A Mission Simulating the Search for Life on Mars with Automated Drilling, Sample Handling, and Life Detection Instruments Performed in the Hyperarid Core of the Atacama Desert, Chile.

We report on a field demonstration of a rover-based drilling mission to search for biomolecular evidence of life in the arid core of the Atacama Desert, Chile. The KREX2 rover carried the Honeybee Robotics 1 m depth The Regolith and Ice Drill for Exploration of New Terrains (TRIDENT) drill and a robotic arm with scoop that delivered subsurface fines to three flight prototype instruments: (1) The Signs of Life Detector (SOLID), a protein and biomolecule analyzer based on fluorescence sandwich microarray immunoassay; (2) the Planetary In Situ Capillary Electrophoresis System (PISCES), an amino acid analyzer based on subcritical water extraction coupled to microchip electrophoresis analysis; and (3) a Wet Chemistry Laboratory cell to measure soluble ions using ion selective electrodes and chronopotentiometry. A California-based science team selected and directed drilling and sampling of three sites separated by hundreds of meters that included a light-toned basin area showing evidence of aqueous activity surrounded by a rocky desert pavement. Biosignatures were detected in basin samples collected at depths ranging from 20 to 80 cm but were not detected in the surrounding area. Subsurface stratigraphy of the units drilled was interpreted from drill sensor data as fine-scale layers of sand/clay sediments interspersed with layers of harder material in the basins and a uniform subsurface composed of course-to-fine sand in the surroundings. The mission timeline and number of commands sent to accomplish each activity were tracked. The deepest sample collected (80 cm) required 55 commands, including drilling and delivery to three instruments. Elapsed time required for drilling and sample handling was less than 3 hours to collect sample from 72 cm depth, including time devoted to recovery from a jammed drill. The experiment demonstrated drilling, sample transfer technologies, and instruments that accomplished successful detection of biomolecular evidence of life in one of the most biologically sparse environments on Earth.

Submersible Capillary Electrophoresis Analyzer: A Proof-of-Concept Demonstration of an In Situ Instrument for Future Missions to Ocean Worlds.

We report here the first fully automated capillary electrophoresis (CE) system that can be operated underwater. The system performs sample acquisition and analysis by coupling CE to contactless conductivity detection. Using 5 M acetic acid as the background electrolyte (BGE), inorganic cations and amino acids at concentrations as low as 5.2 µM can be separated and identified. This technology could be augmented to include a variety of other detection modes. This system serves as an early prototype for potential future underwater explorers on ocean worlds of the outer solar system such as Europa or Enceladus. This work documents the first step in the development of this general-purpose technology platform.

A gravity-independent single-phase electrode reservoir for capillary electrophoresis applications.

Capillary electrophoresis (CE) holds great promise as an in situ analytical technique for a variety of applications. However, typical instrumentation operates with open reservoirs (e.g., vials) to accommodate reagents and samples, which is problematic for automated instruments designed for space or underwater applications that may be operated in various orientations. Microgravity conditions add an additional challenge due to the unpredictable position of the headspace (air layer above the liquid) in any two-phase reservoir. One potential solution for these applications is to use a headspace-free, flow-through reservoir design that is sealed and connected to the necessary reagents and samples. Here, we demonstrate a flow-through high-voltage (HV) reservoir for CE that is compatible with automated in situ exploration needs, and which can be electrically isolated from its source fluidics (in order to prevent unwanted leakage current). We also demonstrate how the overall system can be rationally designed based on the operational parameters for CE to prevent electrolysis products generated at the electrode from entering the capillary and interfering with the CE separation. A reservoir was demonstrated with a 19 mm long, 1.8 mm inner diameter channel connecting the separation capillary and the HV electrode. Tests of these reservoirs integrated into a CE system show reproducible CE system operation with a variety of background electrolytes at voltages up to 25 kV. Rotation of the reservoirs, and the system, showed that their performance was independent of the direction of the gravity vector.

A hydrodynamic injection approach for capillary electrophoresis using rotor-stator valves.

Sample injection is a critical step in a capillary electrophoresis (CE) analysis. Electrokinetic injection is the simplest approach and is often selected for implementation in portable CE instruments. However, in order to minimize the effect of sample matrix upon the results of a CE analysis, hydrodynamic injection is preferred. Although portable CE instruments with hydrodynamic injection have been reported, injection has always been performed at the grounded end of the capillary. This simplifies fluidic handling but limits coupling with electrochemical detectors and electrospray ionization-mass spectrometry (ESI-MS). We demonstrated previously that injection at the high-voltage (HV) end of the capillary could be performed using an HV-compatible rotary injection valve (fixed-volume injection). However, the mismatch between the bore sizes of the channels on the rotor-stator valve and the separation capillary caused peak tailing and undesired mixing, impairing analytical performance. In this work, we present an HV-compatible hydrodynamic injection approach that overcomes the issues associated with the fixed-volume injection approach reported previously. The performance of the CE instrument was demonstrated by analyzing a mixture of 13 amino acids by CE coupled to laser-induced fluorescence, which showed relative standard deviations for peak area and migration time below 5% and 1%, respectively, for triplicate analysis. Additionally, replicate measurements of a mixture of amino acids, peptides, nucleobases, and nucleosides by CE coupled to electrospray ionization-mass spectrometry (CE-ESI-MS) were performed to evaluate peak tailing, and results were similar to those obtained with a commercial CE-ESI-MS setup.

A voltage trade study for the design of capillary electrophoresis instruments for spaceflight.

Capillary electrophoresis (CE) systems have undergone extensive development for spaceflight applications. A flight-compatible high voltage power supply and the necessary voltage isolation for other energized components can be large contributors to both the volume and mass of a CE system, especially if typical high voltage levels of 25-30 kV are used. Here, we took advantage of our custom CE hardware to perform a trade study for simultaneous optimization of capillary length, high voltage level, and separation time, without sacrificing method performance. A capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4 D) method recently developed by our group to target inorganic cations and amino acids relevant to astrobiology was used as a test case. The results indicate that a 50 cm long capillary with 15 kV applied voltage (half of that used in the original method) can be used to achieve measurement goals while minimizing instrument size.

A Novel and Sensitive Method for the Analysis of Fatty Acid Biosignatures by Capillary Electrophoresis-Mass Spectrometry.

Fatty acids are a well-established class of compounds targeted as biosignatures for future missions to look for evidence of life on ocean worlds such as Europa and Enceladus. In order to establish their abiotic or biotic origin, we need to separate and quantify fatty acids to determine their relative abundances within a sample. In this study, we demonstrate the high potential of capillary electrophoresis coupled to mass spectrometry (CE-MS) for the efficient separation and sensitive detection of a wide variety of fatty acids. Three derivatization strategies were evaluated to allow the detection of fatty acids by positive ionization mode MS. Furthermore, CE-MS conditions were optimized to provide maximum separation efficiencies and detection sensitivities for the analysis of saturated and unsaturated fatty acids with even- and odd-numbered carbon chain lengths. Optimum separation and detection were obtained using a background electrolyte of 2 M acetic acid in 45% acetonitrile, after derivatization of the fatty acids with 2-picolylamine or N,N-diethylethylenediamine. The limits of detection for the derivatized fatty acids using the optimized method ranged from 25 to 250 nM. The optimized method was also used for the analysis of fatty acids in cell cultures and natural samples. Two distinctive biosignatures were obtained for the microorganisms Halobacillus halophilus and Pseudoalteromonas haloplanktis. In addition, multiple fatty acids were detected in a natural sample from Mono Lake, California.

Detection of Biosignatures by Capillary Electrophoresis Mass Spectrometry in the Presence of Salts Relevant to Ocean Worlds Missions.

Capillary electrophoresis (CE) is a promising liquid-based technique for in situ chemical analysis on ocean worlds that allows the detection of a wide range of organic molecules relevant to the search for life. CE coupled with mass spectrometry (MS) is particularly valuable as it also enables the discovery of unknown compounds. Here we demonstrate that CE coupled to MS via electrospray ionization (ESI) can readily analyze samples containing up to half the saturation levels of salts relevant to ocean worlds when using 5 M acetic acid as the separation media. A mixture containing amino acids, peptides, nucleobases, and nucleosides was analyzed in the presence of two salts, NaCl and MgSO4, based on their relevance to Europa and Enceladus. We demonstrate here CE-MS limits of detection for these organics ranging from 0.05 to 1 µM (8 to 89 ppb) in the absence of salts. More importantly, we demonstrate here for the first time that organics in the low micromolar range (1-50 µM) are detected by CE-MS in the presence of 3 M NaCl without desalting, preconcentration, or derivatization. This demonstration highlights how CE-MS is uniquely suited for organic analysis on future missions to ocean worlds.

Autonomous CE Mass-Spectra Examination for the Ocean Worlds Life Surveyor.

Ocean worlds such as Europa and Enceladus are high priority targets in the search for past or extant life beyond Earth. Evidence of life may be preserved in samples of surface ice by processes such as deposition from active plumes, hydrofracturing, or thermal convection. Terrestrial life produces unique distributions of organic molecules that translate into recognizable biosignatures. Identification and quantification of these organic compounds can be achieved by separation science such as capillary electrophoresis coupled to mass spectrometry (CE-MS). However, the data generated by such an instrument can be multiple orders of magnitude larger than what can be transmitted back to Earth during an ocean world's mission. This requires onboard science data analysis capabilities that summarize and prioritize CE-MS observations with limited computational resources. In response, the autonomous capillary electrophoresis mass-spectra examination (ACME) onboard science autonomy system was created for application to the ocean world's life surveyor (OWLS) instrument suite. ACME is able to compress raw mass spectra by two to three orders of magnitude while preserving most of its scientifically relevant information content. This summarization is achieved by the extraction of raw data surrounding autonomously identified ion peaks and the detection and parameterization of unique background regions. Prioritization of the summarized observations is then enabled by providing estimates of scientific utility, including presence of key target compounds, and the uniqueness of an observation relative to previous observations.

Capillary electrophoresis method for analysis of inorganic and organic anions related to habitability and the search for life.

In situ missions of exploration require analytical methods that are capable of detecting a wide range of molecular targets in complex matrices without a priori assumptions of sample composition. Furthermore, these methods should minimize the number of reagents needed and any sample preparation steps. We have developed a method for the detection of metabolically relevant inorganic and organic anions that is suitable for implementation on in situ spaceflight missions. Using 55 mM acetic acid, 50 mM triethylamine, and 5% glycerol, more than 21 relevant anions are separated in less than 20 min. The method is robust to sample ionic strength, tolerating high concentrations of background salts (up to 900 mM NaCl and 300 mM MgSO4 ). This is an important feature for future missions to ocean worlds. The method was validated using a culture of Escherichia coli and with high salinity natural samples collected from Mono Lake, California.

Automated Capillary Electrophoresis System Compatible with Multiple Detectors for Potential In Situ Spaceflight Missions.

The in situ search for chemical signatures of life on extraterrestrial worlds requires automated hardware capable of performing detailed compositional analysis during robotic missions of exploration. The use of electrophoretic separations in this search is particularly powerful, enabling analysis of a wide range of soluble organic compounds potentially indicative of life, as well as inorganic compounds that can serve as indicators of habitability. However, to detect this broad range of compounds with a single electrophoresis instrument, a combination of different detection modes is required. For detection of any ionizable species, including organic compounds that do not participate in terrestrial biology (i.e., "unknown unknowns"), mass spectrometry (MS) is essential. Inorganic ions, or any dissolved charged species present, can be analyzed using capacitively coupled contactless conductivity detection (C4D). Additionally, for the trace analysis of compounds of key interest to astrobiology (particularly, amino acids), laser-induced fluorescence (LIF) detection holds unique promise, due to the fact that it has the highest demonstrated sensitivity of any form of detection. Here, we demonstrate a fully automated, portable capillary electrophoresis analyzer that is capable of all these modes of detection. The prototype system developed here addresses the three most significant challenges for doing electrophoretic separations: precise sample injection, HV isolation, and automation of all operational steps. These key challenges were successfully addressed with the use of custom-designed rotor-stator valves with optimized operational sequences incorporating gas purging steps, rinses, and HV application.

Fully Automated Microchip Electrophoresis Analyzer for Potential Life Detection Missions.

There are a variety of complementary observations that could be used in the search for life in extraterrestrial settings. At the molecular scale, patterns in the distribution of organics could provide powerful evidence of a biotic component. In order to observe these molecular biosignatures during spaceflight missions, it is necessary to perform separation science in situ. Microchip electrophoresis (ME) is ideally suited for this task. Although this technique is readily miniaturized and numerous instruments have been developed over the last 3 decades, to date, all lack the automation capabilities needed for future missions of exploration. We have developed a portable, automated, battery-powered, and remotely operated ME instrument coupled to laser-induced fluorescence detection. This system contains all the necessary hardware and software interfaces for end-to-end functionality. Here, we report the first application of the system for amino acid analysis coupled to an extraction unit in order to demonstrate automated sample-to-data operation. The system was remotely operated aboard a rover during a simulated Mars mission in the Atacama Desert, Chile. This is the first demonstration of a fully automated ME analysis of soil samples relevant to planetary exploration. This validation is a critical milestone in the advancement of this technology for future implementation on a spaceflight mission.

Long-term thermal stability of fluorescent dye used for chiral amino acid analysis on future spaceflight missions.

Future spaceflight missions focused on life detection will carry with them new, state-of-the-art instrumentation capable of highly selective and sensitive organic analysis. CE-LIF is an ideal candidate for such a mission due to its high separation efficiency and low LODs. One perceived risk of utilizing this technique on a future mission is the stability of the chemical reagents in the spaceflight environment. Here, we present an investigation of the thermal stability of the fluorescent dye (5-carboxyfluorescein succinimidyl ester) used for amino acid analysis. The dye was stored at 4, 25, and 60°C for 1 month, 6 months, 1 year, and 2 years. When stored at 4°C for 2 years, 25°C for 6 months, or 60°C for 1 month there was no effect on CE-LIF assay performance due to dye degradation. Beyond these time points, while the dye degradation begins to interfere with the analysis, it is still possible to perform the analysis and achieve the majority of amino acid biosignature science goals described in the science definition team report for the potential Europa Lander mission. This work indicates that thermal control of the dye at ≤4°C will be needed during transit on future spaceflight missions to maintain dye stability.

Patterning and Modeling Three-Dimensional Microfluidic Devices Fabricated on a Single Sheet of Paper.

This work describes a method to fabricate three-dimensional paper microfluidic devices in one step, without the need of stacking layers of paper, glue, or tape. We used a nontransparent negative photoresist that allows patterning selectively (vertically) the paper, creating systems of two or three layers, including channels. To demonstrate the capabilities of this methodology, we designed, fabricated, and tested a six-level diluter. The performance of the device was also simulated using a simple numerical model implemented in the program PETSc-FEM. The resulting µPAD is small (1.6 cm × 2.2 cm), inexpensive, requires low volumes of sample (5 µL), and is able to perform mixing and dilution in 2 min.

Stability of reagents used for chiral amino acid analysis during spaceflight missions in high-radiation environments.

The search for biosignatures on spaceflight missions requires in situ instrumentation capable of highly selective and sensitive organic analyses. To this end, CE-LIF is a uniquely promising technique, capable of determining the type, abundance, and chirality of amino acids present in environmental samples at nanomolar concentrations. However, this type of assay requires several reagents that have not yet been used on spaceflight missions. A key concern, particularly for future missions to Europa, is the survivability of these critical components for CE separation and LIF detection under high levels of radiation. Here we present an investigation of the chemical stability of the reagents and associated fused silica capillary after a total ionizing dose of 300 krad, exceeding the predicted total ionizing dose for the potential Europa Lander Mission payload by two-fold. Neither the fused silica capillary nor the fluorescent dye (5-carboxyfluorescein succinimidyl ester) showed significant change in performance following irradiation. Following the irradiation of the pre-mixed background electrolyte, both migration time and resolution were affected. However, when the reagents (sodium tetraborate, sodium taurocholate, and γ-cyclodextrin) and the acetonitrile solution were irradiated separately and mixed afterwards, there was no change in the separation performance.

Analysis of inorganic cations and amino acids in high salinity samples by capillary electrophoresis and conductivity detection: Implications for in-situ exploration of ocean worlds.

With growing interest in exploring ocean worlds, such as Europa and Enceladus, there is a fundamental need to develop liquid-based analytical techniques capable of handling high salinity samples while performing both bulk and trace species measurements. In this context, CE with capacitively coupled contactless conductivity detection (CE-C4 D) has tremendous potential. One of its advantages is that this combination allows the detection of a wide number of charged species (both organic and inorganic) without the need of derivatization. Amino acids are an example of organic targets that are powerful biosignatures in the search for life beyond Earth. Simultaneous information on the inorganic cations in a sample helps with assessing the habitability of an extraterrestrial environment, as well as providing sample context for any measurements of trace amino acids. In this work, we present a series of flight-compatible methods capable of simultaneously measuring inorganic cations and amino acids in samples of varying salinity by CE-C4 D. Regardless of the sample total salinity, 5.0 M acetic acid was selected as the optimum BGE. The methods were evaluated by analyzing natural samples of low and high salinity from Hot Creek Gorge, Mono Lake, and Santa Monica beach. Prospects for mission implementation are also discussed.

Extraction of amino acids from aerogel for analysis by capillary electrophoresis. Implications for a mission concept to Enceladus' Plume.

Ocean worlds like Europa and Enceladus in the outer solar system are prime targets in the search for life beyond Earth. Enceladus is particularly interesting due to the presence of a water plume ejecting from the south polar region. The recent discovery of H2 in the plume, in addition to the presence of previously observed organic compounds, highlights the possibility of life in this moon. The plume provides materials from the underlying ocean that could be collected simply by flying through it. The presence of the plume means that material from the ocean is available for collection during a flyby, without the need for landing or complex sample handling operations such as scooping or drilling. An attractive approach to preserve the organics in particles collected during flyby encounters would be to utilize silica aerogel, the material used to collect particles at hypervelocity during the Stardust mission. Here we demonstrate amino acids can be extracted from aerogel simply by adding water. This simple liquid extraction method could be implemented during a mission prior to analysis with a liquid-based technique like capillary electrophoresis.

Enhanced Resolution of Chiral Amino Acids with Capillary Electrophoresis for Biosignature Detection in Extraterrestrial Samples.

Amino acids are fundamental building blocks of terrestrial life as well as ubiquitous byproducts of abiotic reactions. In order to distinguish between amino acids formed by abiotic versus biotic processes it is possible to use chemical distributions to identify patterns unique to life. This article describes two capillary electrophoresis methods capable of resolving 17 amino acids found in high abundance in both biotic and abiotic samples (seven enantiomer pairs d/l-Ala, -Asp, -Glu, -His, -Leu, -Ser, -Val and the three achiral amino acids Gly, ß-Ala, and GABA). To resolve the 13 neutral amino acids one method utilizes a background electrolyte containing γ-cyclodextrin and sodium taurocholate micelles. The acidic amino acid enantiomers were resolved with γ-cyclodextrin alone. These methods allow detection limits down to 5 nM for the neutral amino acids and 500 nM for acidic amino acids and were used to analyze samples collected from Mono Lake with minimal sample preparation.

Implementation of microchip electrophoresis instrumentation for future spaceflight missions.

We present a comprehensive discussion of the role that microchip electrophoresis (ME) instrumentation could play in future NASA missions of exploration, as well as the current barriers that must be overcome to make this type of chemical investigation possible. We describe how ME would be able to fill fundamental gaps in our knowledge of the potential for past, present, or future life beyond Earth. Despite the great promise of ME for ultrasensitive portable chemical analysis, to date, it has never been used on a robotic mission of exploration to another world. We provide a current snapshot of the technology readiness level (TRL) of ME instrumentation, where the TRL is the NASA systems engineering metric used to evaluate the maturity of technology, and its fitness for implementation on missions. We explain how the NASA flight implementation process would apply specifically to ME instrumentation, and outline the scientific and technology development issues that must be addressed for ME analyses to be performed successfully on another world. We also outline research demonstrations that could be accomplished by independent researchers to help advance the TRL of ME instrumentation for future exploration missions. The overall approach described here for system development could be readily applied to a wide range of other instrumentation development efforts having broad societal and commercial impact.

Analysis of thiols by microchip capillary electrophoresis for in situ planetary investigations.

Microchip capillary electrophoresis with laser-induced fluorescence detection (µCE-LIF) enables sensitive analyses of a wide range of analytes employing small volumes of sample and reagent (nL to µL) on an instrument platform with minimal mass, volume, and power requirements. This technique has been used previously in the analysis of amino acids and other organic molecules of interest in the fields of astrobiology and planetary science. Here, we present a protocol for the analysis of thiols using µCE-LIF. This protocol utilizes Pacific Blue C5-maleimide for fluorescent derivatization of thiols, enabling limits of detection in the low nM range (1.4-15 nM). Separations are conducted in micellar electrokinetic chromatography mode with 25 mM sodium dodecyl sulfate in 15 mM tetraborate, pH 9.2. This method allows analysis of 12 thiols in less than 2 min following a labeling step of 2 h. A step-by-step protocol, including tips on microchip capillary electrophoresis, is described here.

Getting started with open-hardware: development and control of microfluidic devices.

Understanding basic concepts of electronics and computer programming allows researchers to get the most out of the equipment found in their laboratories. Although a number of platforms have been specifically designed for the general public and are supported by a vast array of on-line tutorials, this subject is not normally included in university chemistry curricula. Aiming to provide the basic concepts of hardware and software, this article is focused on the design and use of a simple module to control a series of PDMS-based valves. The module is based on a low-cost microprocessor (Teensy) and open-source software (Arduino). The microvalves were fabricated using thin sheets of PDMS and patterned using CO2 laser engraving, providing a simple and efficient way to fabricate devices without the traditional photolithographic process or facilities. Synchronization of valve control enabled the development of two simple devices to perform injection (1.6 ± 0.4 µL/stroke) and mixing of different solutions. Furthermore, a practical demonstration of the utility of this system for microscale chemical sample handling and analysis was achieved performing an on-chip acid-base titration, followed by conductivity detection with an open-source low-cost detection system. Overall, the system provided a very reproducible (98%) platform to perform fluid delivery at the microfluidic scale.