Integrated high performance microfluidic organic analysis instrument for planetary and space exploration.

The exploration of our solar system to characterize the molecular organic inventory will enable the identification of potentially habitable regions and initiate the search for biosignatures of extraterrestrial life. However, it is challenging to perform the required high-resolution, high-sensitivity chemical analyses in space and in planetary environments. To address this challenge, we have developed a microfluidic organic analyzer (MOA) instrument that consists of a multilayer programmable microfluidic analyzer (PMA) for fluidic processing at the microliter scale coupled with a microfabricated glass capillary electrophoresis (CE) wafer for separation and analysis of the sample components. Organic analytes are labeled with a functional group-specific (e.g. amine, organic acid, aldehyde) fluorescent dye, separated according to charge and hydrodynamic size by capillary electrophoresis (CE), and detected with picomolar limit of detection (LOD) using laser-induced fluorescence (LIF). Our goal is a sensitive automated instrument and autonomous process that enables sample-in to data-out performance in a flight capable format. We present here the design, fabrication, and operation of a technology development unit (TDU) that meets these design goals with a core mass of 3 kg and a volume of <5 L. MOA has a demonstrated resolution of 2 × 105 theoretical plates for relevant amino acids using a 15 cm long CE channel and 467 V cm-1. The LOD of LIF surpasses 100 pM (0.01 ppb), enabling biosignature detection in harsh environments on Earth. MOA is ideally suited for probing biosignatures in potentially habitable destinations on icy moons such as Europa and Enceladus, and on Mars.

Operation of a programmable microfluidic organic analyzer under microgravity conditions simulating space flight environments.

A programmable microfluidic organic analyzer was developed for detecting life signatures beyond Earth and clinical monitoring of astronaut health. Extensive environmental tests, including various gravitational environments, are required to confirm the functionality of this analyzer and advance its overall Technology Readiness Level. This work examines how the programmable microfluidic analyzer performed under simulated Lunar, Martian, zero, and hypergravity conditions during a parabolic flight. We confirmed that the functionality of the programmable microfluidic analyzer was minimally affected by the significant changes in the gravitational field, thus paving the way for its use in a variety of space mission opportunities.

Science Objectives for Flagship-Class Mission Concepts for the Search for Evidence of Life at Enceladus.

Cassini revealed that Saturn's Moon Enceladus hosts a subsurface ocean that meets the accepted criteria for habitability with bio-essential elements and compounds, liquid water, and energy sources available in the environment. Whether these conditions are sufficiently abundant and collocated to support life remains unknown and cannot be determined from Cassini data. However, thanks to the plume of oceanic material emanating from Enceladus' south pole, a new mission to Enceladus could search for evidence of life without having to descend through kilometers of ice. In this article, we outline the science motivations for such a successor to Cassini, choosing the primary science goal to be determining whether Enceladus is inhabited and assuming a resource level equivalent to NASA's Flagship-class missions. We selected a set of potential biosignature measurements that are complementary and orthogonal to build a robust case for any life detection result. This result would be further informed by quantifications of the habitability of the environment through geochemical and geophysical investigations into the ocean and ice shell crust. This study demonstrates that Enceladus' plume offers an unparalleled opportunity for in situ exploration of an Ocean World and that the planetary science and astrobiology community is well equipped to take full advantage of it in the coming decades.

Optimization of Fluorescence Labeling of Trace Analytes: Application to Amino Acid Biosignature Detection with Pacific Blue.

Fluorescence labeling of biomolecules and fluorescence detection platforms provide a powerful approach to high-sensitivity bioanalysis. Reactive probes can be chosen to target specific functional groups to enable selective analysis of a chosen class of analytes. Particularly, when targeting trace levels of analytes, it is important to optimize the reaction chemistry to maximize the labeling efficiency and minimize the background. Here, we develop methods to optimize the labeling and detection of Pacific Blue (PB)-tagged amino acids. A model is developed to quantitate labeling kinetics and completeness in the circumstance where analyte labeling and reactive probe hydrolysis are in competition. The rates of PB hydrolysis and amino acid labeling are determined as a function of pH. Labeling kinetics and completeness as a function of PB concentration are found to depend on the ratio of the hydrolysis time to the initial labeling time, which depends on the initial PB concentration. Finally, the optimized labeling and detection conditions are used to perform capillary electrophoresis analysis demonstrating 100 pM sensitivities and high-efficiency separations of an 11 amino acid test set. This work provides a quantitative optimization model that is applicable to a variety of reactive probes and targets. Our approach is particularly useful for the analysis of trace amine and amino acid biosignatures in extraterrestrial samples. For illustration, our optimized conditions (reaction at 4 °C in a pH 8.5 buffer) are used to detect trace amino acid analytes at the 100 pM level in an Antarctic ice core sample.

Quantitative evaluation of the feasibility of sampling the ice plumes at Enceladus for biomarkers of extraterrestrial life.

Enceladus, an icy moon of Saturn, is a compelling destination for a probe seeking biosignatures of extraterrestrial life because its subsurface ocean exhibits significant organic chemistry that is directly accessible by sampling cryovolcanic plumes. State-of-the-art organic chemical analysis instruments can perform valuable science measurements at Enceladus provided they receive sufficient plume material in a fly-by or orbiter plume transit. To explore the feasibility of plume sampling, we performed light gas gun experiments impacting micrometer-sized ice particles containing a fluorescent dye biosignature simulant into a variety of soft metal capture surfaces at velocities from 800 m ⋅ s-1 up to 3 km ⋅ s-1 Quantitative fluorescence microscopy of the capture surfaces demonstrates organic capture efficiencies of up to 80 to 90% for isolated impact craters and of at least 17% on average on indium and aluminum capture surfaces at velocities up to 2.2 km ⋅ s-1 Our results reveal the relationships between impact velocity, particle size, capture surface, and capture efficiency for a variety of possible plume transit scenarios. Combined with sensitive microfluidic chemical analysis instruments, we predict that our capture system can be used to detect organic molecules in Enceladus plume ice at the 1 nM level-a sensitivity thought to be meaningful and informative for probing habitability and biosignatures.

Method for detecting and quantitating capture of organic molecules in hypervelocity impacts.

Enceladus is a prime candidate in the solar system for in-depth astrobiological studies searching for habitability and life because it has a liquid water ocean with significant organic content and ongoing cryovolcanic activity. The presence of ice plumes that jet up through fissures in the ice crust covering the sub-surface ocean, enables remote sampling and in-situ analysis via a fly-by mission. However, capture and transport of organic materials to organic analyzers presents distinctive challenges as it is unknown whether, and to what extent, organic molecules imbedded in ice particles can be captured and survive hypervelocity impacts. This manuscript provides a fluorescence microscopic method to parametrically determine the amount of an organic fluorescent tracer dye, Pacific Blue™ (PB) deposited on a metallic surface. This method can be used to measure the capture and survival outcomes of terrestrial hypervelocity impact experiments where an ice projectile labeled with Pacific Blue impacts a soft metal surface. This work is an important step in the advancement of instruments like the Enceladus Organic Analyzer for detecting biosignatures in an Enceladus plume fly-by mission. An apparatus consisting of a substrate humidification shroud coupled with an epifluorescence microscope with CCD detector is developed to measure the intensity of quantitatively deposited Pacific Blue droplets under controlled humidity. Calibration curves are produced that relate the integrated fluorescence intensity of humidified PB droplets on metal foils to the number of PB molecules deposited. To demonstrate the utility of this method, our calibrations are used to analyze and quantitate organic capture and survival (up to 11% capture efficiency) following ice particle impacts at a velocity of 1.7 km/s on an aluminum substrate.

Venus, an Astrobiology Target.

We present a case for the exploration of Venus as an astrobiology target-(1) investigations focused on the likelihood that liquid water existed on the surface in the past, leading to the potential for the origin and evolution of life, (2) investigations into the potential for habitable zones within Venus' present-day clouds and Venus-like exo atmospheres, (3) theoretical investigations into how active aerobiology may impact the radiative energy balance of Venus' clouds and Venus-like atmospheres, and (4) application of these investigative approaches toward better understanding the atmospheric dynamics and habitability of exoplanets. The proximity of Venus to Earth, guidance for exoplanet habitability investigations, and access to the potential cloud habitable layer and surface for prolonged in situ extended measurements together make the planet a very attractive target for near term astrobiological exploration.

Fabrication of high-quality glass microfluidic devices for bioanalytical and space flight applications.

Microfabricated glass microfluidic and Capillary Electrophoresis (CE) devices have been utilized in a wide variety of applications over the past thirty years. At the Berkeley Space Sciences Laboratory, we are working to further expand this technology by developing analytical instruments to chemically explore our solar system. This effort requires improving the quality and reliability of glass microfabrication through quality control procedures at every stage of design and manufacture. This manuscript provides detailed information on microfabrication technology for the production of high-quality glass microfluidic chips in compliance with industrial standards and space flight instrumentation quality control.•The methodological protocol provided in this paper includes the scope of each step of the manufacturing process, materials and technologies recommended and the specific challenges that often confront the process developer.•Types and sources of fabrication error at every stage have been identified and their solutions have been proposed and verified.•We present robust and rigorous manufacturing and quality control procedures that will assist other researchers in achieving the highest possible quality glass microdevices using the latest apparatus in a routine and reliable fashion.

Monitoring transient cell-to-cell interactions in a multi-layered and multi-functional allergy-on-a-chip system.

We have developed a highly integrated lab-on-a-chip containing embedded electrical microsensors, µdegassers and pneumatically-actuated micropumps to monitor allergic hypersensitivity. Rapid antigen-mediated histamine release (e.g. s to min) and resulting muscle contraction (<30 min) is detected by connecting an immune compartment containing sensitized basophile cells to a vascular co-culture model.

Resonance Raman Characterization of Tetracene Monomer and Nanocrystals: Excited State Lattice Distortions With Implications For Efficient Singlet Fission.

The characterization of specific phonon modes and exciton states that lead to efficient singlet fission (SF) may be instrumental in the design of the next generation of high-efficiency photovoltaic devices. To this end, we analyze the absolute resonance Raman (RR) cross sections for tetracene (Tc) both as a monomer in solution and as a crystalline solid in an aqueous suspension of nanocrystals. For both systems, a time-dependent wavepacket model is developed that is consistent with the absolute RR cross sections, the magnitude of the absorption cross sections, and the vibronic line shapes of the fluorescence. In the monomer, the intramolecular reorganization energy is between 1500 and 1800 cm-1 and the solvent reorganization energy is 70 cm-1. In nanocrystals, the total reorganization is diminished to less than 600 cm-1. The lowest energy exciton has an estimated intramolecular reorganization energy between 300 and 500 cm-1 while intermolecular librational phonons have a reorganization energy of about 130 cm-1. The diminished reorganization energy of the nanocrystal is interpreted in the context of the delocalization of the band-edge exciton onto about ∼7 molecules. When electron and electron-hole correlations are included within many-body perturbation theory, the polarized absorption spectra of crystalline Tc are calculated and found to be in agreement with experiment. The low-lying exciton states and optically active phonons that contribute to the polarized crystal absorption are identified. The likely role of coherent exciton phonon evolution in the SF process is discussed.

Rapid and Fully Microfluidic Ebola Virus Detection with CRISPR-Cas13a.

Highly infectious illness caused by pathogens is endemic especially in developing nations where there is limited laboratory infrastructure and trained personnel. Rapid point-of-care (POC) serological assays with minimal sample manipulation and low cost are desired in clinical practice. In this study, we report an automated POC system for Ebola RNA detection with RNA-guided RNA endonuclease Cas13a, utilizing its collateral RNA degradation after its activation. After automated microfluidic mixing and hybridization, nonspecific cleavage products of Cas13a are immediately measured by a custom integrated fluorometer which is small in size and convenient for in-field diagnosis. Within 5 min, a detection limit of 20 pfu/mL (5.45 × 107 copies/mL) of purified Ebola RNA is achieved. This isothermal and fully solution-based diagnostic method is rapid, amplification-free, simple, and sensitive, thus establishing a key technology toward a useful POC diagnostic platform.

Excited-state vibrational dynamics toward the polaron in methylammonium lead iodide perovskite.

Hybrid organic-inorganic perovskites have attractive optoelectronic properties including exceptional solar cell performance. The improved properties of perovskites have been attributed to polaronic effects involving stabilization of localized charge character by structural deformations and polarizations. Here we examine the Pb-I structural dynamics leading to polaron formation in methylammonium lead iodide perovskite by transient absorption, time-domain Raman spectroscopy, and density functional theory. Methylammonium lead iodide perovskite exhibits excited-state coherent nuclear wave packets oscillating at ~20, ~43, and ~75 cm-1 which involve skeletal bending, in-plane bending, and c-axis stretching of the I-Pb-I bonds, respectively. The amplitudes of these wave packet motions report on the magnitude of the excited-state structural changes, in particular, the formation of a bent and elongated octahedral PbI64- geometry. We have predicted the excited-state geometry and structural changes between the neutral and polaron states using a normal-mode projection method, which supports and rationalizes the experimental results. This study reveals the polaron formation via nuclear dynamics that may be important for efficient charge separation.

Difference Bands in Time-Resolved Femtosecond Stimulated Raman Spectra of Photoexcited Intermolecular Electron Transfer from Chloronaphthalene to Tetracyanoethylene.

The time-resolved femtosecond stimulated Raman spectra (FSRS) of a charge transfer (CT) excited noncovalent complex tetracyanoethylene:1-chloronaphthalene (TCNE:ClN) in dichloromethane (DCM) is reported with 40 fs time resolution. In the frequency domain, five FSRS peaks are observed with frequencies of 534, 858, 1069, 1392, and 1926 cm-1. The most intense peaks at 534 and 1392 cm-1 correspond to fundamentals while the features at 858, 1069, and 1926 cm-1 are attributed to a difference frequency, an overtone and a combination frequency of the fundamentals, respectively. The frequency of the 1392 cm-1 fundamental corresponding to the central C═C stretch of TCNE•- is red-shifted from the frequency of the steady state radical due to the close proximity and electron affinity of the countercation. The observation of a FSRS band at a difference frequency is analyzed. This analysis lends evidence for alternative nonlinear pathways of inverse Raman gain scattering (IRGS) or vertical-FSRS (VFSRS) which may contribute to the time-evolving FSRS spectrum on-resonance. Impulsive stimulated Raman measurements of the complex show coherent oscillations of the stimulated emission with frequencies of 153, 278, and 534 cm-1. The 278 cm-1 mode corresponds to Cl bending of the dichloromethane solvent. The center frequency of the 278 cm-1 mode is modulated by a frequency of ∼30 cm-1 which is attributed to the effect of librational motion of the dichloromethane solvent as it reorganizes around the nascent contact ion pair. The 153 ± 15 cm-1 mode corresponds to an out-of-plane bending motion of TCNE. This motion modulates the intermolecular separation of the contact ion pair and thereby the overlap of the frontier orbitals which is crucial for rapid charge recombination in 5.9 ± 0.2 ps. High time-frequency resolution vibrational spectra provide unique molecular details regarding charge localization and recombination.

Microfluidic System for Detection of Viral RNA in Blood Using a Barcode Fluorescence Reporter and a Photocleavable Capture Probe.

A microfluidic sample preparation multiplexer (SPM) and assay procedure is developed to improve amplification-free detection of Ebola virus RNA from blood. While a previous prototype successfully detected viral RNA following off-chip RNA extraction from infected cells, the new device and protocol can detect Ebola virus in raw blood with clinically relevant sensitivity. The Ebola RNA is hybridized with sequence specific capture and labeling DNA probes in solution and then the complex is pulled down onto capture beads for purification and concentration. After washing, the captured RNA target is released by irradiating the photocleavable DNA capture probe with ultraviolet (UV) light. The released, labeled, and purified RNA is detected by a sensitive and compact fluorometer. Exploiting these capabilities, a detection limit of 800 attomolar (aM) is achieved without target amplification. The new SPM can run up to 80 assays in parallel using a pneumatic multiplexing architecture. Importantly, our new protocol does not require time-consuming and problematic off-chip probe conjugation and washing. This improved SPM and labeling protocol is an important step toward a useful POC device and assay.

Feasibility of Detecting Bioorganic Compounds in Enceladus Plumes with the Enceladus Organic Analyzer.

Enceladus presents an excellent opportunity to detect organic molecules that are relevant for habitability as well as bioorganic molecules that provide evidence for extraterrestrial life because Enceladus' plume is composed of material from the subsurface ocean that has a high habitability potential and significant organic content. A primary challenge is to send instruments to Enceladus that can efficiently sample organic molecules in the plume and analyze for the most relevant molecules with the necessary detection limits. To this end, we present the scientific feasibility and engineering design of the Enceladus Organic Analyzer (EOA) that uses a microfluidic capillary electrophoresis system to provide sensitive detection of a wide range of relevant organic molecules, including amines, amino acids, and carboxylic acids, with ppm plume-detection limits (100 pM limits of detection). Importantly, the design of a capture plate that effectively gathers plume ice particles at encounter velocities from 200 m/s to 5 km/s is described, and the ice particle impact is modeled to demonstrate that material will be efficiently captured without organic decomposition. While the EOA can also operate on a landed mission, the relative technical ease of a fly-by mission to Enceladus, the possibility to nondestructively capture pristine samples from deep within the Enceladus ocean, plus the high sensitivity of the EOA instrument for molecules of bioorganic relevance for life detection argue for the inclusion of EOA on Enceladus missions. Key Words: Lab-on-a-chip-Organic biomarkers-Life detection-Planetary exploration. Astrobiology 17, 902-912.

Critical Role of Methylammonium Librational Motion in Methylammonium Lead Iodide (CH3NH3PbI3) Perovskite Photochemistry.

Raman and photoluminescence (PL) spectroscopy are used to investigate dynamic structure-function relationships in methylammonium lead iodide (MAPbI3) perovskite. The intensity of the 150 cm-1 methylammonium (MA) librational Raman mode is found to be correlated with PL intensities in microstructures of MAPbI3. Because of the strong hydrogen bond between hydrogens in MA and iodine in the PbI6 perovskite octahedra, the Raman activity of MA is very sensitive to structural distortions of the inorganic framework. The structural distortions directly influence PL intensities, which in turn have been correlated with microstructure quality. Our measurements, supported with first-principles calculations, indicate how excited-state MA librational displacements mechanistically control PL efficiency and lifetime in MAPbI3-material parameters that are likely important for efficient photovoltaic devices.

High-performance detection of somatic D-loop mutation in urothelial cell carcinoma patients by polymorphism ratio sequencing.

UNLABELLED: Utilizing a polymorphism ratio sequencing platform, we performed a complete somatic mutation analysis of the mitochondrial D-loop region in 14 urothelial cell carcinomas. A total of 28 somatic mutations, all heteroplasmic, were detected in 8 of 14 individuals (57.1 %). Insertion/deletion changes in unstable mono- and dinucleotide repeat segments comprise the most pervasive class of mutations (9 of 28), while two recurring single-base substitution loci were identified. Seven variants, mostly insertion/deletions, represent population shifts from a heteroplasmic germline toward dominance in the tumor. In four cases, DNA from matched urine samples was similarly analyzed, with all somatic variants present in associated tumors readily detectable in the bodily fluid. Consistent with previous findings, mutant populations in urine were similar to those detected in tumor and in three of four cases were more prominent in urine. KEY MESSAGES: PRS accurately detects high mtDNA mutations in UCCs and their body fluids. mtDNA mutations are universally heteroplasmic and often appear at low levels. The PRS technology could be a viable approach to develop mitochondrial biomarkers.

Femtosecond Stimulated Raman Exposes the Role of Vibrational Coherence in Condensed-Phase Photoreactivity.

Femtosecond spectroscopy has revealed coherent wave packet motion time and time again, but the question as to whether these coherences are necessary for reactivity or merely a consequence of the experiment has remained open. For diatomic systems in the gas phase, such as sodium iodide, the dimensionality of the system requires coordinated atomic motion along the reaction coordinate. Coherent dynamics are also readily observed in condensed-phase multidimensional systems such as chromophores in proteins and solvated charge transfer dimers. Is precisely choreographed nuclear motion (i.e., coherence) required for reactivity in these systems? Can this coherence reveal anything about the reaction coordinate? In this Account, we describe our efforts to tackle these questions using femtosecond stimulated Raman spectroscopy (FSRS). Results of four exemplary systems are summarized to illustrate the role coherence can play in condensed-phase reactivity, the exploitation of vibrational coherence to measure vibrational anharmonicities, and the development of two-dimensional FSRS (2D-FSRS). We begin with rhodopsin, the protein responsible for vertebrate vision. The rhodopsin photoreaction is preternaturally fast: ground-state photoproduct is formed in less than 200 fs. However, the reactively important hydrogen out-of-plane motions as well as various torsions and stretches remain vibrationally coherent long after the reaction is complete, indicating that vibrational coherence can and does survive reactive internal conversion. Both the ultrashort time scale of the reaction and the observed vibrational coherence indicate that the reaction in rhodopsin is a vibrationally coherent process. Next we examine the functional excited-state proton transfer (ESPT) reaction of green fluorescent protein. Oscillations in the phenoxy C-O and imidazolinone C═N stretches in the FSRS spectrum indicated strong anharmonic coupling to a low-frequency phenyl wagging mode that gates the ESPT reaction. In this case, the coherence revealed not only itself but also the mode coupling that is necessary for reactivity. Curious as to whether vibrational coherence is a common phenomenon, we examined two simpler photochemical systems. FSRS studies of the charge transfer dimer tetramethylbenzene:tetracyanoquinodimethane revealed many vibrational oscillations with high signal-to-noise ratio that allowed us to develop a 2D-FSRS technique to quantitatively measure anharmonic vibrational coupling for many modes within a reacting excited state. Armed with this technique, we turned our attention to a bond-breaking reaction, the cycloreversion of a cyclohexadiene derivative. By means of 2D-FSRS, the vibrational composition of the excited-state transition state and therefore the reaction coordinate was revealed. In aggregate, these FSRS measurements demonstrate that vibrational coherences persist for many picoseconds in condensed phases at room temperature and can survive reactive internal conversion. Moreover, these coherences can be leveraged to reveal quantitative anharmonic couplings between a molecule's normal modes in the excited state. These anharmonic couplings are the key to determining how normal modes combine to form a reaction coordinate. It is becoming clear that condensed-phase photochemical reactions that occur in 10 ps or less require coordinated, coherent nuclear motion for efficient reactive internal conversion.

Femtosecond Stimulated Raman Spectroscopy.

Femtosecond stimulated Raman spectroscopy (FSRS) is an ultrafast nonlinear optical technique that provides vibrational structural information with high temporal (sub-50 fs) precision and high spectral (10 cm(-1) ) resolution. Since the first full demonstration of its capabilities ≈15 years ago, FSRS has evolved into a mature technique, giving deep insights into chemical and biochemical reaction dynamics that would be inaccessible with any other technique. It is now being routinely applied to virtually all possible photochemical reactions and systems spanning from single molecules in solution to thin films, bulk crystals and macromolecular proteins. This review starts with an historic overview and discusses the theoretical and experimental concepts behind this technology. Emphasis is put on the current state-of-the-art experimental realization and several variations of FSRS that have been developed. The unique capabilities of FSRS are illustrated through a comprehensive presentation of experiments to date followed by prospects.

End-to-end automated microfluidic platform for synthetic biology: from design to functional analysis.

BACKGROUND: Synthetic biology aims to engineer biological systems for desired behaviors. The construction of these systems can be complex, often requiring genetic reprogramming, extensive de novo DNA synthesis, and functional screening. RESULTS: Herein, we present a programmable, multipurpose microfluidic platform and associated software and apply the platform to major steps of the synthetic biology research cycle: design, construction, testing, and analysis. We show the platform's capabilities for multiple automated DNA assembly methods, including a new method for Isothermal Hierarchical DNA Construction, and for Escherichia coli and Saccharomyces cerevisiae transformation. The platform enables the automated control of cellular growth, gene expression induction, and proteogenic and metabolic output analysis. CONCLUSIONS: Taken together, we demonstrate the microfluidic platform's potential to provide end-to-end solutions for synthetic biology research, from design to functional analysis.