Research Articles
ASTROBIOLOGY Volume 11, Number 1, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/ast.2010.0501
SOLID3: A Multiplex Antibody Microarray-Based Optical Sensor Instrument for In Situ Life Detection in Planetary Exploration Vı´ctor Parro,1 Graciela de Diego-Castilla,1 Jose´ A. Rodrı´guez-Manfredi,1 Luis A. Rivas,1 Yolanda Blanco-Lo´pez,1 Eduardo Sebastia´n,1 Julio Romeral,1 Carlos Compostizo,2 Pedro L. Herrero,2 Adolfo Garcı´a-Marı´n,2 Mercedes Moreno-Paz,1 Miriam Garcı´a-Villadangos,1 Patricia Cruz-Gil,1 Vero´nica Peinado,1 Javier Martı´n-Soler,1 Juan Pe´rez-Mercader,1,* and Javier Go´mez-Elvira1
Abstract
The search for unequivocal signs of life on other planetary bodies is one of the major challenges for astrobiology. The failure to detect organic molecules on the surface of Mars by measuring volatile compounds after sample heating, together with the new knowledge of martian soil chemistry, has prompted the astrobiological community to develop new methods and technologies. Based on protein microarray technology, we have designed and built a series of instruments called SOLID (for ‘‘Signs Of LIfe Detector’’) for automatic in situ detection and identification of substances or analytes from liquid and solid samples (soil, sediments, or powder). Here, we present the SOLID3 instrument, which is able to perform both sandwich and competitive immunoassays and consists of two separate functional units: a Sample Preparation Unit (SPU) for 10 different extractions by ultrasonication and a Sample Analysis Unit (SAU) for fluorescent immunoassays. The SAU consists of five different flow cells, with an antibody microarray in each one (2000 spots). It is also equipped with an exclusive optical package and a charge-coupled device (CCD) for fluorescent detection. We demonstrated the performance of SOLID3 in the detection of a broad range of molecular-sized compounds, which range from peptides and proteins to whole cells and spores, with sensitivities at 1–2 ppb (ng mL1) for biomolecules and 104 to 103 spores per milliliter. We report its application in the detection of acidophilic microorganisms in the Rı´o Tinto Mars analogue and report the absence of substantial negative effects on the immunoassay in the presence of 50 mM perchlorate (20 times higher than that found at the Phoenix landing site). Our SOLID instrument concept is an excellent option with which to detect biomolecules because it avoids the high-temperature treatments that may destroy organic matter in the presence of martian oxidants. Key Words: Planetary exploration—SOLID instrument—In situ automated analysis—Life-detection chips—Antibody microarrays—Environmental monitoring. Astrobiology 11, 15–28.
the scheduled Mars Science Laboratory. One of the most relevant discoveries from the Phoenix lander was the detection of a relatively high concentration of perchlorate (ClO4 ) at the landing site, around 2.4 mM (Hecht et al., 2009). This is considerably higher than the concentration found in terrestrial analogues, like the Atacama Desert, where perchlorate concentration is in the 0.1 mM range (Ericksen, 1981; Michalski et al., 2004; Parro et al., unpublished results). It is known that the presence of perchlorate at high temperature destroys organic matter, and this can be a reasonable
1. Introduction
T
o date, the technology used to detect organic molecules on Mars has been based on the detection of volatile compounds by gas chromatography–mass spectrometry after a ramp of heating (up to 7508C) or a direct pyrolytic process (1000–11008C). This is the case for the gas chromatograph– mass spectrometer instruments on board the pioneer Viking missions (Klein, 1974), the recent Phoenix lander (Hoffman et al., 2008), and the Sample Analysis at Mars instrument on
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Centro de Astrobiologı´a (INTA-CSIC), Madrid, Spain. SENER Ingenierı´a y Sistemas SA, Las Arenas, Spain. *Present address: Department of Earth and Planetary Sciences, Harvard University, Cambridge, Massachusetts, USA.
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16 explanation for the negative results obtained in the Viking and Phoenix missions, as has been suggested (Aubrey et al., 2009; Navarro-Gonza´lez et al., 2010). Consequently, to detect organic compounds and biomolecules, it is necessary to develop new and robust technologies compatible with the martian soil chemistry and independent from the type and properties of the substrate. One of the most promising technologies is based on immunosensors. Immunological techniques are the basis for the development of many biosensor devices (for a review see Marquette and Blue, 2006; Wang, 2006), as the recently developed antibody microarraybased biosensors. DNA and protein microarray technology allows the covalent binding of thousands of probes in a small area (few square centimeters) on a solid support (Ekins, 1998; MacBeath and Schreiber, 2000; Kusnezow et al., 2003; Weller, 2005), which constitutes a bio-affinity biosensor. Instrumentation that uses two-dimensional array immunosensors for rapid analysis of complex samples for environmental purposes has been reported (Rodrı´guez-Mozaz et al., 2004; Taitt et al., 2004; Tschmelak et al., 2005). In fact, antibodies or antibody-like molecules have also been proposed during the last few years as sensors for the detection of biomarkers in astrobiology (Steele et al., 2001; Parro et al., 2005; Schweitzer et al., 2005), and instrumentation based on antibody microarrays was positively considered for the ESA ExoMars Mission (http://exploration.esa.int/science-e/www/object/ index.cfm?fobjectid¼46048). We have previously reported the Signs Of LIfe Detector (SOLID) concept (Parro et al., 2005), an antibody microarray-based biosensor for in situ analysis. A field prototype (SOLID2) was successfully tested during the MARTE project, a NASA-CAB Mars drilling simulation experiment (Parro et al., 2008b; Stoker et al., 2008). Herein, we present the construction and testing of the SOLID3 instrument, which incorporates substantial improvements with respect to the previous prototypes, as follows: (1) lower mass, from 15 kg of SOLID2 to 7 kg; (2) a new modular lab-on-a-chip-based concept; (3) an improved and specially designed optical package, based on total internal reflection (TIRF) and portable charge-coupled device (CCD) capture; (4) the capacity to perform both sandwich and competitive microarray immunoassays; (5) full automation of processing and analysis of soil, mud, ground rocks or crushed ice, and so on. All these improvements make SOLID3 a unique instrument for in situ life detection in planetary exploration as well as for environmental monitoring on Earth. Additionally, we checked the robustness of the system in the analysis of samples by sandwich immunoassay in the presence of a perchlorate concentration more than 10 times higher than that found in the Phoenix landing site. 2. SOLID3 Design: The Latest Version of the SOLID Concept The SOLID instrument concept is based on the automation of microarray immunoassays and is devoted to the detection of biomolecules and other compounds in planetary exploration (Parro et al., 2005, 2006, 2008a, 2008b). The SOLID1 version (Parro et al., 2005) consisted of a breadboard that allowed us to test the proof of concept, which was validated with the fully automatic field-tested version SOLID2 during the course of a Mars drilling simulation campaign, the MARTE project (Parro et al., 2006, 2008a; Stoker et al., 2008).
PARRO ET AL. To gain flexibility, the new instrument version (SOLID3) was divided into two main units that are functionally and physically separate: the Sample Preparation Unit (SPU) for sample allocation, homogenization, and further processing; and the Sample Analysis Unit (SAU), which bears an antibody microarray biosensor for the immunological assays following lab-on-a-chip concepts (Fig. 1). 2.1. The Sample Preparation Unit (SPU) The current SPU prototype consists of 10 extraction cells (Fig. 1), each one capable of processing 0.1–0.5 g of solid (ground) material and up to 2 mL of extraction buffer or another liquid sample. A functional diagram with the main parts is shown in Fig. 2. Once a sample (S) has been loaded through the sampling port (4) of each extraction cell, the sonicator (1) fits into the extraction chamber (3) and moves the membrane forward (2), leaving behind the sampling port and sealing the chamber. The sample is compressed and hermetically confined into a smaller volume before adding 2 mL of extraction buffer from a supply reservoir (6) in the opposite direction through the filter (5). The sonicator is activated, and several ultrasonication cycles can be done. A linear actuator pushes forward the sonicator horn, which acts as a plunger, forcing the sample to pass through a 15-micron filter (5) to remove any large particulate matter. The filtrate goes to the so-called recirculation chamber (8) and then leaves the SPU to flood either one of the antibody chambers (11) or one of the microarray flow cells (12), depending on the type of immunoassay, in the SAU. The filtrate constitutes the crude extract that can be directly analyzed by the SAU either by a sandwich microarray immunoassay (SMI) or a competitive microarray immunoassay (CMI). Both the SPU and SAU have pumps and a set of valves that allow the sample to reach the appropriate place and also allow recirculation through different circuits. The total weight of the instrument (SPU plus SAU) is 7 kg, and the instrument can be transported in a suitcase bearing 12 V rechargeable batteries. The peak of energy consumption is next to 90 W just for 1 min periods during ultrasonication and 45 W for image acquisition, which is less than 10 W the average for most of the operating time. 2.2. The Sample Analysis Unit (SAU) The current SAU consists of five flow cells that accommodate the same number of antibody microarrays, as well as the motor, pumps, fluidics, valves, and so on, necessary to perform both sandwich and competitive immunoassays (Figs. 1 and 2). The analyte-containing solution supplied from the SPU, from another instrument, or from manual inoculation is incubated with the antibody microarray in each flow cell to carry out either sandwich or competitive immunoassays (see below and Materials and Methods). After the assay is completed, the Signal Readout Module subsystem is activated; a laser beam enters by the front edge of the microarray support and is transmitted by TIRF, using the same support (the glass slide with spotted antibodies) as a waveguide (Fig. 2C, 2D). The light excites the fluorochromes on the spots, and the fluorescence signal is captured through a microlens array by a CCD. Because space exploration requires miniaturization and low-mass instrumentation, we designed a specific optical package in order to capture a relatively big surface area with
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FIG. 1. SOLID3 instrument concept. (A) The instrument is divided into three functional units: (i) the Sample Preparation Unit; (ii) the Sample Analysis Unit; and (iii) the Control Unit. (B) Photograph of the SPU showing the 10 different extraction chambers. (C) Three-dimensional models of both SPU (left) and SAU (right). (D) Details of the laser, the fiber optic bundle for conducting the light to the side edge of the antibody microarray support (left of the image), and the optical package (pinhole array, microlens array, and filters) set up onto a commercial CCD. The numbers indicate (1) sampling port of the extraction cells; (2) ultrasonicator; (3) electronics; (4) linear actuator; (5) laser; (6) fiber optic bundle; (7) optics for laser beam; (8) optic package (supports, spacers, microlens array); (9) one out of the five microarrays which fits with the corresponding flow cell in the fluidic module (not shown); (10) the commercial CCD camera with its electronics and hardware. the minimal mass allowable (Fig. 2C). In addition, the compact optical package also allows for a more compact and lighter SAU (around 1.0 kg). The antibodies are printed on a specially designed and built solid support, a 75270.15 mm glass piece chemically activated with epoxy groups for covalent binding of the antibodies. By using serial dilutions of known antibody concentrations, we estimated the sensitivity to be near to the conventional microarray scanners, that is, approximately 1 molecule of fluorochrome per square micron (Fig. 3). The light was homogeneously transmitted through the waveguide, showing a homogeneous excitation pattern on the whole printed area (2535 mm). 3. Materials and Methods 3.1. Samples, antigens and antibody production, purification and labeling The antibodies used in this work, their purification with protein-A, and their labeling with fluorescence have been
reported previously (Parro et al., 2005; Ferna´ndez-Calvo et al., 2006; Rivas et al., 2008). The antigens used in this work are a 15-amino-acid-long peptide (VEAIIKPFKLDAVKE) of the GlnB protein from the iron-oxidizing bacterium Leptospirillum ferrooxidans (Ferna´ndez-Calvo et al., 2006), the 10 kDa protein thioredoxin (Sigma Cat. No. T0910), and spores from Bacillus subtilis MB11 (our laboratory strain collection). To test the effect of perchlorates on the immunoassay, we used a sample from a well-known filamentous biofilm from Rı´o Tinto (Moreno-Paz et al., 2010). To test SOLID3 performance, we used a semidried phyllosilicate-rich crust from the river bank with recent sediments (Parro et al., 2011). SOLID3 was also tested (not shown) with a halite-rich and nitrate- and perchlorate-containing powder sample obtained from 2 m deep in the Salar Grande (Atacama Desert, Chile). 3.2. Microarray design and construction The SOLID3 microarray was designed and constructed with a MicroGrid II TAS arrayer (BioRobotics, Genomic Solutions, UK). Up to five parallel microarrays were printed
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FIG. 2. Functional diagrams of both SPU (A) and SAU (B). Sample (S); sampling port (4); sonicator (1); extraction chamber (3); membrane (2); buffer supply reservoir (6); filter (5); recirculation chamber (8); antibody chambers (11); microarray flow cells (12); waste deposits which are separated by a membrane from the fresh buffer supply reservoir (top) (7, 9, and 10). (C) The signal readout module consisting of a laser, focus optics, a fiber optic to illuminate the side front of the microarray support, which acts as a waveguide to propagate light and excite the fluorochromes, and an optical package comprising a pinhole array spacer, a microlens array, and a CCD sensor. (D) Scheme showing how light (arrow) propagates through a waveguide (hatched) and excites the fluorochromes retained by the antibodies (Y) right on the surface.
so that they fit into the five different flow cells on the SOLID3 SAU. Antibodies were printed at 1 mg mL1 in protein printing buffer (Whatman, Schleicher & Schuell, Sanford, ME, USA) on a specially designed glass substrate (dimensions 75270.15 mm) activated with Superepoxy chemistry (Arrayit Corporation, CA, USA). For CMI (see below), the antigen or the hapten conjugate instead of the antibody was printed at 0.5 mg mL1 in the same buffer and conditions. We printed a 15–amino acid peptide called GlnB1, whose sequence is VEAIIKPFKLDAVKE, with multiple replicate (20) spots per array. 3.3. Sandwich microarray immunoassay (SMI) in SOLID3 Up to 0.5 g of soil sample was loaded into the homogenizing chamber of the SPU, and then 2 mL of extractionincubation buffer TBST-RR (0.4 M Tris-HCl pH 8, 0.3 M NaCl, 0.1% Tween 20) was added. Then the sonicator horn pushed forward a ring membrane, closed the chamber, and advanced to the sonication position. After one or several cycles of sonication (depending on the type of sample to be processed), the linear actuator pushed the sample through a filtering system (from 2- to 15-micron pore size can be used). At that point, the filtrate is delivered to a recirculation chamber, from which it could be manually collected, or directly injected to the SAU. In the latter case, the filtrated sample floods one of the flow cells and contacts one of the antibody microarrays. An internal recirculation circuit allows sample to be recirculated for up to 1 h to enhance the reaction kinetics between antigens and spotted antibodies. After the
incubation time, the remaining sample is discarded into the waste deposit, and the microarray cell is washed out with incubation buffer to remove the nonbound sample. Fresh buffer is injected to the auxiliary chambers to dissolve the preloaded lyophilized fluorescent antibodies; then this solution is incubated with the microarray by recirculation during 1 h. An additional wash removes the excess of fluorescent antibodies and leaves the microarray ready for fluorescent detection by excitation with a laser beam via TIRF through the glass support, which acts as a waveguide. The fluorescent signal is captured by a high-performance cooled CCD camera (Alta U16, Apogee Instruments, Roseville, CA, USA) and stored as an image in fits format that can be processed and analyzed by conventional microarray software. As a blank control, one of the microarrays is incubated only with buffer and developed with fluorescent antibodies. The obtained image is used as a blank to measure any unspecific fluorescent signals. 3.4. Competitive microarray immunoassay (CMI) In the competitive immunoassay, we used antigens, conjugates, or target molecules (0.5 mg mL1) as immobilized capturing probes. Titrated fluorescent antibodies were used as tracers, and the antigens, conjugates, or target molecules as competitors. To determine the calibration curves, all five microarrays on the SAU were printed with the corresponding antigens and in multiple replicate spots. Each microarray is used for a single assay so that, out of five microarrays, one is used as the control and the four remaining to assay four different antigen dilutions. When it is necessary to assay
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chamber and set to recirculate for 10 min at 0.15 mL min1 before washing with 2 mL of PBST-B buffer for 5 min at 0.4 mL min1. The optical subsystem was powered on, and a CCD image was captured. For the blank control, that is, the assay that gave the 100% signal intensity in each spot, the same procedure was followed but in the absence of antigens or competitors in a parallel chamber. The images were processed, the spot intensities of the microarrays determined, and the data analyzed by GenePix 6.0 pro Software (Molecular Devices, Sunnyvale, CA, USA). Every experiment was repeated at least twice; and, in general, the standard deviation was no more than 10–15% of the highest signal. 3.5. Sample preparation by the Sample Preparation Unit (SPU) Up to 0.5 g of soil or ground rock or ice were loaded into one of the 10 homogenizing chambers through the loading port. Then the sonicator horn moved forward so that it displaced a close membrane-ring that confined the sample in a hermetic chamber. The extraction buffer (2 mL of TBST-RR) was injected in the opposite direction of the exit, and the ultrasonicator was powered on to perform 3 to 101 min cycles of ultrasonication. The sonicator horn and membranering, acting as a piston, pushed forward the sample, which was filtrated through 2- to 15-micron pore size filter. The filtrate can be directly injected to either the lyophilized antibodies or to one of the microarray chambers of the SAU (depending on whether a CMI or a SMI, respectively, has to be done) and set to recirculate between SAU and SPU (through the recirculation chamber), or just inside the SAU. 3.6. Ultrasonication tests
FIG. 3. Testing the SOLID3 signal readout module. (Upper panel) A 10 s exposure image showing a whole biochip (five flow cells) fully printed with a fluorescent antibody concentration gradient in all five microarrays (rows 1 to 5). The yellow rectangle delimits microarray 1 and flow cell 1. The concentration gradient was replicated eight times, one for each of the eight sub-arrays composing each of the microarrays. (Middle) An example of sub-array (1014 spots) showing the gradient pattern obtained by direct printing of an Alexa-647 labeled antibody: (from right to left) 30 mg mL1 (two first columns), 15 mg mL1 (third and fourth), and 7.5, 3.7, 1.8, 0.9, 0.45, 0.23, 0.12, 0.06, and 0.03 mg mL1 for each the remaining columns, respectively. (Bottom) The fluorescent intensity along the gradient (line at the bottom) was quantified and plotted (dynamic range from 0–65,000). Spots of the four first columns on the right are saturated in this image, showing similar signals in the plot.
more than four dilutions, new microarrays must be used. The assays were performed as follows: serial dilutions of target molecules were incubated with their corresponding fluorescent antibody or with an antibody mixture in a total volume of 1 mL of PBST-B buffer [1phosphate-buffered saline (PBS), 0.01% Tween 20, 1% BSA] for 10 min. Then they were manually injected to an already flooded SOLID3 SAU microarray
The SOLID3 SPU sonication efficiency was tested by measuring the lysing effect on vegetative bacterial cells (Escherichia coli) or resistant cells (Bacillus subtilis spores). Up to 108 cells or spores per mL were diluted in 2 mL 1 PBS buffer and loaded into a homogenizing chamber. Then the sonicator was moved forward to close the chamber and continued until the sonication position. Several 1 min sonication cycles were applied: 0 (control), 3, 6, and 10 at different sonicator power (40% and 70%). Cell lysis or irreversible damage on spores was checked by viable counts on LB plates after plating serial dilutions and 12–14 h of incubation at 378C. Additionally, 10% sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE) was performed, and the gel stained as described (Laemmli, 1970) to test protein integrity. 3.7. Effect of perchlorate on the performance of sandwich immunoassays A biofilm sample from Rı´o was used (a) to test the effect of the presence of perchlorate anions on frozen extracts and (b) to test the effect of perchlorate during ultrasonication and the consequent heating (up to 70–808C). For the first treatment, 20 mg of wet biofilm were resuspended in 1 mL of TBST-RR buffer and sonicated (330 s at maximal power in a MISONIX XL with a small probe). The homogenate was centrifuged (2000g for 5 min at 48C) to remove particulate matter and the supernatant (the extract) recovered. Aliquots (15 mL) of this extract were prepared in the presence of 0, 6, or 50 mM sodium perchlorate and then frozen (208C) for 45 days until
20 assayed in LDCHIP200 by sandwich immunoassay as described (Rivas et al., 2008). In the second treatment, 20 mg of biofilm was resuspended in 1 mL of buffer TBST-RR with 0, 6, or 50 mM sodium perchlorate, sonicated (330 s cycles, reaching a temperature of 608C in the second cycle and 70–808C in the third cycle), and then centrifuged (2000g for 5 min at 48C) to remove particulate matter. The supernatants (the extracts) were assayed by sandwich immunoassay as described above. 3.8. Image processing and analysis The images were analyzed and quantified with Genepix Pro Software (Genomic Solutions). The final fluorescent intensity (FI) for each antibody spot was calculated by applying the following equation: FI ¼ (F635 B)sample (F635 B)blank, where F635 B is the fluorescent intensity at 635 nm minus the local background (B) of each spot as quantified by the software (GenePix Pro). As a blank sample, we used only buffer. Spots having obvious defects or those duplicated spots whose standard deviation from their mean was 0.15 times higher than their mean signal intensity value were not considered for quantifications. The limit of detection was assigned to the minimal concentration of the analyte that gave a significant and reproducible fluorescent signal, in the corresponding antibody spots, with respect to the sample with no added analyte, that is, at least twice the fluorescent intensity of the blank control for a SMI and a reproducible loss of intensity around 5% for a competitive immunoassay. 4. Results 4.1. SOLID3 operations: ultrasonication tests for sample preparation We initially considered two alternative procedures for sample homogenization, extraction, and/or lysis of biologi-
PARRO ET AL. cal material: (1) heating the sample at 1008C at 1.2 bar pressure or (2) ultrasonication with consequent temperature (up to 808C) and pressure (1.2 bar) increases. There is abundant scientific literature reporting good performance of sonication for organic extraction from solid samples. In addition, we did a simple lysing test by treating 108 Escherichia coli cells as follows: (1) autoclaving at 1208C at 1.2 bar for 30 min, or (2) 31 min ultrasonication cycles into the bath of a commercial sonicator at maximal power (a MISONIX XL, Qsonica, Newtown, CT, USA). After checking the protein integrity by electrophoresis in a SDS-PAGE gel (Fig. 4A), the presence of clear and neat bands in the sonicated sample, in contrast to a diffuse smear in the autoclaved one, indicated that proteins and other biopolymers were better preserved in the former than in the latter. The sonication efficiency of the SOLID3-SPU was tested by its ability to lyse bacterial vegetative cells and to damage or impair germination of spores (see Materials and Methods). Two distinct biological sources were chosen for the assays: the Gram-negative bacterium Escherichia coli and the highly resistant spores of the Gram-positive Bacillus subtilis. The sonicator strength was high enough to lyse 108 E. coli cells completely and irreversibly kill more than 50% of 108 B. subtilis spores after 31 min sonication cycles (Fig. 4B). 4.2. Performance of the SOLID3 SAU for automated sandwich and competitive microarray immunoassays We tested the SAU in CMI by the detection of small compounds, such as the 15–amino acid peptide GlnB1 (Fig. 5A), and in SMI by the detection of a 10 kDa protein (bacterial thioredoxin) and B. subtilis spores (Fig. 5B). For CMI, serial dilutions of the peptide were preincubated with the corresponding fluorescent antibody and then injected to one of the reaction chambers of the SPU. After sample processing by the SAU (see Materials and Methods), the images corresponding to the different peptide dilutions were analyzed,
FIG. 4. Ultrasonication efficiency by SOLID3. (A) Ultrasonication versus heat extraction. Cells from E. coli were autoclaved, 1208C, 1.2 atm for 30 min (1) or sonicated (2), and the lysates were fractionated in an SDS-PAGE gel. (B) The efficiency of the SPU sonicator was tested by its ability to lyse B. subtilis spores, destroy them, or both, after several 1 min sonication cycles at two different power sets: 40% (upper) and 70% (lower). Mr, relative molecular mass ladder, from 15 kDa (lowest band) to 212 kDa (uppermost band).
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FIG. 5. Test of SOLID3 in competitive and sandwich microarray immunoassays. (A) A calibration curve was done with a 15–amino acid peptide by the competitive microarray immunoassay. (Right panel) a cartoon showing how the competitive immunoassay works: (1) in a first assay without competitor, fluorescent antibodies bind to immobilized hapten conjugates, giving a 100% fluorescent intensity (vertical bars); (2) in a test sample, the antigens compete with immobilized conjugates to bind to fluorescent antibodies, with the corresponding loss of signal in the spot (3). (B) and (C) Calibration curves for sandwich microarray immunoassay for the detection of the 10 kDa protein thioredoxin (B) and B. subtilis spores (C). The cartoon on right shows how antigens bind to printed capturing Abs (1) and fluorescent antibodies sandwich the captured antigens (2).
quantified, and plotted (Fig. 5A). The limit of detection was around 1 ng mL1, which is similar to that we previously reported by a manual procedure (Ferna´ndez-Calvo et al., 2006). The SMI was performed by testing serial dilutions of thioredoxin and B. subtilis spores and assayed as described in Materials and Methods (Fig. 5B, 5C). The limits of detection were 0.2–1.0 ng mL1 of protein and 104 to 105 spores per milliliter. These results were, again, in agreement with those detected with manual methods (Ferna´ndez-Calvo et al., 2006; Rivas et al., 2008). 4.3. Testing SOLID3 with samples from a Mars analog environment The Rı´o Tinto system is considered a good Mars analogue mainly in those aspects concerning mineralogy and energy sources for a hypothetical biota on the Red Planet (Ferna´ndez-Remolar et al., 2005, 2008). Samples from Rı´o Tinto were directly analyzed with SOLID3 and LDCHIP200 (Rivas et al.,
2008). Up to 0.5 g of semidried phyllosilicate-rich crust from the river bank was loaded into one of the homogenizing chambers of the SPU and then run automatically. Two negative controls were additionally run: C1, without sample, only buffer; and C2, consisting of the same amount of sediment sample but previously subjected to 2508C for 12 h. The resulting images were analyzed and quantified (Fig. 6). We obtained positive signals corresponding to antibodies against the bacteria usually found in this ecosystem, such us Acidithiobacillus ferrooxidans, A. thiooxidans, or Leptospirillum spp. strains, among other positive signals (Fig. 6). The results are in good agreement with other results obtained after manual experimentation (Rivas et al., 2008). The fluorescent signals either completely disappeared or were substantially reduced in the heat-treated sample, which indicates that fluorescence was indeed due to molecules whose structure was destroyed by heat and consequently not recognized by the antibodies. Additionally, SOLID3 has been recently tested in a field campaign in the Atacama Desert (Chile), one of the best Mars
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FIG. 6. Test of SOLID3 with environmental samples from a Mars analogue. Up to 0.5 g of a phyllosilicate-rich sample from the river bed were loaded into one of the homogenizing chambers of the SOLID3 SPU and subjected to 31 min sonication cycles in 2 mL of TBST-RR buffer (see Materials and Methods). Then the sample was filtered (15 microns) and injected to one of the microarray chambers on SOLID3 SAU for a sandwich microarray immunoassay (see text for explanation) with a 200antibody-containing microarray, LDCHIP200 (Rivas et al., 2008). (Upper panel) Several positive spots were detected corresponding to antibodies raised against Rı´o Tinto natural extracts or isolated microorganisms: 1, Rı´o Tinto sediment extract (A138); 2, Leptospirillum ferrooxidans whole cell extract (A139); 3, 4, Acidithiobacillus ferrooxidans (A183, A186); 5, dinitrophenol derivatives; 6, 7, extracts from Rı´o Tinto sediments (IC2C3 and IC4C1, respectively); 8, L. ferriphilum extracellular material (IVE2S1); 9, A. thiooxidans S100 cellular extract (IVE4S100); 10, A. albertensis whole cells (IVE5C1); 11, A. albertensis S100 extract (IVE5S100); 12, Psychroserpens burtonensis extracellular material (IVF4S1); 13, 14, Burkholderia fungorum cellular debris and EDTA-extracted extracellular material, respectively (IVI4C2, IVI4S2). Most of the fluorescent signals clearly disappeared when the same amount of sample was heated to 2508C for 12 h prior to the assay (lower panel), indicating that the antigens responsible for these signals were destroyed after heat treatment. The antigenic extracts and antibody codes (in parentheses) were described in Rivas et al., 2008. analog environments. The instrument was loaded with 0.5 g of a halite-rich and nitrate- and perchlorate-containing powder sample from the subsurface (2 m depth) and automatically run for a SMI with the now-called LDCHIP300 (Life Detector CHIP with 300 antibodies). We detected some positive signals, which indicates the presence of molecular biomarkers in the samples (to be published elsewhere). 4.4. Compatibility of SOLID3 with the perchlorate chemistry found on Mars We have demonstrated the feasibility of antibody microarray-based instrumentation for automatic in situ analysis of liquid and solid environmental samples. SOLID3 can play a critical role in the analysis of martian samples in light of the recent discoveries by NASA’s Phoenix lander of large amounts of perchlorate salts in the martian north pole soil (Hecht et al., 2009). It is known that perchlorate destroys organic matter at high temperatures, and therefore protocols and instrumentation for the analysis of samples at low temperatures, like SOLID, would be necessary for biomarker detection on Mars. We have tested the capacity of our immunodetection system for functioning in the presence of 2–3fold and 20-fold (50 mM) the martian (Phoenix landing site) concentration of perchlorate anions (Materials and Methods). We first tested the effect of the presence of perchlorate in an environmental extract under freezing temperatures. No significant differences in the fluorescent signal nor in the immunoprofile were observed with respect to the positive control in the absence of perchlorate (Fig. 7A, 7C). In another
experiment designed to test the effect of perchlorate during ultrasonication and the consequent increase of temperature (up to 70–808C), perchlorate was added to the sample, then ultrasonicated for 330 s cycles, with measured temperatures next to 808C, and finally analyzed by LDCHIP200. Again, no substantial differences were obtained at the different perchlorate concentrations with respect to the positive control (Fig. 7B, 7C), which confirms the compatibility of the SOLID-LDCHIP immunoassay with this critical component of the martian soil chemistry. 5. Discussion 5.1. SOLID3: an instrument for in situ life detection In this work, we have reported the fabrication and performance of SOLID3, a biochip-based instrument to detect biomolecules in planetary exploration. The main improvements of SOLID3 with respect to the other versions are (1) Versatility. The separation into two physical and functional units allows the SAU to receive and analyze samples either from the SPU or from other sample-preparation systems available in a hypothetical mission. (2) Lower mass. We reduced the mass from more than 15 kg for the previous version (SOLID2) to 7 kg for the whole SOLID3 assemblage (SPU plus SAU), 1 kg being approximately the mass of the SAU. This significant mass reduction makes the SOLID system highly attractive for planetary missions. (3) The ability to perform sandwich and competitive immunoassays. The former is applicable to the detection of high-molecular-weight and multiepitope-containing molecules,
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FIG. 7. Test of the effect of perchlorate anions on the sandwich immunoassay. (A) A whole extract from a biofilm sample from Rı´o Tinto was obtained by ultrasonication and incubated with different sodium perchlorate concentrations at 208C for 45 days. Then the samples were assayed with the sandwich microarray immunoassay in a LDCHIP200 (see Materials and Methods) by triplicate. The bars correspond to the fluorescent signals of 12 different antibodies after assaying the extract in the absence (gray bars) or in the presence of 6 mM (dotted bars) or 50 mM (hatched bars) perchlorate. (B) Perchlorate was added to the biofilm sample then ultrasonicated for preparation of the extract and assayed as above. Antibodies showing positive fluorescent signals corresponded, as expected, to those raised against Rı´o Tinto sediment samples or biofilms as well as to bacterial acidophiles: IC1S1 and IC7C1, sediments from different locations; IC3C1, IC3C3, and IC4C1, cellular fraction from different biofilms; IVE3C.., IVE4C.., IVE5C1, IVE6C1, and IVI4C2, cellular fractions from Acidithiobacillus ferrooxidans, A. thiooxidans, A. albertensis, A. caldus, and Burkholderia fungorum, respectively (Rivas et al., 2008). (C) Comparison of the effect of perchlorate in frozen extracts (dotted) or in ultrasonicated samples (hatched). The bars represent the sum of the intensities of all 12 antibodies of (A) and (B) as a percentage with respect to the sum obtained in the absence of perchlorate.
and the latter is for small-molecular-weight analytes. (4) Compact lab-on-a-chip-based design of the SAU in which the antibody support, the fluidics, and the optics form a compact device for reducing mass and volume. (5) An improved optical system. The laser light excites the fluorochrome by TIRF, using the biochip support as a waveguide, which renders cleaner images than the direct excitation system used in SOLID1 and SOLID2. The SOLID SPU sampling port is just a hole (5 mm in diameter actual size) whose final dimensions and configuration can be adapted to the sample loading system. The
basic idea for a flight model is to maintain either a lateral open hole or just the entire diameter (ca. 20 mm) of the homogenization chamber to receive the sample. In the case of a ‘‘sticky’’ and granulate sample (>1 mm), the latter option would be more appropriate, the chamber in a vertical orientation. Whichever the case, the chamber is sealed by the rings around the sonicator tip, which act as a piston when entering the chamber. Once sealed, liquid extraction buffer can be added and confined in the homogenization chamber during ultrasonication.
24 One important component of the SOLID concept is the homogenization and extraction of the sample to be analyzed by an ultrasonication device. Ultrasonication has been used for many years as a preferred method of cell lysis for the extraction of macromolecules like nucleic acids, protein, or extracellular polymeric substances (EPS), and for organic extraction from geological samples. Our results indicate that ultrasonication is more efficient than only heating because, in addition to its ability to lyse cells, it homogenizes the samples by destroying aggregated materials. Additionally, the increase in temperature and pressure as a consequence of the sonication may contribute to the extraction efficiency. Our SPU system bears appropriate temperature and pressure sensors to keep these parameters below 808C and 2 bar, respectively. The SOLID3 sonicator makes homogeneous suspensions from soil or ground samples, lyses vegetative cells, and irreversibly damages spores in a few ultrasonication cycles. One of the strengths of the SOLID system assay is that, in most of the cases, it is not necessary to lyse cells for positive reactions. Most of the target antigens for our antibodies lie on extracellular locations (cell walls, exopolymeric substances, or organomineral particles), while the rest correspond to intracellular targets of metabolically active vegetative cells, not dormant spores. For sandwich and competitive immunoassays, SOLID3 allows detection of different biomolecules, from peptides to whole spores, with detection limits in the range of few parts per billion (ng mL1), which is in good agreement with the manual procedures (Ferna´ndez-Calvo et al., 2006). Additionally, we have tested SOLID3 with natural samples from terrestrial analogues to Mars-like Rı´o Tinto sediments (Fig. 6) and salt-rich subsurface samples from the Atacama Desert (to be published elsewhere). Different positive reactions were obtained in antibodies against cellular and environmental extracts and other compounds. These reactions were absent (Fig. 6) when the samples were previously subjected to a heat treatment at 2508C for 12 h, which indicates that antibodies were in fact recognizing biological molecules whose three-dimensional structure was lost by heat denaturation. Because we have an extensive collection of antibodies against microorganisms and biofilms from extremely acidic environments, another interesting application of the antibody microarray is the monitoring of industrial bioleaching processes as well as environmental monitoring of acid mine drainage. The detection of a relatively high concentration of perchlorate in the Phoenix landing site on Mars, together with the known destructive effect of the organic matter by perchlorate at high temperatures, raised the question as to whether our immunoassay was compatible with this martian chemistry. Our results show no apparent effects on the sandwich immunoassay after using more than 20-fold the martian perchlorate concentration in the incubation buffer, nor at freezing (208C), nor during ultrasonication (with temperatures near 808C).
PARRO ET AL. will render common molecules that eventually could be good targets to search for life. Antibodies can be highly specific and capable of distinguishing between two enantiomers (Hiasa and Moriyama, 2006). However, they can also show a relaxed specificity to recognize similar structures, for example, to bind hydrophobic stretches from different biopolymers (Helmerhorst et al., 1998). We believe that all ranges of specificities are suitable in an antibody microarray for astrobiology. Therefore, we suggest two strategies for target selection (Fig. 8): (i) a direct approach, in which well-known molecules (including metabolites or compounds detected in space and in meteorites) from those listed in Parnell et al. (2007) and Parro et al. (2008a) are used as antigens to produce antibodies; and (ii) a shotgun strategy, where we apply biochemical extraction and fractionation to environmental samples and use these extracts to produce antibodies (Parro et al., 2008a; Rivas et al., 2008; Parro and Mun˜oz-Caro, 2010). The shotgun strategy is critical because we produce highly specific and reactive antibodies against the actual environmental molecules, which very often are slightly modified with respect to the laboratory or commercial counterparts. Chemical modifications, such as acylations, methylations, and amination, in natural samples can make useless those antibodies raised against apparently the same molecules isolated in the laboratory from other sources. Obviously, additional efforts are necessary to identify the exact environmental molecule that is being recognized by the antibody. The more we know about how living matter is degraded or preserved on Earth, as well as under different extraterrestrial environments like Mars, the better for selecting a list of target molecules for antibody production. Our results with antibodies against natural extracts indicate that they recognized polymeric material mainly composed of polysaccharides and proteinaceous material (Rivas et al., 2008; Parro et al., 2008b, 2011). Polysaccharides or their multiple combinations with proteins and lipids are excellent targets for searching for
Terrestrial analogues: Acid and metal rich Hydrothermal Antarctic permafrost
Environmental extracts from soil, sediments, biofilms, rocks, etc. Characterization Environmental biomolecules Commerical Abs against known biomarkers and organics
Microbial type strains and environmental isolates
Fractionation Antibody production
Microarrays Laboratory tests
Proteins, Polysaccharides, Nucleic acids, Other known derivatives
Prebiotics, extraterrestrial organics, aa, etc.
Instrument implementation (SOLID)
5.2. Target molecular biomarkers for antibody production We hypothesize that microorganisms living under similar environmental conditions share similar molecular mechanisms to deal with such environments. These mechanisms
In situ analysis
FIG. 8. Strategies for target selection and antibody production for life detection in astrobiology (see text, Section 5.2, for explanation).
GC-MS, gas chromatograph–mass spectrometer; MOMA, Mars Organic Molecule Analyser; MSL, Mars Science Laboratory; PAHs, polycyclic aromatic hydrocarbons; SAM, Sample Analysis at Mars.
Microscope imager Microscopic structures
Solid surfaces
Microstructures
Cells, colonies, etc.
Parro et al., 2005; Ferna´ndez-Calvo et al., 2006 Pullan et al., 2008 100 Da to kDa From aa, nt, to whole cells
Laser desorption time of flight TOF-MS Ab microarrays/SOLID Laser desorption
Bioaffinity
Laser desorption LD-MS/MOMA Laser desorption
Liquid suspension
Todd et al., 2007 500–5000 Da
Solid surfaces IR, Raman spectrometers
Desorb neutral and ionized molecules Ionized molecules
Oligo and small polymers
Evans-Nguyen et al., 2008 500–2000 Da
Edwards et al., 2003 Any
Aa, nucleobases, PAHs, etc. Chemical bonds, pigments, etc. Oligo and small polymers Volatiles Capillary electrophoresis/Urey
Water extraction þ volatilization Spectrometric
Volatiles GC-MS/Viking, Cassini-Huygens, SAM (MSL)
Elemental, gases, PAHs, aliphatic hydrocarbons, etc.
Elemental to 500 Da [12–220 (Viking) 2–146 (Cassini)] Up to 500–600 Da
Novotny et al., 1975 (Viking); Niemann et al., 2005 (Cassini) Aubrey et al., 2008
25
Heating and pyrolysis
Size Target Sample Instrumentation
To date and mainly due to the simplicity in sample preparation, the instruments devoted to organic detection on space missions have been based on the analysis of the volatile compounds released after sample heating or pyrolysis (Table 1). However, volatilization processes have their own constraints, and relatively complex compounds cannot be detected. Bioaffinity-based assays, like antigen-antibody reaction, have a niche in space science (Table 1). Immunoassays have never been used in planetary exploration, so several issues, such as the antibody stability under space conditions or the planetary protection constraints, have to be addressed beforehand. For several years, we have performed experiments to test the antibody stability, either printed or fluorescently labeled, under high doses of gamma radiation, temperature cycles, or long-term storage (up to 5 years). Our results (De Diego-Castilla et al., unpublished data) indicate that antibodies are relatively robust molecules when treated and stored under appropriate conditions. Particularly, printed antibodies are stable for at least 1 year when stored dry
Method
5.3. Antibodies for planetary exploration
Table 1. Methodology and Instrumentation Used or Proposed for Organic Detection in Planetary Exploration
extant life in planetary exploration. Living organisms usually respond to environmental parameters (sudden changes in temperature, water availability, interaction with mineral substrates, etc.), by producing EPS like polysaccharides, lipopolysaccharides, or anionic polymers (teichoic and teichuronic acids) (Ophir and Gutnick, 1994). In addition, microorganisms produce high-molecular-weight pigments (melanin and carotenoid derivatives) to protect themselves against solar radiation and oxidative stresses. Such polymeric pigments are good targets as biomarkers of an extant microbial community (Gorbushina et al., 2002). Finally, metastable products like benzenecarboxylic acids or phthalic acid, suggested by Benner et al. (2000) to be present at the martian surface, must be considered as a target for antibody production. Depending on the molecular size and the number of sites to bind to the antibody (epitope), a sandwich or a competitive immunoassay must be performed. For those multiepitopecontaining polymers (proteins, polysaccharides, cells, organomineral complexes, etc.) a sandwich assay is the most appropriate, while for single epitope or small molecules (amino acids, nucleobases, short peptides, steroid compounds, etc.), a competitive immunoassay is the right way to go. Consequently, we have focused on terrestrial analogues for Mars, and we have produced polyclonal antibodies against extracts from the acidic, iron-, and sulfur-rich environments, hydrothermal environments, permafrost, and so on. Samples were taken from water, sediments, mineral deposits (sulfate precipitates, jarosite, hematite, etc.), rocks, and the subsurface during drill campaigns. At the same time, we are performing biochemical fractionation from pure bacterial cultures so that we can produce antibodies against different kinds of macromolecules (EPS, anionic polymers, cell wall components, etc). We have also produced antibodies against pure bacterial cultures (type collections) isolated from cold (Artic and Antarctic), hydrothermal, saline, dry environments, and so on. We currently have a collection of nearly 400 antibodies, including all mentioned categories. For further details and extensive discussion see Parnell et al. (2007), Parro et al. (2008a), Rivas et al. (2008), and Parro and Mun˜ozCaro (2010).
Reference
ANTIBODY MICROARRAY-BASED INSTRUMENT FOR ASTROBIOLOGY
26 or lyophilized at ambient temperature, while lyophilized or vacuum-dried fluorescent antibodies retain more than 80% of their functionality after 48 months of storage at ambient temperature. Concerning the planetary protection concerns, the only method considered until now has been heat treatment at 1108C to reduce as much as possible the biological contamination. Antibodies, as any other mesophilic protein, are inactivated or denatured at high temperatures in liquid solution. Whether they can survive at 1108C is yet to be discerned. We know that multiple cycles (208C ? 258C ? 508C) for up to 1 year do not affect the performance of lyophilized antibodies. We are currently performing tests with higher temperatures. If antibodies cannot survive at 1108C, another option would be to sterilize them by filtration (0.22 microns) and print in clean rooms under sterile conditions and on sterilized supports. Additionally, we intend to sterilize the printed arrays by radiation system, given that our printed antibodies can support high gamma radiation dose (De Diego-Castilla et al., unpublished data). For an astrobiological mission, we propose a routine protocol that includes three analyses with LDCHIP in SOLID: (1) a control analysis with no sample, only buffer, to check false positives either from contamination or from the contribution of fluorescent antibodies to the background; (2) the sample analysis itself (using 0.1–0.5 g of ground material); and (3) one extra control with a preheated sample at more than 2508C to check whether the positive reactions are in fact a consequence of the binding of high-molecularweight compounds and not of unspecific interaction with minerals present in the samples (Fig. 6). Heat would destroy the structure of biological polymers; consequently, the antigen-antibody interaction would not take place, and no signal would be detected.
6. Conclusion The simplicity in sample preparation makes instruments devoted to the analysis of volatiles the most suitable for the detection of organics on space missions (Table 1). Although the performance and the sensitivity of the instruments (most of them gas chromatograph–mass spectrometers) are of high quality, the main constraints have to do with the low extraction efficiency and the modification that some compounds may suffer during sample heating. For example, only a small portion of the organic matter present in meteorites is extractable, in that most of it is retained in the form of insoluble polymeric organic matter (Sephton et al., 2004). However, those methods, based on the bioaffinity properties, that is, the ability of biological polymers (e.g., antibodies, enzymes) to specifically bind to other molecules, do not need the target molecules to be fully separated from their matrix. These systems require a liquid solution or suspension in which it is the capturing molecule (the antibody) that binds to the targets, even if it is forming part of a big, complex macromolecular structure (macromolecules, organic-mineral particles, etc.). Immunological assays can bind a wide range of molecules (amino acid size, even metal-ion chelator complexes) to whole cells, or biofilm debris, in a liquid solution/ suspension at low temperatures. The SOLID instrument fits in this category, and we believe that it should be supported for future astrobiological missions.
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Address correspondence to: Victor Parro Centro de Astrobiologı´a (INTA-CSIC) Carretera de Ajalvir km 4 Torrejo´n de Ardoz 28850 Madrid Spain E-mail:
[email protected] Submitted 17 June 2010 Accepted 7 October 2010
