Detecting Nonvolatile Life- and Nonlife-Derived Organics in a Carbonaceous Chondrite Analogue with a New Multiplex Immunoassay and Its Relevance for Planetary Exploration

Abstract

Potential martian molecular targets include those supplied by meteoritic carbonaceous chondrites such as amino acids and polycyclic aromatic hydrocarbons and true biomarkers stemming from any hypothetical martian biota (organic architectures that can be directly related to once living organisms). Heat extraction and pyrolysis-based methods currently used in planetary exploration are highly aggressive and very often modify the target molecules making their identification a cumbersome task. We have developed and validated a mild, nondestructive, multiplex inhibitory microarray immunoassay and demonstrated its implementation in the SOLID (Signs of Life Detector) instrument for simultaneous detection of several nonvolatile life- and nonlife-derived organic molecules relevant in planetary exploration and environmental monitoring. By utilizing a set of highly specific antibodies that recognize D- or L- aromatic amino acids (Phe, Tyr, Trp), benzo[a]pyrene (B[a]P), pentachlorophenol, and sulfone-containing aromatic compounds, respectively, the assay was validated in the SOLID instrument for the analysis of carbon-rich samples used as analogues of the organic material in carbonaceous chondrites or even Mars samples. Most of the antibodies enabled sensitivities at the 1-10 ppb level and some even at the ppt level. The multiplex immunoassay allowed the detection of B[a]P as well as aromatic sulfones in a water/methanol extract of an Early Cretaceous lignite sample (c.a., 140 Ma) representing type IV kerogen. No L- or D-aromatic amino acids were detected, reflecting the advanced diagenetic stage and the fossil nature of the sample. The results demonstrate the ability of the liquid extraction by ultrasonication and the versatility of the multiplex inhibitory immunoassays in the SOLID instrument to discriminate between organic matter derived from life and nonlife processes, an essential step toward life detection outside Earth.

Keywords: D- and L- aromatic amino acids; Kerogen type IV; Life detection; Molecular biomarkers; Multiplex inhibitory/competitive immunoassay; Planetary exploration.

FIG. 1.

Molecular structures of the organic compounds used in this work as haptens or free analytes. All analytes were conjugated to different proteins (BSA, KLH, or OVA) for printing on epoxy-activated glass slides. BSA, bovine serum albumin; KLH, keyhole limpet hemocyanin; OVA, ovalbumin.

FIG. 2.

Multiplex inhibitory microarray immunoassays (MIMI). (A) MAAM device and SOLID instrument used for the inhibitory microarray immunoassays (Section 2). (B) Scheme showing how the inhibitory immunoassay works: Top: Antibodies are incubated with the hapten conjugate (HC) microarray without competitor/analyte as control for no inhibition. Antibodies (Abs) are captured by their corresponding printed hapten conjugates. The Abs not bound to conjugates are washed out, while those retained produce an image after incubation with Alexa 647-labeled protein A and laser-induced fluorescence excitation. The image represents 100% of FSI for each Ab-conjugate pair. Bottom: In a test sample, a mixture of antibodies is incubated with a liquid extract of natural soil sample so that the organic analytes present in the sample compete with the immobilized hapten conjugates on the microarray for binding to the antibodies. The analyte/antibody interaction is revealed as above. The competition of the analyte in the sample will reduce the fluorescence intensity of the corresponding spot in a proportional manner to its concentration. Observed signals are normalized to 100% by using the following formula: FSI = A/A₀ × 100 where A₀ is the fluorescence in the absence of analyte. (C) Calibration curves for each antibody were carried out. Example of a calibration curve of an inhibitory immunoassay for pentachlorophenol (right) and the corresponding microarray images used for quantification (left). FSI, fluorescence signal intensity; MAAM, multiarray analysis module; SAU, Sample Analysis Unit; SOLID, Signs of Life Detector; SPU, Sample Preparation Unit.

FIG. 3.

Standard calibration curves obtained by single inhibitory immunoassay for each analyte/antibody pair. (A–J) Calibration curves showing the base 10 logarithms of the concentration of free analytes in solution versus the percentage of inhibition (% Inhibition). Percentage of inhibition was calculated by using the following formula: (1 − A/A₀) × 100; where A₀ and A are the fluorescence signals observed in the absence and presence of the inhibitor, respectively. Results represent averages of six tests performed on each of three different arrays. Error bars represent the standard deviation of three experiments. Sigmoidal curves have been fitted to a four-parameter logistic function for determining the IC₅₀ (Table 1). Serial dilutions of analytes were diluted in PBST buffer (Tween 20, 0.01%) with the exception of B[a]P, which was diluted in PBST and 10% methanol (see Section 2 for details). In (B, G), filled triangles (▴) indicate the percentage of inhibition corresponding to the analyte phthalylsulfathiazole, and open triangles (▵) indicate the percentage of inhibition obtained for the analyte sulfamethazine for both, anti-phthalylsulfathiazole and anti-sulfamethazine antibodies.

FIG. 4.

Multiplex inhibitory microarray immunoassays (MIMI). (A) The fluorescence obtained in each hapten conjugate spot after incubation with the 5-antibody mixture, without inhibitors, was quantified and normalized as 100% fluorescence (white bars, no inhibition). (A–E) Sequential and specific antibody binding inhibition by using the mixture of five antibodies and their corresponding inhibitors/analytes added in a stepwise manner as indicated by asterisks (*). Inhibitors/analytes were added in two different concentrations to account for each IC₅₀ (hatched bars) and IC₁₀₀ (gray bars). (F, G) MIMI using seven and nine antibodies, respectively. ATZ, atrazine; PCP, pentachlorophenol; PSTZ, phthalylsulfathiazole; SMZ, sulfamethazine; FINA, finasteride; D-aa, D-Phe; L-aa, L-Phe; B[a]P, Benzo[a]pyrene; and ModA peptide.

FIG. 5.

Detecting organics and aromatic amino acids with chiral selectivity by inhibition immunoassays in spiked soil samples. Antarctic soil samples were doped with IC₅₀, IC₇₅, and IC₁₀₀ concentrations (open symbols) of free L-Phe (A), D-Phe (B), and atrazine (C), extracted, and analyzed by inhibition immunoassay with the corresponding antibody. Parallel assays with the same compounds and concentration were performed as controls without soil extract (closed symbols).

FIG. 6.

MIMI for the detection of small organic compounds by using both MAAM and SOLID devices. (A) Normalized fluorescence intensity of a series of twofold dilutions of an ECL extract assayed in the MAAM device. A mixture of six fluorescent antibodies (anti-sulfamethazine, anti-phthalylsulfathiazole, anti-L-AA 18.3, anti-D-AA, anti-ModA peptide, and anti-B[a]P-5G1) at their optimal concentration was mixed with different amounts of the ECL extract and assayed for inhibition of binding to the hapten conjugate microarray (Section 2). The results are the average of two replicate experiments. (B) The plot displays the MIMI performed with 50 μl of ECL extract in SOLID instrument by using a mixture of six antibodies indicated above (see Section 2). Hatched bars, no sample controls (100% of FSI); gray bars, loss of signal after MIMI with ECL extract. Arrows show inhibition effects with B[a]P and phthalylsulfathiazole of about 50% and 100%, respectively.

FIG. 7.

Detection of the B[a]P in the ECL sample by GC-MS analysis. The retention time at 25.7 min in the chromatogram and the mass spectrum (insert) are indicated.