In order to gain flexibility, SOLID has been divided into two main functional and physically separate units: the sample preparation unit (SPU) for sample allocation, homogenization, further processing, etc., and the sample analysis unit (SAU), bearing the antibody microarray biosensor for the immunological assays. Current design of the instrument is able to run 10 samples by using five single arrays. Considering this block scheme, the mass distribution is: SPU around 5,5 Kg and SAU less than 2 Kg.
The Sample Preparation Unit (SPU)
The SPU prototype consists of 10 extraction cells, each one capable to process from 0.1 to 1 g of solid material and up to 2 ml of extraction buffer or another liquid sample. The cell has a loading port, which is sealed after the advance of a sonication horn. For each measurement, up to 1 g of soil, ground rock or ice is loaded into the extraction cell through the loading port. Then, the sonicator horn moves forward so that it displaces a close membrane-ring confining the sample in a hermetic chamber. The extraction buffer is injected from the opposite direction of the exit flow, and the ultrasonicator is powered on to perform from 3 to 10 cycles of ultrasonication (1 min each). The sonicator horn and membrane-ring acting as a piston push forward the sample, which is filtrated through 5-15 microns pore size filter. The filtrate can then be directly injected to the microarray chamber of the SAU, where it will be analyzed by a sandwich microarray immunoassay (SMI) or competitive immunoassay. After the first sample has been analyzed, the SPU pipes and valves are rinsed to avoid cross-contamination between samples and obstruction of the valves by particulate matter.
SOLID3 instrument. (left) Sample Preparation Unit (SPU) showing 10 different extraction cells for 0.5-1.0 g sample. (right) The Sample Analysis Unit (SAU) showing electronics, pump, liquid deposits over a module with 5 flow cells, each one for one microarray analysis.
The Sample Analysis Unit (SAU)
The current configuration of the SAU consists of one analysis module, equipped with 5 flow cells, which allocate one antibody microarrays each, as well as the motor, pumps, fluidics, valves, etc., necessary to perform the analysis. The analyte-containing solution from the SPU is incubated with the antibody microarray in the flow cell to perform a sandwich microarray immunoassay, or a competitive microarray immunoassay. In both cases, 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 total internal reflexion using the same support as a waveguide. The light excites the fluorochromes on the spots and the fluorescence signal is captured through a micro-lens array by a CCD device. Because the instrument was initially conceived for space exploration, we designed a specific optical package in order to capture a relatively big surface area with the minimal mass allowable. In addition, the compact optical package also allows a compact and light SAU.
Two tests are required before the analysis of the sample:
- Testing for the image capturing process. A picture of all the microarrays is taken for two purposes: i) to check the image capturing system (laser and CCD), and ii) to estimate the optical background of the image.
- To conduct a blank control by measuring the contribution of the fluorescent antibodies to the background of the image. For this purpose one of the microarrays is incubated just with buffer (no sample) and revealed with fluorescent antibodies. The image thus obtained is used as a base line to eliminate all the unspecific fluorescent signals.
Due to the modular design of SOLID, the SAU can consist of any number of flow cells, and this can be adjusted according to payload limitations and science objectives. We have chosen an instrument design based on one analysis module with 5 flow cells, which is capable of conducting two types of analyses (sandwich microarray immunoassay and competitive microarray immunoassay). Under this design, after a sample has been analyzed, the cells can be washed with buffer solution once more to remove any traces of the original sample, and the cell can be loaded with a new sample.
HOW SOLID WORKS
Due to its modular design, SOLID3 can perform 2 different types of analysis within the same SAU module: a Sandwich microarray immunoassay (SMI), and a Competitive microarray immunoassay (CMI). It can switch from one type to the other depending on scientific goals and instrument performance: CMI to detect extinct life molecular biomarkers and other small molecules, and SMI to detect extant life molecular complexes (proteins, EPS, nucleic acids, whole cells…).
Sandwich microarray immunoassay (SMI) in SOLID3.
After the sample has been filtered, the filtrate leaves the SPU and is directly injected to cell1 in the SAU. Here, the filtrated sample floods one of the flow cells and contact the antibody microarrays. An internal recirculation circuit allows the sample to be recirculated for up to 1 h, in order speed 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 non-bound sample. Then, buffer is injected to the auxiliary chambers filled with lyophilized fluorescent antibodies, let to dissolve, flood the microarray chamber, and set for recirculation for up to 1h. An additional wash remove the excess of fluorescent antibodies and leaves the microarray ready for fluorescent detection by excitation with a laser beam by total internal reflexion (TIRF) through the glass support which acts as a waveguide. The fluorescent signal is captured by a high sensitive CCD device and store a .fits image that can be processed and analyze by conventional microarray software. As a blank control, one of the microarrays are just incubated with buffer and revealed with fluorescent antibodies. The obtained image is used as a baseline to eliminate all the unspecific fluorescent signals.
Scheme for a sandwich microarray immunoassay.
A positive signal in an SMI indicates:
i) Detection of a molecule with the same or highly related structure to that used to produce the antibody, and
ii) This molecule is part of a bigger complex, either as a structural component of another molecule or polymer, or just captured by amorphous macromolecules, aggregates or even mineral particles.
Whether this result can be considered as a direct or indirect sign of life would depend on the type and number of different molecules detected. Just one positive may not be taken as a final proof for the presence of bio-molecules, but several positives with overlapping specificities in several antibodies would represent clear evidence of life.
Competitive microarray immnuno-assay (CMI).
This immunoassay format is necessary for the detection of small size molecules such as, for example, those molecular biomarkers for extinct life (hopanes and derivatives, carotanes, etc) and, in general, all molecules with just one site (epitope) to bind to the antibody. For the CMI, what it is usually printed in the microarray is: antigens, conjugates or target molecules at 0.5 mg/ml as immobilized capturing probes. Previously titrated fluorescent antibodies are used as tracers, and the antigens, conjugates or target molecules as competitors. Calibration curves must be previously obtained in the laboratory as follows: serial dilutions of target molecules are incubated with their corresponding fluorescent antibody or with an antibody mixture in a total volume of 1ml of PBST-B buffer (1 x PBS, 0.01% Tween 20, 1% BSA) for 10 minutes. Then, they are manually injected to an already flooded SOLID’s SAU microarray chamber and set to recirculate for 10 minutes at 0.15 ml min-1 before washing with 2 ml of PBST-B buffer for 5 min at 0.4 ml min-1. The optical subsystem is powered on and a CCD image is captured. For the blank control, that is, the assay that gives the 100% signal intensity in each spot, the same procedure is followed but without antigens or competitors in a parallel chamber. The images are processed, the spot intensities of the microarrays determined, and the data analyzed by microarray Software. For actual test samples, CMI is done by passing the filtrate from SPU through the lyophilized fluorescent antibody chamber and set to recirculate for 10 min before incubating for another 10 min to the cell 2 microarray chamber. After washing, an image is captured by CCD.
Functional diagram of both the SPU (A) and the SAU (B). Once a sample (S) has been loaded through the sampling port (4), the sonicator (1) fits into the extraction chamber (3) and moves forward the membrane (2), leaving behind the sampling hole. The sample in compressed and hermetically confined in a smaller volume before adding 2 ml of extraction buffer from deposit (6) in opposite direction through the filter (5). The sonicator is activated and several sonication cycles can be done. Then the sonicator horn moves forward to force the sample to pass through the filter. The filtrate then goes to the so called recirculation chamber (8) and then leaves the SPU to flood the microarray flow cells (12). 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. (C) The signal read-out 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 pin-holes array spacer, a micro lens array, and a CCD sensor. (D) Scheme showing how light (arrow) propagates through a waveguide (hatched) and excite the fluorochromes retained by the antibodies (Y) right on the surface
We have recently report the production of an antibody to mellitic acid and the development of a fluorescent inhibition microarray immunoassay (IMI) to detect this substance to a limit of 5 ppb (ng mL-1). We used the anti-mellitic antibody to detect mellitic acid in drill core samples obtained from different depths in the Atacama Desert (Chile), a highly relevant terrestrial analogue for Mars.
SENSITIVY
We have demonstrated the performance of SOLID for the detection of a broad range of molecular size compounds, from the amino acid size, peptides, proteins, to whole cells and spores, with sensitivities at 1-2 ppb (ng ml-1) for biomolecules and 104-5 cells ml-1 (Rivas et al., 2008; Parro et al., 2011). The sensitivity is more dependent from the quality of the antibody than the instrumentation itself.
Determining the sensitivity of different immunoassay with SOLID 3: competitive (A) and sandwich (B,C).
FIELD CAMPAIGNS
Both, LDCHIP (Life Detector Chip) and SOLID have been tested in the laboratory and during several field campaigns. Especially relevant was the work with SOLID2 prototype during the 2005 campaign of the MARTE (Mars Analogue Research Technology Experiment) project in Rio Tinto (Stoker et al., 2008, Parro et al., 2008; Parro et al., 2011). LDChip300 and SOLID3 were tested during the “AtacaMars2009” field campaign to the Atacama Desert in July 2009. The LDChip300 detected high molecular weight biological compounds in 0.5 g of a 2 m deep drill core (Parro et al., 2011; Fernández-Remolar et al., 2013). Similarly, SOLID3 was tested in Deception Island (Antarctica) in two field campaigns (2010 and 2012) with permafrost, superficial samples (soils and rocks) (Blanco et al., 2012), and hydrothermal samples. Finally, a new version of SPU containing a single, reusable, extraction cell has been recently tested in a field campaign to the Canadian Arctic, particularly to the McGill Arctic Research Station (MARS). The SOLID detected psychrophile microbes in sulfate rich springs and the Arctic permafrost.
