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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Harnessing Heterologous Bacterial Two-Component Systems as Biosensors to Address Challenges in Fermentation Scale-Up

Scaling up bacterial fermentation from bench to industrial scale often results in unpredictable performance losses, possibly in part due to changes in microenvironmental conditions such as pH. To investigate this, we developed a suite of pH-sensitive biosensors from bacterial two-component systems (TCSs) that provide a dynamic, fluorescent readout in response to extracellular pH changes. TCSs consist of a periplasmic sensor histidine kinase (HK) that, in response to an extracellular stimulus, autophosphorylates intracellularly and subsequently transfers the phosphate to a cognate response regulator (RR) that modulates transcription of target genes. We utilized three pH-responsive TCSs (referred to here as CVJ1, CVJ30, and CVJ79) and linked their output to GFP. This was achieved by placing the RR promoter upstream of GFP or by constructing a chimeric RR composed of the native receiver domain and the DNA-binding domain of another well-characterized RR with a defined promoter. All components - HK, RR (native or chimeric), and GFP under its corresponding promoter - were cloned into a broad-host-range plasmid. Sensors were validated in Escherichia coli and Pseudomonas putida, including the muconic acid-producing strain P. putida TL207. All three biosensors successfully reported pH, with fluorescence (normalized to optical density) correlating strongly with media pH. Among the native sensors, CVJ79 showed the most robust performance while CVJ1 also performed best in its native form; CVJ30 exhibited improved functionality as a chimera, suggesting that modular RR design can enhance compatibility in some heterologous hosts. Further, CVJ79 was activated by alkaline conditions, while CVJ30 responded to acidic environments. Notably, CVJ1 was induced by high pH in wild-type E. coli and P. putida, but low pH in TL207. The observed differences in sensor activation between strains - particularly the divergent response of CVJ1 - suggest that host-specific regulatory pathways may influence how cells perceive and adapt to pH stress. Moving forward, these biosensors can be used to guide the rational design of more robust strains, optimize process conditions in real time, and inform strategies to minimize physiological heterogeneity during scale-up. Integrating these tools into high-throughput screening and bioreactors will be a key step toward improving predictability and performance in industrial bioprocesses.

09 BIOMASS FUELS↗
DOE OSTI2025

A Sensitive Electrochemical Cholinesterase-Inhibiting Biosensor for Organophosphorus Pesticides Based on Ti 3 C 2 TX MXene Quantum Dots

Organophosphorus pesticides (OPs) pose significant environmental and health risks due to their widespread use and toxicity, primarily by inhibiting acetylcholinesterase. Traditional detection methods are often slow and costly, highlighting the urgent need for advanced, sensitive, and accessible technologies. This study developed a highly sensitive electrochemical cholinesterase-inhibiting biosensor for OP pesticides, utilizing Ti 3 C 2 T x MXene Quantum Dots (MQDs), which was synthesized via a hydrothermal method. The biosensor’s performance was characterized using electrochemical impedance spectroscopy, differential pulse voltammetry (DPV), and cyclic voltammetry. DPV proved to be the optimal technique, exhibiting an ultralow detection limit of 1 × 10 −17 M and a wide linear range (10 −14 –10 −8 M) for chlorpyrifos (a model OP) with an estimated inhibition constant of 62 nM. The biosensor demonstrated high selectivity for OPs (chlorpyrifos, acephate, glyphosate) over a non-target pyrethroid (permethrin), confirmed by distinct electrochemical signatures and compared to in vitro cholinergic activity assays in bean beetle homogenates. The enhanced performance is attributed to the high surface-to-volume ratio, quantum confinement effects, and superior conductivity of the MQDs, as well as the robust enzyme immobilization facilitated by glutaraldehyde cross-linking and a chitosan matrix. This work presents a promising platform for rapid, sensitive, and selective detection of OP pesticides, with potential applications in environmental monitoring and public health protection.

Chemistry↗
NASA NTRS2016

BioSentinel: Biosensors for Deep-Space Radiation Study

The BioSentinel mission will be deployed on NASA's Exploration Mission 1 (EM-1) in 2018. We will use the budding yeast, Saccharomyces cerevisiae, as a biosensor to study the effect of deep-space radiation on living cells. The BioSentinel mission will be the first investigation of a biological response to space radiation outside Low Earth Orbit (LEO) in over 40 years. Radiation can cause damage such as double stand breaks (DSBs) on DNA. The yeast cell was chosen for this mission because it is genetically controllable, shares homology with human cells in its DNA repair pathways, and can be stored in a desiccated state for long durations. Three yeast strains will be stored dry in multiple microfluidic cards: a wild type control strain, a mutant defective strain that cannot repair DSBs, and a biosensor strain that can only grow if it gets DSB-and-repair events occurring near a specific gene. Growth and metabolic activity of each strain will be measured by a 3-color LED optical detection system. Parallel experiments will be done on the International Space Station and on Earth so that we can compare the results to that of deep space. One of our main objectives is to characterize the microfluidic card activation sequence before the mission. To increase the sensitivity of yeast cells as biosensors, desiccated yeast in each card will be resuspended in a rehydration buffer. After several weeks, the rehydration buffer will be exchanged with a growth medium in order to measure yeast growth and metabolic activity. We are currently working on a time-course experiment to better understand the effects of the rehydration buffer on the response to ionizing radiation. We will resuspend the dried yeast in our rehydration medium over a period of time; then each week, we will measure the viability and ionizing radiation sensitivity of different yeast strains taken from this rehydration buffer. The data obtained in this study will be useful in finalizing the card activation sequence for this mission.

Yeast↗
NASA NTRS2017

Building Better Biosensors for Exploration into Deep-Space, Using Humanized Yeast

1.BioSentinel is 1 of 13 secondary payloads hitching a ride beyond Low Earth Orbit on Exploration Mission 1 (EM-1), set to launch from NASAs Space Launch System in 2019. EM-1 is our first opportunity to investigate the effects of the deep space environment on a eukaryotic biological system, the budding yeast S. cerevisiae. Though separated by a billion years of evolution we share hundreds of genes important for basic cell function, including responses to DNA damage. Thus, yeast is an ideal biosensor for detecting typesextent of damage induced by deep-space radiation.We will fly desiccated cells, then rehydrate to wake them up when the automated payload is ready to initiate the experiment. Rehydration solution contains SC (Synthetic Complete) media and alamarBlue, an indicator for changes in growth and metabolism. Telemetry of LED readings will then allow us to detect how cells respond throughout the mission. The desiccation-rehydration process can be extremely damaging to cells, and can severely diminish our ability to accurately measure and model cellular responses to deep-space radiation. The aim of this study is to develop a better biosensor: yeast strains that are more resistant to desiccation stress. We will over-express known cellular protectants, including hydrophilin Sip18, the protein disaggregase Hsp104, and thioredoxin Trx2, a responder to oxidative stress, then measure cell viability after desiccation to determine which factors improve stress tolerance. Over-expression of SIP18 in wine yeast starter cultures was previously reported to increase viability following desiccation stress by up to 70. Thus, we expect similar improvements in our space-yeast strains. By designing better yeast biosensors we can better prepare for and mitigate the potential dangers of deep-space radiation for future missions.This work is funded by NASAs AES program.

biosensor↗
NASA NTRS2017

Biosensor Integration Development ExMC/Canadian Space Agency Collaboration

In support of the NASA Human Research Program Exploration Medical Capability (ExMC) Element, NASA Ames Research Center (ARC) established a collaborative effort with the Canadian Space Agency (CSA). The collaboration focuses on leveraging CSA capability in the areas of biosensors and decision support that will augment future development of such components for Exploration Missions. The CSA advancement of biosensors enables NASA to focus on the integration and data management associated with these types of components through the system currently under development by the Medical Data Architecture (MDA) project. This approach has enabled the establishment of a successful collaborative working relationship between ExMC and CSA.Applying lessons learned from the fiscal year 2016 (FY16) Human Exploration Research Analog (HERA) campaign, CSA and NASA ARC developed a solution to provide real-time feedback to researchers who monitor the collection of vital signs data from a wearable Astroskin garment. The advances in the interfaces included the development of an iPad application (by CSA) to wirelessly forward the vital signs data to the MDA system, which collected the vital signs data through a receiver developed by NASA ARC. The development of these interfaces aims to provide communications between the Astroskin and the MDA system such that data may be seamlessly collected, stored and retrieved by the MDA. The first steps towards this goal were demonstrated in FY16. In FY17, ExMC will complete the first in a series of test beds that establishes a system to automate collection and management of vital sign data from the Astroskin, and other sources of data, to provide information for a crewmember to make medical decisions. In addition, the MDA Test Bed 1 will enable CSA to evaluate and optimize biosensor advancement and facilitate decision support algorithm development.

Vital signs↗
NASA NTRS2017

Biosensor Integration Development ExMC/Canadian Space Agency Collaboration

In support of the NASA Human Research Program Exploration Medical Capability (ExMC) Element, NASA Ames Research Center (ARC) established a collaborative effort with the Canadian Space Agency (CSA). The collaboration focuses on leveraging CSA capability in the areas of biosensors and decision support that will augment future development of such components for Exploration Missions. The CSA advancement of biosensors enables NASA to focus on the integration and data management associated with these types of components through the system currently under development by the Medical Data Architecture (MDA) project. This approach has enabled the establishment of a successful collaborative working relationship between ExMC and CSA.Applying lessons learned from the fiscal year 2016 (FY16) Human Exploration Research Analog (HERA) campaign, CSA and NASA ARC developed a solution to provide real-time feedback to researchers who monitor the collection of vital signs data from a wearable Astroskin garment. The advances in the interfaces included the development of an iPad application (by CSA) to wirelessly forward the vital signs data to the MDA system, which collected the vital signs data through a receiver developed by NASA ARC. The development of these interfaces aims to provide communications between the Astroskin and the MDA system such that data may be seamlessly collected, stored and retrieved by the MDA. The first steps towards this goal were demonstrated in FY16. In FY17, ExMC will complete the first in a series of test beds that establishes a system to automate collection and management of vital sign data from the Astroskin, and other sources of data, to provide information for a crewmember to make medical decisions. In addition, the MDA Test Bed 1 will enable CSA to evaluate and optimize biosensor advancement and facilitate decision support algorithm development.

Astroskin↗
DOE OSTI2024

Biosensor and machine learning-aided engineering of an amaryllidaceae enzyme

A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimer’s medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from the low-yielding daffodil Narcissus pseudonarcissus. Here, we propose an efficient biosensor-machine learning technology stack for biocatalyst development, which we apply to engineer an Amaryllidaceae enzyme in Escherichia coli. Directed evolution is used to develop a highly sensitive (EC 50 = 20 μM) and specific biosensor for the key Amaryllidaceae alkaloid branchpoint 4’-O-methylnorbelladine. A structure-based residual neural network (MutComputeX) is subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which are rapidly screened with the biosensor. Functional enzyme variants are identified that yield a 60% improvement in product titer, 2-fold higher catalytic activity, and 3-fold lower off-product regioisomer formation. A solved crystal structure elucidates the mechanism behind key beneficial mutations.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Sensitivity measurements for a 250 MHz quartz shear-horizontal surface acoustic wave biosensor under liquid viscous loading

Surface acoustic wave (SAW) devices have been used in biochemical assays due to their high sensitivity. The device sensitivity is a function of changes in the density and viscosity of the liquid. Here, we studied the effect of fluid viscosity using a 250 MHz quartz shear-horizontal (SH)-SAW biosensor by monitoring different concentrations of binary aqueous/glycerol solutions. In this study, the sensitivity of the biosensor was determined by fitting the data to models derived from perturbation theory. Measurements in water were used as the reference. For a 0% to 50% glycerol solution, an 87°–204° separation in the phase shift was observed. The slope of the plot of the phase shift vs (ηρ)0.5 was used to indicate the sensor’s sensitivity. The sensitivity for our 250 MHz quartz SH-SAW sensors was calculated to be 3.7×10−3m2sKg. The corresponding mass sensitivity was determined to be 9.25 × 105m2Kg. The limit of detection was calculated to be 36 picograms (pg), while the limit of quantification or LOQ was calculated to be 109 pg. Traditionally, liquid phase measurements have been challenging for SAW devices because liquids dampen the vibrating sensors severely. This problem has been largely solved using a transverse (shear) wave instead of the more popular longitudinal or Rayleigh waves. Liquid measurements are now possible using transverse waves, also known as shear waves, because transverse waves are only minimally attenuated by liquids. Shear-horizontal SAW sensors (SH-SAW) show great promise as label-free biosensors because of their ability to handle liquid samples. However, the viscosity of the liquid still induces loading effects and can be measured when the liquid is loaded onto the SH-SAW propagating surface (delay line). When the liquid above the delay line is perturbed by physical or chemical changes, such as binding to a receptor, it alters the propagating acoustic wave. The SH-SAW device can measure these changes in liquid properties as a change in the wave’s phase compared to the original wave. The device’s phase shift was recorded as a function of the changes in the density and viscosity of the binary glycerol solution and used to determine the sensitivity in the linear dynamic range of responses.

Materials Science↗
DOE OSTI2025

State-dependent motion of a genetically encoded fluorescent biosensor

Genetically encoded biosensors can measure biochemical properties such as small-molecule concentrations with single-cell resolution, even in vivo. Despite their utility, these sensors are “black boxes”: Very little is known about the structures of their low- and high-fluorescence states or what features are required to transition between them. We used LiLac, a lactate biosensor with a quantitative fluorescence-lifetime readout, as a model system to address these questions. X-ray crystal structures and engineered high-affinity metal bridges demonstrate that LiLac exhibits a large interdomain twist motion that pulls the fluorescent protein away from a “sealed,” high-lifetime state in the absence of lactate to a “cracked,” low-lifetime state in its presence. Understanding the structures and dynamics of LiLac will help to think about and engineer other fluorescent biosensors.

Rosen, Paul C. (ORCID:000000017414454X)↗
DOE OSTI2022

A sensitive and specific genetically-encoded potassium ion biosensor for in vivo applications across the tree of life

Potassium ion (K + ) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K + biosensors are promising tools to further improve our understanding of K + -dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded fluorescent K + biosensor, GINKO1, in the K + -bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K + biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K + dynamics in multiple model organisms, including bacteria, plants, and mice.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1996

Medical and surgical applications of space biosensor technology

Researchers in space life sciences are rapidly approaching a technology impasse. Many of the critical questions on the impact of spaceflight on living systems simply cannot be answered with the limited available technologies. Research subjects, particularly small animal models like the rat, must be allowed to function relatively untended and unrestrained for long periods to fully reflect the impact of microgravity and spaceflight on their behavior and physiology. These requirements preclude the use of present hard-wired instrumentation techniques and limited data acquisition systems. Implantable sensors and miniaturized biotelemetry are the only means of capturing the fundamental and critical data. This same biosensor and biotelemetry technology has direct application to Earth-based medicine and surgery. Continuous, on-line data acquisition and improved measurement capabilities combined with the ease and flexibility offered by automated, wireless, and portable instruments and data systems, should provide a boon to the health care industry. Playing a key role in this technology revolution is the Sensors 2000! (S2K!) Program at NASA Ames Research Center. S2K!, in collaboration with space life sciences researchers and managers, provides an integrated capability for sensor technology development and applications, including advanced biosensor technology development, spaceflight hardware development, and technology transfer and commercialization. S2K! is presently collaborating on several spaceflight projects with dual-use medical applications. One prime example is a collaboration with the Fetal Treatment Center (FTC) at the University of California at San Francisco. The goal is to develop and apply implantable chemical sensor and biotelemetry technology to continuously monitor fetal patients during extra-uterine surgery, replacement into the womb, through birth and beyond. Once validated for ground use, the method will be transitioned to spaceflight applications to remotely monitor key biochemical parameters in flight animals. Successful application of NASA implantable biosensor and biotelemetry technologies should accelerate the advancement of this and other modern medical procedures while furthering the exploration of life in space.

NASA Discipline General Space Life Sciences↗
NASA NTRS2001

Fluidics cube for biosensor miniaturization

To create a small, portable, fully automated biosensor, a compact means of fluid handling is required. We designed, manufactured, and tested a "fluidics cube" for such a purpose. This cube, made of thermoplastic, contains reservoirs and channels for liquid samples and reagents and operates without the use of any internal valves or meters; it is a passive fluid circuit that relies on pressure relief vents to control fluid movement. We demonstrate the ability of pressure relief vents to control fluid movement and show how to simply manufacture or modify the cube. Combined with the planar array biosensor developed at the Naval Research Laboratory, it brings us one step closer to realizing our goal of a handheld biosensor capable of analyzing multiple samples for multiple analytes.

NASA Discipline Life Sciences Technologies↗
NASA NTRS2017

Identification of Novel Desiccation-Tolerant S. cerevisiae Strains for Deep Space Biosensors

NASA's BioSentinel mission, a secondary payload that will fly on the Space Launch System's first Exploration Mission (EM-1), utilizes the budding yeast S. cerevisiae to study the biological response to the deep space radiation environment. Yeast samples are desiccated prior to launch to suspend growth and metabolism while the spacecraft travels to its target heliocentric orbit beyond Low Earth Orbit. Each sample is then rehydrated at the desired time points to reactivate the cells. A major risk in this mission is the loss of cell viability that occurs in the recovery period following the desiccation and rehydration process. Cell survival is essential for the detection of the biological response to features in the deep space environment, including ionizing radiation. The aim of this study is to mitigate viable cell loss in future biosensors by identifying mutations and genes that confer tolerance to desiccation stress in rad51, a radiation-sensitive yeast strain. We initiated a screen for desiccation-tolerance after rehydrating cells that were desiccated for three years, and selected various clones exhibiting robust growth. To verify retention of radiation sensitivity in the isolated clones - a crucial feature for a successful biosensor - we exposed them to ionizing radiation. Finally, to elucidate the genetic and molecular bases for observed desiccation-tolerance, we will perform whole-genome sequencing of those rad51 clones that exhibit both robust growth and radiation sensitivity following desiccation. The identification and characterization of desiccation-tolerant strains will allow us to engineer a biological model that will be resilient in face of the challenges of the deep space environment, and will thus ensure the experimental success of future biosensor missions.

S. cerevisiae↗
NASA NTRS2021

BioSensor Users' Guide

The BioSensor is a fully autonomous 3-color LED-based spectrophotometer paired with a fluidics system that supports microbes in liquid culture. Originally developed for the BioSentinel CubeSat mission to study the response of a wild type and mutant strain of yeast to the deep space environment, the BioSensor consists of a series of 16-well, independently plumbed fluidics cards. The cards utilize filters to allow the fluidic system to administer reagents while constraining the microbes in their wells. Heaters on each card incubate to the appropriate growth temperature once the experiment begins. During the active experiment, the LED/detector system measures the transmission of light through each well at three specific wavelengths, similar to a standard benchtop spectrophotometer. The transmission/absorbance kinetics curves for each well are telemetered back to Earth, along with temperature data, for analysis on the ground. The BioSensor is being upgraded from its original CubeSat free-flyer interface to be a secondary payload on lunar landers, Gateway, and other BLEO applications, while maintaining the same functionality and science utility for future experiments.

Matthew Lera↗
NASA NTRS2021

BioSensor Users' Guide

The BioSensor is a fully autonomous 3-color LED-based spectrophotometer paired with a fluidics system that supports microbes in liquid culture. Originally developed for the BioSentinel CubeSat mission to study the response of a wild type and mutant strain of yeast to the deep space environment, the BioSensor consists of a series of 16-well, independently plumbed fluidics cards. The cards utilize filters to allow the fluidic system to administer reagents while constraining the microbes in their wells. Heaters on each card incubate to the appropriate growth temperature once the experiment begins. During the active experiment, the LED/detector system measures the transmission of light through each well at three specific wavelengths, similar to a standard benchtop spectrophotometer. The transmission/ absorbance kinetics curves for each well are telemetered back to Earth, along with temperature data, for analysis on the ground. The BioSensor is being upgraded from its original CubeSat free-flyer interface to be a secondary payload on lunar landers, Gateway, and other BLEO applications, while maintaining the same functionality and science utility for future experiments.

Matthew Lera↗
DOE OSTI2025

Light-triggered electrochemical biosensor using singlet oxygen for self-powered operation and glucose detection

This study introduces a light-activated sensing strategy that integrates photosensitization with electrochemical detection. The sensor employs Eosin Y, a photosensitizer that generates singlet oxygen ( 1 O 2 ) via type II photosensitization. Immobilized within a thin polymer matrix on a carbon working electrode, Eosin Y produces 1 O 2 , under green light (520 nm) illumination, initiating a redox process that yields a measurable current. To incorporate biosensing capabilities and enable self-powered operation, this 1 O 2 – mediated process was coupled with glucose oxidase (GOx) to construct a fully operational glucose biosensor. The addition of glucose reverses the current flow by causing GOx to compete for electrons, with the resulting current magnitude correlating with glucose concentration providing a sensitive measure of glucose. The biosensor, as proof-of-principle, demonstrated excellent performance over a range of glucose concentrations (0–73 mM), achieving a detection limit (LOD) of 2.8 mM for steady state photocurrent under oxygen-saturated conditions. This platform leverages light and 1 O 2 as stimuli for tunable, on-demand signal control, offering a novel approach for adaptive, real-time biosensing technologies.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Biosensor-driven strain engineering reveals key cellular processes for maximizing isoprenol production in Pseudomonas putida

Synthetic biology generates vast combinatorial designs, yet high-throughput analytical methods to screen them are poorly matched to interrogate this search space. We address this challenge by developing a biosensor-driven, growth-coupled selection strategy in Pseudomonas putida for isoprenol, a potential aviation fuel precursor. We found and characterized a noncanonical signaling pathway, revealing a functional and physical complex between a hybrid histidine kinase and an alcohol dehydrogenase, whose activity is tuned by heterodimerization. Leveraging this biosensor in a pooled CRISPRi library selection, we identified key host limitations. Iterative combinatorial strain engineering derived from these hits yielded a 36-fold titer increase to ~900 milligrams per liter. Integrated omics analysis revealed that metabolic rewiring toward amino acid catabolism was crucial for this improvement. This observation was found to be beneficial by technoeconomic analysis. Our modular workflow provides a powerful strategy for optimizing complex heterologous pathways and uncovering emergent host biology.

CRISPRi↗
DOE OSTI2022

Biosensor and optogenetics for systems biology of yeast branched-chain alcohol production and tolerance

In this project we combined synthetic biology, systems biology, protein engineering and metabolic engineering to study, control, and improve the production of branched chain alcohols (BCAs), a class of advanced biofuels preferred by the DOE, in the yeast Saccharomyces cerevisiae. This involved the development and application of optogenetic systems as a new modality of dynamic control of native and engineered metabolic pathways, using light as inducible or repressible agent. The optogenetic systems include gene circuits for light control of gene expression, as well as light-assembled synthetic organelles and photo-switchable protein binders to control metabolic and protein function with light at the protein level. In addition, we developed the first genetically encoded biosensor for BCA production in yeast, which we used to design high throughput assays to identify highly productive strains, pathways, and enzymes. We also showed that this biosensor can be functionally co-expressed with optogenetic circuits in the same strain, raising the possibility of establishing, for the first time, computer-interfaced closed-loop controls of engineered metabolic pathways. The yeast gene deletion library was also utilized to conduct the first systems-level study on BCA toxicity in yeast, which uncovered key fundamental principles of yeast sensitivity and tolerance to these alcohols, allowing us to design highly tolerant strains with increased BCA production. This project, thus comprises the development of several new technologies, which we integrated to make new discoveries on the dynamics of BCA production and their mechanisms of cellular toxicity and tolerance, as well as to establish new paradigms to engineer and control metabolic pathways and microbial fermentations with light, for the production of BCAs and other products of interest to the DOE.

2-metyl-1-butanol↗