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At least 217 records · Page 12

Carrier-assisted One-pot Sample Preparation for Targeted Proteomics Analysis of Small Numbers of Human Cells

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent limitations (e.g., low multiplex and unavailability of antibodies for new proteins). Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative because it is antibody-free, high multiplex, and has high specificity and quantitation accuracy. Recent advances in MS instrumentation make MS-based targeted proteomics possible for multiplexed quantification of highly abundant proteins in single cells. However, there is a technical challenge for effective processing of single cells with minimal sample loss for MS analysis. To address this issue, we have recently developed a convenient protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM for targeted proteomics analysis of small numbers of human cells. This method capitalizes on using the combined excessive exogenous protein as a carrier and low-volume one-pot processing to greatly reduce surface adsorption losses as well as high-specificity LC-SRM to effectively address the increased dynamic concentration range due to the addition of exogeneous carrier protein. Its utility has been demonstrated by accurate quantification of most moderately abundant proteins in small numbers of cells (e.g., 10-100 cells) and highly abundant proteins in single cells. The easy-to-implement features and no need for specific devices make this method readily accessible to most proteomics laboratories. In this study, we have provided a detailed protocol for cLC-SRM analysis of small numbers of human cells including cell sorting, cell lysis and digestion, LC-SRM analysis, and data analysis. Further improvements in detection sensitivity and sample throughput are needed towards targeted single-cell proteomics analysis. We anticipate that cLC-SRM will be broadly applied to biomedical research and systems biology with the potential of facilitating precision medicine.

60 APPLIED LIFE SCIENCES↗

Compact, low power, high resolution ADC per pixel for large area pixel detectors

A compact ADC circuit can include one or more comparators, and a serial DAC (Digital-to-Analog) circuit that provides a signal to the comparator (or comparators). In addition, the ADC circuit can include a serial DAC redistribution sequencer that can provide a plurality of signals as input to the serial DAC circuit and is subject to a redistribution cycle and which receives as input a signal from a data multiplexer whose input connects electronically to an output of the comparator. The circuit can further include an ADC code register that provides an ADC output that connects electronically to the output of the comparator and the input to the data multiplexer. Shared logic circuitry for sharing common logic between pixels can be included, wherein the shared logic circuitry connects electronically to the data multiplexer and the ADC code register, wherein the shared logic circuitry promotes area and power savings for the pixel detector circuit.

Zimmerman, Tom↗

Gated ring oscillator with constant dynamic power consumption

A pure digital ring oscillator with constant power consumption as oscillation frequency is adjusted. Circuit topology includes a multiplexer implemented in NAND gates and a delay element positioned after a path selection NAND gate of that multiplexer such that delay element transistors may not toggle if the non-delaying signal path is selected. Assuming a delay element oscillation frequency f and a total capacitance C, and also assuming a plurality N of delay gates each characterized by a propagation delay t1 and a capacitance C1 such that C=C1*N, the ring oscillator of the present invention is characterized by a C value that is proportional to N and an f value that is inversely proportional to N. Furthermore, each of the N delay gates as well as the input and output gates of the multiplexer are characterized by a common capacitance-to-propagation delay ratio=C1/t1.

Wu, Jinyuan↗

Gated ring oscillator with constant dynamic power consumption

A pure digital ring oscillator with constant power consumption as oscillation frequency is adjusted. Circuit topology includes a multiplexer implemented in NAND gates and a delay element positioned after a path selection NAND gate of that multiplexer such that delay element transistors may not toggle if the non-delaying signal path is selected. Assuming a delay element oscillation frequency f and a total capacitance C, and also assuming a plurality N of delay gates each characterized by a propagation delay t1 and a capacitance C1 such that C=C1*N, the ring oscillator of the present invention is characterized by a C value that is proportional to N and an f value that is inversely proportional to N. Furthermore, each of the N delay gates as well as the input and output gates of the multiplexer are characterized by a common capacitance-to-propagation delay ratio=C1/t1.

Wu, Jinyuan↗

Compact, low power, high resolution ADC per pixel for large area pixel detectors

A compact ADC circuit can include one or more comparators, and a serial DAC (Digital-to-Analog) circuit that provides a signal to the comparator (or comparators). In addition, the ADC circuit can include a serial DAC redistribution sequencer that can provide a plurality of signals as input to the serial DAC circuit and is subject to a redistribution cycle and which receives as input a signal from a data multiplexer whose input connects electronically to an output of the comparator. The circuit can further include an ADC code register that provides an ADC output that connects electronically to the output of the comparator and the input to the data multiplexer. Shared logic circuitry for sharing common logic between pixels can be included, wherein the shared logic circuitry connects electronically to the data multiplexer and the ADC code register, wherein the shared logic circuitry promotes area and power savings for the pixel detector circuit.

Fahim, Farah↗

Enhanced Laser-Induced Graphene Microfluidic Integrated Sensors (LIGMIS) for On-Site Biomedical and Environmental Monitoring

The convergence of microfluidic and electrochemical biosensor technologies offers significant potential for rapid, in-field diagnostics in biomedical and environmental applications. Traditional systems face challenges in cost, scalability, and operational complexity, especially in remote settings. Addressing these issues, laser-induced graphene microfluidic integrated sensors (LIGMIS) are presented as an innovative platform that integrates microfluidics and electrochemical sensors both comprised of laser-induced graphene. This study advances the LIGMIS concept by resolving issues of uneven fluid transport, increased hydrophobicity during storage, and sensor biofunctionalization challenges. Key innovations include Y-shaped reservoirs for consistent fluid flow, hydrophilic polyethyleneimine coatings to maintain wettability, and separable microfluidic and electrochemical components enabling isolated electrode nanoparticle metallization and biofunctionalization. Multiplexed electrochemical detection of the neonicotinoid imidacloprid and nitrate ions in environmental water samples yields detection limits of 707 nm and 10 -5.4 m with wide sensing ranges of 5–100 µm and 10 -5 –10 -1 m, respectively. Similarly, uric acid and calcium ions are detected in saliva, demonstrating detection limits of 217 nm and 10 -5.3 m with sensing ranges of 10–50 µm, and 10 -5 –10 -2.5 m, respectively. Overall, this biosensing demonstrates the capability of the LIGMIS platform for multiplexed detection in biologically complex solutions, with applications in environmental water quality monitoring and oral cancer screening.

environmental monitoring↗

Characterization of Aliased Noise in the Advanced ACTPol Receiver

Advanced ACTPol is the second-generation polarization-sensitive upgrade to the 6m aperture Atacama Cosmology Telescope (ACT), which increased detector count and frequency coverage compared to the previous ACTPol receiver. Advanced ACTPol utilizes a new two-stage time-division multiplexing readout architecture based on superconducting quantum interference devices to achieve a multiplexing factor as high as 64 (rows), fielding a 2012 detector camera at 150/220 GHz and two 90/150 GHz cameras containing 1716 detectors each. In a time-domain system, aliasing introduces noise to the readout. Here in this work we present a figure of merit to measure this noise contribution and present measurements of the aliased noise fraction of the Advanced ACTPol receiver as deployed.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗

BICEP Array: 150 GHz Detector Module Development

The Background Imaging of Cosmic Extragalactic Polarization (BICEP)/Keck (BK) collaboration is currently leading the quest for the highest-sensitivity measurements of the polarized cosmic microwave background (CMB) anisotropies on a degree scale with a series of cryogenic telescopes, of which BICEP Array (BA) is the latest Stage-3 upgrade with a total of ~ 32,000 detectors. The instrument comprises 4 receivers spanning 30–270 GHz, with the low-frequency 30/40 GHz deployed to the South Pole Station in late 2019. The full complement of receivers is forecast to set the most stringent constraints on the tensor-to-scalar ratio r. Building on these advances, the overarching small-aperture telescope concept is already being used as the reference for further Stage-4 experiment design. This paper describes the development of the BICEP Array 150 GHz detector module and its fabrication requirements, with highlights on the high-density time division multiplexing (TDM) design of the cryogenic circuit boards. The low-impedance wiring required between the detectors and the frst stage of superconducting quantum interference device amplifers is crucial to maintaining a stable bias current on the detectors. Here, a novel multilayer FR4 Printed Circuit Board with superconducting traces, capable of reading out up to 648 detectors, is detailed along with its validation tests. An ultra-high-density TDM detector module concept we developed for a CMB-S4-like experiment that allows up to 1920 detectors to be read out is also presented. TDM has been chosen as the detector readout technology for the Cosmic Microwave Background Stage-4 (CMB-S4) experiment based on its proven low-noise performance, predictable costs, and overall maturity of the architecture. The heritage for TDM is rooted in mm- and sub-mm-wave experiments dating back 20 years and has since evolved to support a multiplexing factor of 64x in Stage-3 experiments.

79 ASTRONOMY AND ASTROPHYSICS↗

End-to-End Modeling of the TDM Readout System for CMB-S4

The CMB-S4 experiment is developing next-generation ground-based microwave telescopes to observe the cosmic microwave background with unprecedented sensitivity. This will require an order of magnitude increase in the 100-mK detector count, which, in turn, increases the demands on the readout system. The CMB-S4 readout will use time-division multiplexing (TDM), taking advantage of faster switches and amplifiers in order to achieve an increased multiplexing factor. To facilitate the design of the new readout system, we have developed a model that predicts the bandwidth and noise performance of this circuitry and its interconnections. This is then used to set requirements on individual components in order to meet the performance necessary for the full system. Here, we present an overview of this model and compare the model results to the performance of both legacy and prototype readout hardware.

47 OTHER INSTRUMENTATION↗

Conceptual design of the modular detector and readout system for the CMB-S4 survey experiment

We present the conceptual design of the modular detector and readout system for the Cosmic Microwave Background – Stage four (CMB-S4) ground-based survey experiment. CMB-S4 will map the cosmic microwave background (CMB) and the millimeter-wave sky to unprecedented sensitivity, using 500,000 superconducting detectors observing from Chile and Antarctica to map over 60% of the sky. The fundamental building block of the detector and readout system is a detector module package operated at 100 mK, which is connected to a readout and amplification chain that carries signals out to room temperature. It uses arrays of feedhorn-coupled orthomode transducers (OMT) that collect optical power from the sky onto dc-voltage-biased transition-edge sensor (TES) bolometers. The resulting current signal in the TESs is then amplified by a two-stage cryogenic Superconducting Quantum Interference Device (SQUID) system with a time-division multiplexer to reduce wire count, and matching room-temperature electronics to condition and transmit signals to the data acquisition system. Sensitivity and systematics requirements are being developed for the detector and readout system over wide range of observing bands (20 to 300 GHz) and optical powers to accomplish CMB-S4’s science goals. While the design incorporates the successes of previous generations of CMB instruments, CMB-S4 requires an order of magnitude more detectors than any prior experiment. This requires fabrication of complex superconducting circuits on over 10 m2 of silicon, as well as significant amounts of precision wiring, assembly and cryogenic testing

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli

Regulation of metabolic gene expression is crucial for maximizing bioproduction titers. Recent engineering tools including CRISPR/Cas9, CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) have enabled effective knock-out, knock-down, and overexpression of endogenous pathway genes, respectively, for advanced strain engineering. CRISPRi in particular has emerged as a powerful tool for gene repression through the use of a deactivated Cas9 (dCas9) protein and target guide RNA (gRNA). By constructing gRNA arrays, CRISPRi has the capacity for multiplexed gene downregulation across multiple orthogonal pathways for enhanced bioproduction titers. In this study, we harnessed CRISPRi to downregulate 32 essential and non-essential genes in E. coli strains heterologously expressing either the original mevalonate pathway or isopentenyl diphosphate (IPP) bypass pathway for isoprenol biosynthesis. Isoprenol remains a candidate bioproduct both as a drop-in blend additive and as a precursor for the high-performance sustainable aviation fuel, 1,4-dimethylcyclooctane (DMCO). Of the 32 gRNAs targeting genes associated with isoprenol biosynthesis, a subset was found to vastly improve product titers. Construction of a multiplexed gRNA library based on single guide RNA (sgRNA) performance enabled simultaneous gene repression, yielding a 3 to 4.5-fold increase in isoprenol titer (1.82 ± 0.19 g/L) on M9-MOPS minimal medium. We then scaled the best performing CRISPRi strain to 2-L fed-batch cultivation and demonstrated translatable titer improvements, ultimately obtaining 12.4 ± 1.3 g/L isoprenol. Our strategy further establishes CRISPRi as a powerful tool for tuning metabolic flux in production hosts and that titer improvements are readily scalable with potential for applications in industrial bioproduction.

59 BASIC BIOLOGICAL SCIENCES↗

Salivary Antibodies against Multiple Environmental Pathogens Found in Individuals Recreating at an Iowa Beach

Detecting environmental exposures and mitigating their impacts are growing global public health challenges. Antibody tests show great promise and have emerged as fundamental tools for large-scale exposure studies. Here, we apply, demonstrate and validate the utility of a salivary antibody multiplex immunoassay in measuring antibody prevalence and immunoconversions to six pathogens commonly found in the environment. The study aimed to assess waterborne infections in consenting beachgoers recreating at an Iowa riverine beach by measuring immunoglobulin G (IgG) antibodies against select pathogens in serially collected saliva samples. Results showed that nearly 80% of beachgoers had prior exposures to at least one of the targeted pathogens at the beginning of the study. Most of these exposures were to norovirus GI.1 (59.41%), norovirus GII.4 (58.79%) and Toxoplasma gondii (22.80%) and over half (56.28%) of beachgoers had evidence of previous exposure to multiple pathogens. Of individuals who returned samples for each collection period, 6.11% immunoconverted to one or more pathogens, largely to noroviruses (GI.1: 3.82% and GII.4: 2.29%) and T. gondii (1.53%). Outcomes of this effort illustrate that the multiplex immunoassay presented here serves as an effective tool for evaluating health risks by providing valuable information on the occurrence of known and emerging pathogens in population surveillance studies.

59 BASIC BIOLOGICAL SCIENCES↗

The X-ray Integral Field Unit at the end of the Athena reformulation phase

The Athena mission entered a redefinition phase in July 2022, driven by the imperative to reduce the mission cost at completion for the European Space Agency below an acceptable target, while maintaining the flagship nature of its science return. This notably called for a complete redesign of the X-ray Integral Field Unit (X-IFU) cryogenic architecture towards a simpler active cooling chain. Passive cooling via successive radiative panels at spacecraft level is now used to provide a 50 K thermal environment to an X-IFU owned cryostat. 4.5 K cooling is achieved via a single remote active cryocooler unit, while a multi-stage Adiabatic Demagnetization Refrigerator ensures heat lift down to the 50 mK required by the detectors. Amidst these changes, the core concept of the readout chain remains robust, employing Transition Edge Sensor microcalorimeters and a SQUID-based Time-Division Multiplexing scheme. Noteworthy is the introduction of a slower pixel. This enables an increase in the multiplexing factor (from 34 to 48) without compromising the instrument energy resolution, hence keeping significant system margins to the new 4 eV resolution requirement. This allows reducing the number of channels by more than a factor two, and thus the resource demands on the system, while keeping a 4’ field of view (compared to 5’ before). Here, in this article, we will give an overview of this new architecture, before detailing its anticipated performances. Finally, we will present the new X-IFU schedule, with its short term focus on demonstration activities towards a mission adoption in early 2027.

Peille, Philippe [Centre National d’Etudes Spatial↗

Particle Response of Antenna-Coupled TES Arrays: Results from SPIDER and the Laboratory

Future mm-wave and sub-mm space missions will employ large arrays of multiplexed transition-edge-sensor (TES) bolometers. Such instruments must contend with the high flux of cosmic rays beyond our atmosphere that induce ‘glitches’ in bolometer data, which posed a challenge to data analysis from the Planck bolometers. Future instruments will face the additional challenges of shared substrate wafers and multiplexed readout wiring. Here, we explore the susceptibility of modern TES arrays to the cosmic ray environment of space using two data sets: the 2015 long-duration balloon flight of the SPIDER cosmic microwave background polarimeter, and a laboratory exposure of SPIDER flight hardware to radioactive sources. We find manageable glitch rates and short glitch durations, leading to minimal effect on SPIDER analysis. We constrain energy propagation within the substrate through a study of multi-detector coincidences and give a preliminary look at pulse shapes in laboratory data.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

Hyperspectral X-ray Imaging with TES Detectors for Nanoscale Chemical Speciation Mapping

We are developing an imaging capability (“Hyperspectral X-ray Imaging”) for mapping chemical information (molecular formula, phase, oxidation state, hydration) that is based on ultra-high-resolution X-ray emission spectroscopy with large transition-edge sensor microcalorimeter arrays in the scanning electron microscope. By combining microcalorimeter arrays with hundreds of pixels, high-bandwidth microwave frequency-division multiplexing, and fast digital electronics for near real-time data processing, our goal is to enable measurements using laboratory-scale instrumentation rather than synchrotron beamlines. Our application focus here is on mapping the chemical form of uranium compounds on the nanoscale. Furthermore, we will present our approach to developing the Hyperspectral X-ray Imaging capability, progress toward a 128-pixel microwave multiplexed X-ray fluorescence instrument at LANL, and the path to high-throughput nanoscale chemical mapping.

47 OTHER INSTRUMENTATION↗

Long-term thermal stability and calibration of Type-II fiber Bragg grating array inscribed in radiation-hardened fibers

This paper investigates the long-term thermal stability of Type-II fiber Bragg grating (FBG) arrays, inscribed by femtosecond laser in radiation-hardened fiber, for potential applications as multiplexed sensors in high-temperature energy systems. The thermal stability of FBG sensors was assessed through 16 thermal cycles from room temperature (RT) to 750 ℃ about two months, involving 100 FBG sensors. The results show that the absolute temperature drift of FBG sensors can be reduced to less than 0.4 pm/day after 54 h thermal annealing process at a constant temperature of 800 ℃. As temperature sensors, the FBGs demonstrated stable performance, achieving a standard deviation (STD) of 1.8 pm (corresponding to a temperature resolution of 0.118 ℃) post-annealing. Repeated thermal cycles revealed a random drift of 2.3 pm in the FBG wavelength at RT. Polynomial fitting was explored as a calibration method to convert FBG wavelength shifts into absolute temperature measurements. By optimizing calibration temperature points (RT, 200 ℃, 400 ℃, and 750 ℃), the study shows that cubic polynomial calibration using four points yields an average R2 of 0.9997 and an RMSE of 3.58 ℃ across the entire temperature range (RT to 750 ℃). This approach represents an 11.53-fold improvement over empirical slope calibration and a 1.34-fold improvement over four-point piecewise fitting. The findings indicate that Type-II FBGs inscribed in radiation-hardened fibers can function as accurate temperature sensors, with performance on par with or exceeding that of thermocouples. With their multiplexing capability, robust signal transmission over long lead cables, and immunity to electromagnetic interference, FBG sensors offer a promising alternative to traditional electronic sensors for energy system monitoring.

Dominguez-Ontiveros, Elvis [ORNL] (ORCID:000000018↗

A landing pad system for multicopy gene integration in Issatchenkia orientalis

The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in producing organic acids using a variety of carbon sources. Recently, the development of a genetic toolbox for I. orientalis, including an episomal plasmid, characterization of multiple promoters and terminators, and CRISPR-Cas9 tools, has eased the metabolic engineering efforts in I. orientalis. However, multiplex engineering is still hampered by the lack of efficient multicopy integration tools. To facilitate the construction of large, complex metabolic pathways by multiplex CRISPR-Cas9-mediated genome editing, we developed a bioinformatics pipeline to identify and prioritize genome-wide intergenic loci and characterized 47 gRNAs located in 21 intergenic regions. These loci are screened for guide RNA cutting efficiency, integration efficiency of a gene cassette, the resulting cellular fitness, and GFP expression level. We further developed a landing pad system using components from these well-characterized loci, which can aid in the integration of multiple genes using single guide RNA and multiple repair templates of the user’s choice. We have demonstrated the use of the landing pad for simultaneous integrations of 2, 3, 4, or 5 genes to the target loci with efficiencies greater than 80%. As a proof of concept, we showed how the production of 5-aminolevulinic acid can be improved by integrating five copies of genes at multiple sites in one step. We have further demonstrated the efficiency of this tool by constructing a metabolic pathway for succinic acid production by integrating five gene expression cassettes using a single guide RNA along with five different repair templates, leading to the production of 9 g/L of succinic acid in batch fermentations. Furthermore, this study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in a non-conventional yeast. This landing pad system will be a valuable tool for the metabolic engineering of I. orientalis.

59 BASIC BIOLOGICAL SCIENCES↗