcales Chroococcales Chroococcales Nostocales Oscillatoriales Nostocales Chroococcales Oscillatoriales Nostocales Nostocales Nostocales Nostocales Oscillatoriales Nostocales Oscillatoriales
unknown unknown planktonic planktonic planktonic planktonic benthic benthic benthic benthic benthic planktonic benthic benthic benthic benthic planktonic
a
culture conditions 30 °C, 30 °C, 28 °C, 28 °C, 28 °C, 28 °C, 18 °C, 18 °C, 18 °C, 18 °C, 18 °C, 28 °C, 13 °C, 13 °C, 13 °C, 13 °C, none
continuous light continuous light continuous light continuous light continuous light continuous light 16−8 photoperiod 16−8 photoperiod 16−8 photoperiod 16−8 photoperiod 16−8 photoperiod continuous light 16−8 photoperiod 16−8 photoperiod 16−8 photoperiod 16−8 photoperiod
medium
LODa (cells mL−1)
BG11 and nitrate BG11o BG11 BG11 BG11 BG11o BG11 CHU-D BG11 BG11 BG11o BG11o BG11o BG11o BG11 BG11 none
