Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

1. Preparation of the Immunogens

1. Grow each cyanobacterial strain in the corresponding culture medium under conditions described in Table 1.
   NOTE: Growth medium and culture conditions for each cyanobacteria strain are listed in Table 1. All the cyanobacterial strains, with the exception of K17, belong to Antonio Quesada's group from Autonoma University (Madrid, Spain). The antibody against Planktothrix rubescens was generated from a natural sample of the monospecific bloom of this cyanobacterium from Vilasouto reservoir (northern Spain).
2. Quantify the number of cells using a cell counting chamber by optical microscopy to obtain approximately 10⁸ cells/mL from a late exponential or stationary growth phase culture.
3. Harvest cells from 5 mL of culture by centrifugation at 2,000 x g for 5 min.
4. Discard the supernatant and resuspend the cells in a 10-mL tube with 5 mL of 1x phosphate-buffered saline (1x PBS) to obtain around 10⁸ cells/mL.
5. Homogenize and lyse the cells of the suspension by sonication for 5 cycles, 30 s each, with a 30- to 60-s pause on ice, using a portable, hand-held ultrasonic processor or by dipping the tube into the water bath of a cell disruptor at maximum amplitude (30 kHz).
6. Repeat steps 1.3 – 1.5 for each strain of cyanobacterium.
   NOTE: Sonication produces cell disruption and cellular content release. While proteins and polysaccharides are good immunogens, specific molecules, such as lipids and nucleic acids, do not induce a humoral immunogenic response by themselves. Therefore, they must bind to carriers, such as polysaccharides or proteins, to increase the molecular complexity. In addition, sonication releases intracellular material that can trigger antibody production.

2. Production of Polyclonal Antibodies

1. Prepare the first immunogen dose by mixing 0.5 mL of the ultrasonicated cell lysate obtained in step 1.5 with 0.5 mL of complete Freund's adjuvant. Deliver it to the operator of an animal facility for polyclonal rabbit antibody production.
2. Prepare three more doses as before for further use as memory boosts in the antibody production process by mixing 0.5 mL of the same homogenate/lysate obtained in step 1.5 with 0.5 mL of incomplete Freund's adjuvant. Give them to the animal facility to fulfill the antibody production process.
3. Repeat steps 2.1 and 2.2 for each new antibody production process.
   NOTE: Normally, antibody production is entrusted to specialized animal facilities or companies, because appropriate licensing and training are required to work with animals. The companies supply a relative enzyme-linked immunosorbent assay (ELISA) measurement of the quantity of antigen-specific antibodies present in the serum sample.

3. Antibody Purification

1. Purify the immunoglobulin G (IgG) fraction from both the immune and the pre-immune serum collected in step 2 by protein-A affinity chromatography. Some commercial purification kits, based on cartridge systems, work well; follow the provider instructions.
2. When the purification kit does not provide a desalting system, change the buffer after the elution of the purified antibodies to 0.1x PBS, either by dialysis or by using centrifugal filter devices with a 100-kDa (or lower) membrane pore size.
3. Determine the antibody concentration by measuring the absorbance at 280 nm or by using colorimetric methods, such as Bradford²³, Lowry²⁴, or Bicinchoninic acid (BCA)²⁵.
   NOTE: It is important to avoid including amine groups in the elution buffer (e.g., Tris buffers) because they compete with the antibody for binding to solid surfaces activated with epoxy groups. After purification, it is important to test the antibody activity with ELISA.

4. Fluorescence Antibody Labeling

1. Label the purified antibodies obtained in section 3 with a fluorochrome (e.g., a far-red fluorescent dye) by dissolving a commercial vial containing dye for labeling 1 mg of protein into 100 µL of dimethyl sulfoxide (DMSO). Add 2 µL of the dissolved dye to each antibody preparation at a concentration of 2 mg/mL in a final volume of 50 µL in 50 mM phosphate-buffered saline (pH 8.5).
2. Maintain the labeling reactions under continuous agitation for 1 h at ambient temperature and 1,200 rpm on a vibrating platform.
3. Purify the labeled antibodies by size exclusion chromatography (e.g., using a gel with a fractionation range between 1.5 and 30 kDa trapped into a column), following supplier recommendations.
4. Measure the absorbance at 280 nm and at 650 nm in eluates and calculate the labeling efficiencies following supplier recommendations.
   NOTE: Up to 50 x 50-µL antibody-labeling reactions can be done with a single vial of the fluorescent dye for labeling 1 mg of protein. It is recommended to cover the tubes with aluminum foil or, alternatively, to use opaque 0.5-mL tubes to avoid quenching processes after step 4.3. For IgG antibodies, optimal labeling is achieved with 3-7 mol of the dye per mol of antibody.

5. CYANOCHIP Production

1. Antibodies and controls in printing solution
   1. Prepare 30 µL of each purified antibody in printing solution by mixing each antibody at 1 mg/mL in a commercial 1x protein printing buffer with 0.01% (v/v) Tween 20 (a non-ionic detergent), all as final concentrations.
      NOTE: Alternatively, an antibody solution may contain 20% glycerol, 1% (w/v) sucrose (or trehalose, as a preservative), and 0.01% (v/v) Tween 20 in carbonate buffer (pH 8.5).
   2. Prepare 30 µL of the printing solution as controls: (a) 1x protein printing buffer with 0.01% (v/v) Tween 20, (b) protein-A purified pre-immune serum at 1 mg/mL in 1x protein printing buffer with 0.01% (v/v) Tween 20, and (c) bovine serum albumin (BSA) at 1 mg/mL in 1x protein printing buffer with 0.01% (v/v) Tween 20.
   3. Prepare 30 µL of a fluorescently labeled, purified pre-immune serum at different concentrations (e.g., from 50 µg/mL to 1 µg/mL) in 1x protein printing buffer with Tween 20, as in step 5.1.1. These samples will be used as fluorescent frame markers and for relative fluorescence quantification after spotting.
   4. Add 30 µL per well of the printing solutions prepared in steps 5.1.1, 5.1.2, and 5.1.3 to the highest-quality 384-well microplates for microarray manufacturing, such as polypropylene.
      NOTE: Protein printing buffer increases the quality and stability of the antibodies, and Tween 20 homogenizes spot morphology and builds up protein coupling up. It is recommended to maintain the 384-well microplate at 4 °C before use. Store it at -20 °C for long periods of time. Polypropylene has low DNA, protein, cell extract, and small-molecule intrinsic binding.
2. Antibody printing onto a microscope slide
   NOTE: Print the antibodies onto activated microscope slides by using different array platforms, like contact, split-needle, or non-contact devices, such as piezoelectric printers or "ink jet" technologies. In this work, the CYANOCHIP has been routinely printed by contact using a robotic system (arrayer) capable of spotting nL quantities of the antibodies at the µm scale.
   1. Set the environmental conditions of the printing room to 20 °C and 40 – 50% relative humidity.
   2. Set up the slide substrates (e.g., 75- x 25-mm epoxy-activated microscope glass slides) to perform several identical antibody arrays on each slide.
   3. Spot each purified antibody, including controls and the reference frame, in a triplicate spot pattern; under these conditions, the spots are 180 – 200 µm in diameter.
   4. After printing, leave the slides for at least 30 min at ambient temperature to let them dry, and then store them at 4 °C; for working in the field, the slides can be transported and stored at ambient temperature for several months.
      NOTE: The CYANOCHIP is printed in a triplicate spot microarray format with 3 x 8 identical microarrays per slide or 9 identical arrays in a 1 x 9 microarray format. Each array size must not be higher than the reaction chamber dimensions for a 24-well gasket (usually 7.5 x 6.5 mm in a 3 x 8 hybridization chamber).
   5. Before using the microarray to analyze environmental samples, use FSMI to determine the working dilution for each antibody in a titration curve. For each antibody, use a standard concentration of the corresponding immunogen (10³ – 10⁴ cells/mL) and serial dilutions of the fluorescent antibody (between 1:500 and 1:32,000). The optimal antibody concentration corresponds to 50% of the maximum signal intensity obtained in the titration curve. Also, the sensitivity and specificity for each antibody must be determined, as described in Blanco et al.²².

6. Preparation of Environmental Multianalyte Extracts for the Fluorescent Sandwich Microarray Immunoassay (FSMI)

1. Multianalyte extract from a liquid sample
   1. Take 1 – 100 mL of the liquid sample with a sterile syringe (e.g., water from the shore of a water reservoir); the amount of sample is greatly dependent upon the potential concentration of the targets.
   2. Concentrate the cells by passing the water sample through a 3-µm pore size, 47-mm diameter polycarbonate filter; a cellular concentration between 10³ – 10⁸ cells/mL is desirable for positive detection, but the actual concentration is unknown.
   3. Recover the biomass collected in the filter with 1 mL of a modified Tris-buffered saline, Tween 20-reinforced buffer (TBSTRR; 0.4 M Tris-HCl (pH 8), 0.3 M NaCl, and 0.1% Tween 20) by scraping it with a spatula into a 15-mL tube.
   4. Homogenize and disaggregate by using a hand-held ultrasonic processor, as described in step 1.5, or just by pipetting up and down multiple times; this prepares the sample for analysis by the microarray.
2. Multianalyte extract from a solid sample
   1. Weigh up to 0.5 g of the solid sample (e.g., rock, soil, or sediments) into a 10-mL tube and add up to 2 mL of TBSTRR.
   2. Sonicate by immersing the sonicator probe in the tube, by dipping the tube into the water bath of a powerful sonicator horn, or by using a hand-held sonicator. Perform at least 5 x 30-s cycles at 30 kHz, stopping for 30 s while on ice .
   3. Filter to remove sand, clay, and other coarse material with a 10-mL syringe coupled to a 10- to 12-mm diameter, 5- to 20-µm pore size nylon filter holder. Push the sample through the filter into a 1.5-mL tube. If the filter saturates, agitate the suspension in the syringe and take it to a new one; this prepares the filtrate material for the immunoassay (step 7).
      NOTE: The buffering capacity of TBSTRR depends on the type of sample. It is important to carry out the immunoassay immediately after the preparation of the environmental extract to avoid the effect of enzymatic degradation on the analytes. Alternatively, add protease inhibitors into the environmental extract and freeze at -80 °C until the next step.

7. Fluorescent Sandwich Microarray Immunoassay (FSMI)

1. Blocking the CYANOCHIP
   NOTE: Immediately before use, treat the printed slides to block all free epoxy groups on the slide and to remove the excess of non-covalently bound antibodies.
   1. Immerse the microarray into 0.5 M Tris-HCl (pH 9) with 5% (w/v) BSA solution on a clean surface (e.g., Petri dish or a 50-mL tube) with mild agitation from a rocker platform for 5 min. Alternatively, lay the slide down onto a 100- to 200-µL drop of the above solution with the microarray spots facing it. Leave for 3 – 5 min and then proceed.
   2. Carefully pick up the slide using plastic-tipped forceps; try to avoid touching microarray zones. Eliminate the excess of liquid by softly knocking the slide onto a paper towel. Immerse it in 0.5 M Tris-HCl (pH 8) with 2% (w/v) BSA solution for 30 min with mild agitation from a rocker platform.
   3. Dry the slide by performing a short centrifugation (200 – 300 x g for 1 min) using a commercial microcentrifuge adapted for microscope slides. Alternatively, dry the slide by softly knocking onto a paper towel.
2. Incubation of the multianalyte sample extract with the microarray
   1. Set up the slide in a commercial microarray hybridization cassette with 24 wells for multiple microarrays; follow the provider instructions.
   2. After slide and cassette assembly, pipette up to 50 µL of the sample extract or a dilution of it in TBSTRR into each well of the cassette.
   3. Repeat step 7.2.2 for each sample to be analyzed.
   4. As a blank control, pipette 50 µL of TBSTRR buffer into at least two separate wells of the cassette.
   5. Incubate at ambient temperature for 1 h with mixing by pipetting every 15 min or by leaving it under mild shaking. Alternatively, incubate for 12 h at 4 °C.
      NOTE: Use other incubation gaskets as a function of the microarray pattern. The time and temperature of the incubation in step 7.2.5 are empirical parameters that normally depend on the affinity and binding kinetics of each paired antigen-antibody.
3. Washing
   1. Remove the samples by putting the cassette down and carefully knocking it onto a clean, absorbent paper.
   2. Wash the wells by adding 150 µL of TBSTRR to each one, and eliminate the buffer, as above.
   3. Repeat step 7.3.2 three more times.
4. Incubation with fluorescent detector antibodies
   1. Add 50 µL of an antibody mixture containing the 17 anti-cyanobacterial-strain antibodies, each labeled with the fluorochrome in TBSTRR with 1% (w/v) BSA. Determine the concentration of each fluorescent antibody in the mixture (from 0.7 to 2 µg/mL) by performing titration experiments of each antigen/antibody pair²².
   2. Incubate for 1 h at ambient temperature, as described in step 7.2.5, or for 12 h at 4 °C.
5. Washing out the fluorescent antibodies
   1. Remove the fluorescent unbound antibodies, as in step 7.3.
   2. Disassemble the cassette and immerse the slide into 0.1x PBS (e.g., in a 50-mL tube) for a quick rinse.
   3. Dry the slide as in step 7.1.3.

8. Scanning for Fluorescence

1. Scan the slide for fluorescence at the maximum emission fluorescence peak for far-red fluorescent dye in a scanner for fluorescence. Take several images of the microarrays at different scanning parameters, generally by lowering the laser gain value.
   NOTE: Avoid saturated spots (> 65,000 fluorescence counts)—they are out of scale and may introduce quantification errors.

9. Image Processing and Data Analysis

1. Use a commercial software for image analysis and quantification; the software provides measurements of the fluorescence intensity (FI; median or mean of all pixels from a single spot) for each spot of the whole microarray. Subtract the local background around the spots: FI = FI_spot – FI_{local background}.
2. Save the FI data and open them using a spreadsheet program.
3. Use the values obtained for the blank arrays as the negative control to identify and discard the false positives. Apply the following equation to calculate the FI for each antibody spot: FI = (FI_sample – FI_blank), where FI_sample = FI_spot – FI_{local background} in the microarrays run with sample extracts and FI_blank = FI_spot – FI_{local background} in the blank microarrays.
4. Apply an additional cut-off threshold of 2- to 3-fold the average of the FI of the whole microarray to minimize the probability of false positives; this is especially relevant for poor-quality microarray images and low signal-to-noise ratios.
5. Create plots and/or perform further analysis as necessary.