The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.
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.
The detection and monitoring of microorganisms in complex natural microbial communities are crucial in many fields, including biomedicine, environmental ecology, and astrobiology. Cyanobacteria are prokaryotic microorganisms well-known for their ability to form blooms (excessive proliferation) of cells in fresh water. They are ubiquitous, and many species are able to produce toxins, leading not only to a potential risk for human health, but also to an ecological impact. In this regard, it is essential to develop rapid and sensitive methods for the early detection of cyanobacteria and/or their toxins in soil and water. For this purpose, a multiplex fluorescent sandwich microarray immunoassay (FSMI) has been developed as a tool for water managers to help them in making decisions and, consequently, in implementing proper water management programs.
A diverse range of methods has been developed to detect and identify cyanobacterial cells and cyanotoxins in soil and water, including optical microscopy, molecular biology, and immunological techniques. These methods can vary greatly in the information they provide. Microscopic techniques are based on cell morphology and the detection of in vivo fluorescence from cyanobacterial pigments, such as phycocyanin or chlorophyll a1. Although they are quick and cheap methods for real-time and frequent monitoring that inform about the type and number of cyanobacteria present in a sample, they do not give information about the potential toxicity. In addition, they require a certain level of expertise, considering that it is often very difficult to distinguish between closely related species2. To overcome these limitations, light microscopy must be accompanied by both biological and biochemical screening assays and physicochemical methods for the identification and quantification of cyanotoxins.
Enzyme-linked immunosorbent assays (ELISA), protein phosphate inhibition assays (PPIA), and neurochemical tests in mice are examples of biochemical screening assays for the detection of cyanotoxins. While the first two are rapid and sensitive methodologies, false positives have been described when using ELISA and PPIA tests are restricted to three types of toxins. The mouse bioassay is a qualitative technique with low sensitivity and precision, and special licensing and training is required. In addition, it does not give information about the type of toxins present in a sample. Cyanotoxins can be identified and quantified by other analytical methods, such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), or matrix-assisted laser desorption/ionization time of flight (MALDI-TOF). However, this is only possible if reference standards, which are needed to determine individual toxin concentrations in complex samples, are available3,4. Moreover, these methods are time-consuming; require costly equipment, supplies, and sample preparation; and must be performed by experienced and specialized staff.
Molecular-based methods have been applied for decades to detect, identify, and quantify cyanobacteria and their corresponding cyanotoxins thanks to the sequence information published in the genome databases (e.g., National Center for Biotechnology Information, NCBI). Among these methods are those based on the polymerase chain reaction (PCR), which requires the design of sets of primers for DNA amplification and depend on previous knowledge of DNA sequences of different cyanobacterial species. While gene detection, like the phycocyanin operon, leads to accurate identification at the genus level, some species or strains are undetected with this method. However, toxin-encoding genes, such as those belonging to the microcystin operon, facilitate the identification of toxins in samples where the producers are scarce5. Nonetheless, the detection of toxin markers by PCR does not necessarily imply toxicity in the environment. Furthermore, the set of primers developed to analyze the whole range of species of cyanobacteria and toxin producers present in a sample is still incomplete, and further studies must be done to identify unknown species. Other molecular techniques are non-PCR-based, such as fluorescence in situ hybridization (FISH) and DNA microarrays.
In the last two decades, microarray technology has gained importance in many fields of application, particularly in environmental monitoring. DNA microarrays allow for discrimination between species and analytes4,6,7,8,9,10, but they are considered very laborious and time-consuming tasks that involve multiple steps (e.g., microarray performance, DNA extraction, PCR amplification, and hybridization). For that reason, less time-consuming assays based on antibodies, such as sandwich and competitive immunological microarrays, have become an essential and reliable high-throughput method for the detection of multiple environmental analytes11,12,13. The capability of antibodies to specifically recognize their target compounds and to detect small amounts of analytes and proteins, along with the possibility of producing antibodies against almost any substance, make antibody microarrays a powerful technique for environmental purposes. In addition, the capability of achieving multiple analyses in a single assay, with limits of detection ranging from ppb to ppm, is one of the main advantages of this method14.
Antibody-based biosensors have proved to be sensitive and rapid tools for the detection of a wide range of pathogens and toxins in environmental monitoring15,16,17,18,19,20,21. While DNA methods involve several steps, the antibody-based microarrays only require a small sample preparation that is mainly based on a short lysis step in an appropriate solution buffer. Delehanty and Ligler15 reported the simultaneous detection of proteins and bacterial analytes in complex mixtures based on an antibody sandwich immunoassay capable of detecting a protein concentration of 4 ppb and 104 cfu/mL of cells. Szkola et al.21 have developed a cheap and reliable multiplex microarray for the simultaneous detection of proteotoxins and small toxins, compounds that might be used in biological warfare. They detected concentrations of ricin toxin, with a limit of detection of 3 ppb, in less than 20 min. Recently, the CYANOCHIP, an antibody microarray-based biosensor for the in situ detection of toxic and nontoxic cyanobacteria, has been described22. This microarray allows for the identification of potential cyanobacterial blooms, mostly in aquatic environments, which are difficult to identify microscopically. The limit of detection of the microarray is 102 – 103 cells for most species, turning this biosensor into a cost-effective tool for the multiplex detection and identification of cyanobacteria, even at the species level. All these properties make the antibody microarray technique, and particularly the method presented in this work, a quicker and simpler method compared to the aforementioned techniques.
This work presents two examples of experiments that use an antibody microarray-based biosensor to detect the presence of cyanobacteria in soil and water samples. It is a simple and reliable method based on a sandwich immunoassay format that requires very small sample volumes and very basic sample preparation. The method requires a short time and can be easily performed in the field.
1. Preparation of the Immunogens
2. Production of Polyclonal Antibodies
3. Antibody Purification
4. Fluorescence Antibody Labeling
5. CYANOCHIP Production
6. Preparation of Environmental Multianalyte Extracts for the Fluorescent Sandwich Microarray Immunoassay (FSMI)
7. Fluorescent Sandwich Microarray Immunoassay (FSMI)
8. Scanning for Fluorescence
9. Image Processing and Data Analysis
This work describes a multiplex immunoassay test for the simultaneous identification of the most relevant freshwater cyanobacterial species (Table 1) using the CYANOCHIP antibody microarray. The microarray can be a 3 x 8 microarray format printed onto microscope slides. Each microarray is made up of a set of 17 antibodies printed in a triplicate spot pattern, their corresponding pre-immune antibodies and BSA as negative controls. The microarrays also include a fluorescent frame, using a fluorescently labeled pre-immune antibody to easily localize the microarray pattern22 (Figure 1).
The microarray was tested in the field for the in situ analysis of water samples collected at the shore of the freshwater Lozoya Reservoir, which supplies the city of Madrid. Samples were processed as described above, and the main positive immunoreactions corresponded to antibodies to planktonic cyanobacteria, such as Microcystis spp. (K4 and K5), and to benthic Oscillatoriales, such as Leptolyngbya spp. (K10 and K15). Lower fluorescence signals were obtained from Nostocales (K6 and K12, two planktonic Aphanizomenon spp.), benthic Rivularia sp. (K8), Anabaena sp. (K1), and planktonic Microcystis flos-aquae sp. (K3). Optical microscope observations (not shown) showed a diverse cyanobacterial community within abundant terrigenous debris from the shore. Several species of Anabaena dominated the community, but Microcystis spp. and Pseudanabaena spp. were also present.
Additionally, the microarray was also validated by assaying a dry, nearly 1,000-year-old microbial mat collected in the McMurdo Ice Shelf in Antarctica to test for the presence of cyanobacterial markers. Figure 1 shows highly fluorescent, positive reactions in antibodies produced to benthic cyanobacteria isolated from other Antarctic mats, including Anabaena sp. and Leptolyngbya spp. (K14 and K15, respectively). Additional positive immunoreactions were detected with antibodies to planktonic cyanobacteria, such as Microcystis spp. (K4 and K5), Aphanizomenon spp. (K6 and K12), and Planktothrix rubescens sp. (K17). Low signals were obtained from antibodies to other benthic species isolated in an Antarctic mat, including Tolypothrix sp. (K16) and Anabaena sp. (K1). Fluorescent optical microscopy revealed the presence of cells structurally similar to cyanobacteria and green algae (not shown). No filamentous cyanobacteria were identified. Nevertheless, multiple amorphous fluorescent structures of about 1 µm in size and copious diffuse fluorescence were detected, which could be attributed to the presence of cell remains (broken and dead cells) and extracellular polymeric substances (EPS), respectively. Accordingly, the biochemical analysis showed that the mat consisted of profuse amounts of biopolymers and cell remains (complex biological matter) that could be the targets for the antibodies in the microarray.
Figure 1: Quick and Reliable Multiplex Microarray Immunoassay for Detecting Cyanobacteria. A) Scheme showing the main steps of the FSMI for the analysis of multi-target samples with a CYANOCHIP. B) Schematic of a printing pattern layout (by triplicate) of the anti-cyanobacteria antibody collection: (0) BSA, bovine serum albumin; (X) only printing buffer; (1-17) each of the antibodies as in Table 1 in Blanco et al. 2015 (K1 to K17). From p1 to p17, the corresponding pre-immune antibodies act as controls. The yellow rectangles correspond to a fluorescent spot gradient as a frame reference. C and D) Picture showing the top part of a 1,000-year-old dry microbial mat from the McMurdo Ice Shelf (Antarctica) and the CYANOCHIP image detecting cyanobacteria in this mat, respectively. E) Panoramic view of the Lozoya Reservoir showing transparent water with no visible green particles at the time of sampling. F) microarray image after the in situ analysis of the water sample (modified from reference 22).
Ab code | Immunogen (strain) | Order | Habitat | Medium | Culture conditions |
K1 | Anabaena sp. | Nostocales | unknown | BG11 and nitrate | 30 ºC, continuous light |
K2 | Anabaena sp. | Nostocales | unknown | BG11o | 30 ºC, continuous light |
K3 | Microcystis flos-aquae | Chroococcales | planktonic | BG11 | 28 ºC, continuous light |
K4 | Microcystis novacekii | Chroococcales | planktonic | BG11 | 28 ºC, continuous light |
K5 | Microcystis aeruginosa | Chroococcales | planktonic | BG11 | 28 ºC, continuous light |
K6 | Aphanizomenon ovalisporum | Nostocales | planktonic | BG11o | 28 ºC, continuous light |
K7 | Phormidium sp. | Oscillatoriales | benthic | BG11 | 18 ºC, 16-8 photoperiod |
K8 | Rivularia sp. | Nostocales | benthic | CHU-D | 18 ºC, 16-8 photoperiod |
K9 | Chamaesiphon sp. | Chroococcales | benthic | BG11 | 18 ºC, 16-8 photoperiod |
K10 | Leptolyngbya boryana | Oscillatoriales | benthic | BG11 | 18 ºC, 16-8 photoperiod |
K11 | Tolypothrix distorta | Nostocales | benthic | BG11o | 18 ºC, 16-8 photoperiod |
K12 | Aphanizomenon aphanizomenoides | Nostocales | planktonic | BG11o | 28 ºC, continuous light |
K13 | Nostoc sp. (Antarctica) | Nostocales | benthic | BG11o | 13 ºC, 16-8 photoperiod |
K14 | Anabaena sp. | Nostocales | benthic | BG11o | 13 ºC, 16-8 photoperiod |
K15 | Leptolyngbya sp. | Oscillatoriales | benthic | BG11 | 13 ºC, 16-8 photoperiod |
K16 | Tolypothrix sp. | Nostocales | benthic | BG11 | 13 ºC, 16-8 photoperiod |
K17 | Planktothrix rubescens | Oscillatoriales | planktonic | none | none |
Table 1. List of the Antibodies (Abs) and the Cyanobacterial Strains Used to Produce the CYANOCHIP22.
Here, a multiplex fluorescent sandwich immunoassay using the CYANOCHIP, a 17-antibody microarray for the detection and identification of a wide range of cyanobacterial genera, is described22. These cyanobacteria represent the most frequent benthic and planktonic genera in freshwater habitats, some of them being toxin producers. Recently, the fluorescent sandwich immunoassay format has been used to identify microorganisms and/or bioanalytes in environmental applications26,27,28. The protocol is primarily based on two steps: (i) immobilizing or capturing antibodies to specifically bind analytes from a test sample and (ii) detecting analyte-antibody pairs by using fluorescently labeled antibodies (tracer or detector antibodies). Because the sandwich assay requires at least two accessible antibody binding sites (epitopes) in the analyte for the reaction to take place, any positive fluorescent signal indicates the presence of relatively large and complex oligo- or multimeric analytes that are identical or highly similar to the ones used to produce the capturing antibodies.
Even though this method requires small volumes and basic sample preparation, without the need for special expertise or knowledge, several drawbacks could ruin the assay. Low fluorescent signals or a complete lack thereof may be due to poor antibody purification or poor labeling efficiency. For the isolation of rabbit IgG, protein A is the best choice, because it specifically binds with high efficiency to the Fc region of immunoglobulins. As indicated in section 4, fluorescent antibodies with a labeling range between 3-7 mol of dye per mole of antibody must be used. Furthermore, a lack of fluorescent spots can be explained by the concentration of analytes lying under the limits of detection. In this case, the sample can be concentrated before incubating it with the microarray, or greater amounts can be used for a new extraction. High fluorescent backgrounds are the result of inefficient chip blocking or/and washing steps and of extracts from samples composed of minerals and complex organic matter that stick onto the chip. When complex samples are used as analytes, it is desirable to increase the salt and/or the detergent concentration to favor the specific interaction between analyte-antibody pairs.
In recent years, antibody microarray technology has been developed for environmental applications. Nonetheless, the use of this technique does involve some limitations. Polyclonal antibodies are faster and cheaper to produce and, more importantly, the possibility to bind any target epitope in a complex environmental sample are theoretically higher than with monoclonal antibodies. However, the fact that they can recognize different epitopes increases the number of cross-reactions in FSMI. To gain high specificity, the use of methods to disentangle these cross-reactivity events from the true cognate antigen-antibody reactions by using deconvolution methods26,27, for example, is highly desirable. Basically, the microarray is considered a qualitative biosensor for the multiple detection and classification of cyanobacteria. In this regard, it is essential to determine the limit of detection for each antibody one-by-one to use their optimal working concentrations in the assay. Although this microarray, together with FSMI, is not conceived as a quantitative method, the biosensor implies high sensitivity, because the lower detection limit of most of the antibodies contained in the CYANOCHIP22 is from 102 to 103 cells/mL.
Despite these limitations, this methodology has several advantages against other techniques used in environmental monitoring. Antibody-biosensors allow the possibility of recognizing different molecules simultaneously in a single analysis, the possibility of detecting low concentration of analytes, and the possibility of producing antibodies against almost any substance. Furthermore, the microarray can achieve up to 24 analyses in a single assay, with limits of detection from 102 cells/mL. In comparison with other methods, antibodies can detect living or dead cells, extracellular material, and cellular debris. Although it takes at least 4 h to complete the whole assay, it is faster than other analytical methods applied to environmental monitoring. The CYANOCHIP was originally conceived for the identification of cyanobacterial strains. This biosensor was not formally designed for that purpose, so it can identify potential toxin producers and could be improved in the future by adding antibodies against a wide range of new strains. Biochemical assays, such as ELISA, PPIA, and neurochemical tests in mice, allow the identification of cyanotoxins, but they are restricted to a few known toxins and can give false positives. In addition, using the CYANOCHIP does not require special training, while optical microscopy and mouse bioassays require trained personnel or labor-intensive work with live animals, and they do not give information about the type of toxins present in a sample. Cyanotoxins can also be identified and quantified by other analytical methods, such as HPLC, GC-MS, or MALDI-TOF. In these methods, the sample must be purified, and the lack of reference standards limits the identification of cyanotoxins. Furthermore, analytical methods require costly equipment and supplies and specialized training. Molecular methods are based on DNA extraction from the samples, while FSMI only requires an environmental extract prepared in a couple of very simple steps, without cyanobacteria purification.
The high performance of the CYANOCHIP for the in situ detection of cyanobacteria in freshwater reservoirs makes it a new tool for the early warning of water administrators. In addition, the microarray is also interesting for the field of astrobiology, particularly for searching for microbial markers as evidence of life. The study of extremophiles can help us to understand the origin of life on Earth and how life could survive in the extreme environments present in our solar system and beyond. As several environments on Earth are very similar to places on other planets, such as Mars, it might be possible to find remains of photosynthetic prokaryotes as evidences of extinct life. The microarray was able to identify cyanobacterial markers from living or nonliving cells; from population remains and/or extracellular material in old microbial mats (Figure 1); and from water, soil, and rocks collected in extreme environments, such as the Antarctica, Atacama, the Andean lakes, the High Arctic, or the Rio Tinto area in Spain (not shown). Considering that cyanobacteria are the primeval microorganisms on Earth, there is reason to believe that they might once have lived on other planets.
In conclusion, the fact that CYANOCHIP-FSMI can identify in situ cyanobacterial markers and can even associate them to different phylotypes or groups, and that the microarray covers a broad range of habitats, including those of plankton, benthos, and endoliths, demonstrates that this technique could a tool for environmental monitoring. Future improvements to the microarray could be to increase the number of antibodies to new strains so that relevant phylogenetic groups still pending are included. Additionally, the chip can be implemented with antibodies to specific cyanobacterial compounds, such as toxins or cyanophicin polymer. This is especially relevant for monitoring fresh water reservoirs, pipes, and installations in human facilities. The current microarray and future versions will be very useful in the field of astrobiology, either for life detection or for monitoring human space facilities (e.g., monitoring water reservoirs or life support systems). In fact, we routinely use the microarray in field campaigns to extreme environments as a method for the "on-site" detection of life remains. The microarray is part of the so-called Life Detector Chip (LDChip), a microarray with more than 300 antibodies for the search for life in planetary exploration missions29,30. The microarray alone or as part of the LDChip will be implemented in the Signs of Life Detector (SOLID) instrument29 to validate the SOLID-LDChip concept for planetary exploration in multiple field campaigns to terrestrial analogues. The microarray will provide useful information by identifying cyanobacteria and/or their toxins. By determining the strains, it will give information about the environments and the habitats in which they developed.
The authors have nothing to disclose.
We thank Dr. Antonio Quesada from the Universidad Autónoma de Madrid for providing cyanobacterial strains. This work was funded by the Subdirección General de Proyectos de Investigación of the Spanish Ministerio de Economía y Competitividad (MINECO), grants no. AYA2011-24803 and ESP2014-58494-R.
0.22 mm pore diameter filters | Millipore | GSWP04700 | For preparation of immunogens |
Eppendorf 5424R microcentrifuge | Fisher Scientific | For preparation of immunogens | |
Phosphate buffer saline (PBS) pH 7.4 (10X) | Thermofisher Scientific | 70011036 | 50 mM potassium phosphate, 150 mM NaCl, pH 7.4 |
Ultrasonic processor UP50H | Hielscher | For preparation of immunogens | |
Complete Freund's adjuvant | Sigma-Aldrich | F5881 | Immunopotentiator |
Incomplete Freud's adjuvant | Sigma-Aldrich | F5506 | For boost injections |
Protein A antibody purification kit | Sigma-Aldrich | PURE1A | For isolation of IgG |
Centrifugal filter devices MWCO<100 KDa | Millipore | UFC510096-96K | For isolation of IgG |
Dialysis tubings, benzoylated | Sigma-Aldrich | D7884-10FT | For isolation of IgG |
Illustra Microspin G-50 columns | GE-HealthCare | GE27-5330-02 | For isolation of IgG |
Bradford reagent | Sigma-Aldrich | B6916-500 mL | To quantify the antibody concentration |
MicroBCA protein assay kit | Thermo Scientific | 23235 | To quantify the antibody concentration |
Protein arraying buffer 2X | Whatman (Sigma Aldrich) | S00537 | Printing buffer; 30-40% glycerol in 1X PBS with 0.01% Tween 20 |
Tween 20 | Sigma-Aldrich | P9416 | Non-ionic detergent |
Bovine serum albumin (BSA) | Sigma-Aldrich | A9418 | Control for printing; blocking reagent |
384-wells microplate | Genetix | X6004 | For antibody printing |
Robot arrayer for multiple slides | MicroGrid II TAS arrayer from Digilab | For antibody printing | |
Epoxy substrate glass slides | Arrayit corporation | VEPO25C | Solid support for antibody printing |
Alexa Fluor-647 Succinimidyl-ester | Molecular probes | A20006 | Fluorochrome |
DMSO | Sigma-Aldrich | D8418 | Fluorochrome dissolvent |
Heidolph Titramax vibrating platform shaker | Fisher Scientific | For antibody labeling | |
Illustra Microspin G-50 columns | Healthcare | 27-5330-01 | For purification of labeled antibodies |
Safe seal brown 0,5 ml tubes | Sarstedt | 72,704,001 | For labeled antibodies storage |
Nanodrop 1000 spectrophotometer | Thermo Scientific | To quantify antibody concentration and labeling efficiency | |
3 µm pore size polycarbonate 47 mm diameter filter | Millipore | TMTP04700 | To concentrate cells |
1M Trizma hydrochloride solution pH 8 | Sigma-Aldrich | T3038 | For TBSTRR preparation; to block slides |
Sodium chloride | Sigma-Aldrich | S7653 | For TBSTRR preparation |
20 µm nylon filters | Millipore | NY2004700 | For environmental extract preparation |
10-12 mm filter holders | Millipore | SX0001300 | For environmental extract preparation |
Protease inhibitor cocktail | Sigma-Aldrich | P8340 | For environmental extract storage |
1M Trizma hydrochloride solution pH 9 | Sigma-Aldrich | T2819 | To block slides |
Heidolph Duomax 1030 rocking platform shaker | VWR | To block slides; for incubation processes | |
VWR Galaxy miniarray microcentrifuge | VWR | C1403-VWR | To dry slides |
Multi-Well microarray hybridization cassette | Arrayit corporation | AHC1X24 | Cassette for 24 assays per slide |
GenePix 4100A microarray scanner | Molecular Devices | Scanner for fluorescence | |
GenePix Pro Software | Molecular Devices | Software for image analysis and quantification |