Experimental Protocol for Detecting <em>Cyanobacteria </em>in Liquid and Solid Samples with an Antibody Microarray Chip

Abstract

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-μm filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 μm) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Prokaryotic Community Structure and Metabolisms in Shallow Subsurface of Atacama Desert Playas and Alluvial Fans After Heavy Rains: Repairing and Preparing for Next Dry Period

“…The targets for the antibodies of this study are described in Sánchez-García et al (2018). IgG fraction of each antibody was printed in a triplicate spot-pattern on the surface of epoxy-activated glass slides as described in Blanco et al (2017). To perform the FSMI, all IgGs were fluorescently labeled with Alexa 647, titrated, and used in a mixture that consisted of 181 antibodies to reveal the immunoreactions as reported by Rivas et al (2008).…”

“…Detailed protocol for the analysis of LITA samples with the LDChip200 is described in Blanco et al (2017). In summary, 0.5 g of each protein extract (see previous section) was resuspended in 2 ml of TBSTRR buffer (0.4 M Tris-HCl pH 8, 0.3 M NaCl, 0.1% Tween 20) and was used as a multianalyte-containing sample for the FSMI.…”

Microbiology and Nitrogen Cycle in the Benthic Sediments of a Glacial Oligotrophic Deep Andean Lake as Analog of Ancient Martian Lake-Beds

“…The strains belong to the genera: Brevundimonas , Micrococcus , Frigoribacterium , Frondihabitans , Polaromonas , Sporosarcina , Rhodococcus , Polaromonas , Paenibacillus , Bacillus , Tumebacillus , Dechlorobacter , Azospira , Arcobacter (see Supplementary Table S1 for details). Antibody purification, titration, printing onto microscope slides, antibody fluorescence labeling and multiplex microarray immunoassays were carried out as described in Rivas et al (2008), Parro et al (2011a),and Blanco et al (2017).…”

“…Then, samples were filtered through 8-μm nitrocellulose filters to remove sand and coarse material. 50 μl of each filtrate were used as multianalyte-containing sample for fluorescence sandwich microarray immunoassays (FSMI) as described in previous works (Parro et al, 2011a, 2018; Blanco et al, 2017). As a blank control, only buffer was incubated with the chip instead of sample.…”

Prokaryotic and viral community of the sulfate‐rich crust from Peñahueca ephemeral lake, an astrobiology analogue

“…It contains about 300 antibodies against different molecules including those from cell membranes of archaea and bacteria, extracellular polymers, environmental extracts, proteins, DNA, peptides, exopolysaccharides (EPS) and amino acids. Samples were screened for microbial markers in the field laboratory by fluorescence sandwich microarray immunoassay as previously described Blanco et al, 2017). Briefly, the potential biomarkers present in 0.5 g of sample were extracted by ultrasonication into 2 ml saline buffer (0.1 M Tris-HCl pH 8, 0.1 M NaCl, 0.1% Tween 20), filtered through 10 μm, and then 50 μl of the extract incubated with one out of the nine printed antibody microarrays per microscope slide.…”