A Multiplex Immunosensor for Detecting Perchlorate-Reducing Bacteria for Environmental Monitoring and Planetary Exploration

Abstract

Perchlorate anions are produced by chemical industries and are important contaminants in certain natural ecosystems. Perchlorate also occurs in some natural and uncontaminated environments such as the Atacama Desert, the high Arctic or the Antarctic Dry Valleys, and is especially abundant on the surface of Mars. As some bacterial strains are capable of using perchlorate as an electron acceptor under anaerobic conditions, their detection is relevant for environmental monitoring on Earth as well as for the search for life on Mars. We have developed an antibody microarray with 20 polyclonal antibodies to detect perchlorate-reducing bacteria (PRB) strains and two crucial and highly conserved enzymes involved in perchlorate respiration: perchlorate reductase and chlorite dismutase. We determined the cross-reactivity, the working concentration, and the limit of detection of each antibody individually and in a multiplex format by Fluorescent Sandwich Microarray Immunoassay. Although most of them exhibited relatively high sensitivity and specificity, we applied a deconvolution method based on graph theory to discriminate between specific signals and cross-reactions from related microorganisms. We validated the system by analyzing multiple bacterial isolates, crude extracts from contaminated reactors and salt-rich natural samples from the high Arctic. The PRB detecting chip (PRBCHIP) allowed us to detect and classify environmental isolates as well as to detect similar strains by using crude extracts obtained from 0.5 g even from soils with low organic-matter levels (<103 cells/g of soil). Our results demonstrated that PRBCHIP is a valuable tool for sensitive and reliable detection of perchlorate-reducing bacteria for research purposes, environmental monitoring and planetary exploration.

Immunoanalytical Approach for Detecting and Identifying Ancestral Peptide Biomarkers in Early Earth Analogue Environments

“…We validated the approach depicted above (Figure ) by screening several natural samples with a set of antibodies raised against ancestral resurrected proteins: one biofilm and three samples from different silica sinters from the El Tatio geyser field in Chile, one biofilm and one fumarole regolith sample from two hydrothermal areas in Iceland, one biofilm and two sediment samples from a hydrothermal field in New Zealand, one salt crust sample from a hypersaline perennial spring in the Canadian High Arctic, and one soil sample from a Mediterranean forest near Madrid (Spain; Table S1 and Figure S7). ,− Out of them, sample ET1 , from El Tatio geothermal field, showed positive immunodetection with anti-β-lactamase_PNCA and anti-Trx_LBCA antibodies (Figure ), the antibodies against the theoretically oldest proteins as inferred by ASR (Table ). Later, we demonstrated that analogues to peptides A and B were indeed present in the sample (Figure ).…”

“…Eleven natural samples were tested in this work, corresponding to different environments (hydrothermal, arctic, and mediterranean) and different types (silica precipitates, biofilm, salt crust, sediment, and soil). Most samples have been described elsewhere, ,− and their main features are gathered in Table S1. Crude liquid sample extracts to be analyzed by FSMI were prepared by ultrasonication and filtered (see SI, Experimental Section).…”

“…We have also developed the SOLID (Signs of Life Detector) instrument, capable of obtaining, remotely and automatically, a liquid extract from 0.5 to 1 g of soil, sediments, or ground rock and searching for molecular biomarkers by fluorescence sandwich microarray immunoassay (FSMI) with the LDChip. , The LDChip performance has been demonstrated in many field campaigns, ,− as well as the suitability of antibodies for Mars exploration, ,, and the SOLID-LDChip system has been proposed as a payload instrument for searching for life on Mars in response to ESA and NASA calls …”