Secondary Probe Research Articles

Reduced Non-Specific Binding of Super-Resolution DNA-PAINT Markers by Shielded DNA-PAINT Labeling Protocols.

The DNA-based single molecule super-resolution imaging approach, DNA-PAINT, can achieve nanometer resolution of single targets. However, the approach can suffer from significant non-specific background signals originating from non-specifically bound DNA-conjugated DNA-PAINT secondary antibodies as shown here. Using dye-modified oligonucleotides the location of DNA-PAINT secondary antibody probes can easily be observed with widefield imaging prior to beginning a super-resolution measurement. This reveals that a substantial proportion of DNA probes can accumulate, non-specifically, within the nucleus, as well as across the cytoplasm, of cells. Here, Shielded DNA-PAINT labeling is introduced, a method using partially or fully double-stranded docking strand sequences, prior to labeling, in buffers with increased ionic strength to greatly reduce non-specific interactions in the nucleus as well as the cytoplasm. This new labeling approach is evaluated against various conditions and it is shown that applying Shielded DNA-PAINT can reduce non-specific events approximately five-fold within the nucleus. This marked reduction in non-specific binding of probes during the labeling procedure is comparable to results obtained with unnatural left-handed DNA albeit at a fraction of the cost. Shielded DNA-PAINT is a straightforward adaption of current DNA-PAINT protocols and enables nanometer precision imaging of nuclear targets with low non-specific backgrounds.

Significance of homogeneous conductive network in layered oxide-based cathode for high-rate capability of electric vehicle batteries

The trade-off between battery energy density and power performance is the core problem that puzzles the development of electric vehicles (EVs). Although intensive researches are performed to explore active materials with good dynamics, the heterogeneous reactivity has been identified as an important cause for inferior capability and early death, especially for electrodes characterized with high areal loading and high compacted density. Herein, the heterogeneity and its origination of layered oxide-based (LiNixCoyMn1-x-yO2, NCM) electrodes at high C-rate are investigated through operando X-ray diffraction and ex-situ time-of-flight secondary ion mass spectrometry probe. By introducing Li3V2(PO4)3@G composite as a mixed conductor additive, the heterogeneous reactivity intro-particles are successfully mitigated, enabling NCM electrodes with both high rate capability, high energy density and high cyclability. In detail, the capacity retention at 20C is increased by 2.3 times, and the capacity retention at 0.5C after 160 full cycles is increased by 1.6 times, without electrolyte additive or material modification. This study demonstrates the significance of the homogeneous electronic/ionic transportation network to the rate capability and lifetime of an electrode, and discloses the design strategy of multifunctional additives to enhance the power density of a battery by maximizing the utility of the active particles.

Shorter Peptide Nucleic Acid Probes Improve Affibody-Mediated Peptide Nucleic Acid-Based Pretargeting.

Affibody-mediated PNA-based pretargeting shows promise for HER2-expressing tumor radiotherapy. In our recent study, a 15-mer ZHER2:342-HP15 affibody-PNA conjugate, in combination with a shorter 9-mer [177Lu]Lu-HP16 effector probe, emerged as the most effective pretargeting strategy. It offered a superior tumor-to-kidney uptake ratio and more efficient tumor targeting compared to longer radiolabeled effector probes containing 12 or 15 complementary PNA bases. To enhance the production efficiency of our pretargeting system, we here introduce even shorter 6-, 7-, and 8-mer secondary probes, designated as HP19, HP21, and HP20, respectively. We also explore the replacement of the original 15-mer Z-HP15 primary probe with shorter 12-mer Z-HP12 and 9-mer Z-HP9 alternatives. This extended panel of shorter PNA-based probes was synthesized using automated microwave-assisted methods and biophysically screened in vitro to identify shorter probe combinations with the most effective binding properties. In a mouse xenograft model, we evaluated the biodistribution of these probes, comparing them to the Z-HP15:[177Lu]Lu-HP16 combination. Tumor-to-kidney ratios at 4 and 144 h postinjection of the secondary probe showed no significant differences among the Z-HP9:[177Lu]Lu-HP16, Z-HP9:[177Lu]Lu-HP20, and the Z-HP15:[177Lu]Lu-HP16 pairs. Importantly, tumor uptake significantly exceeded, by several hundred-fold, that of most normal tissues, with kidney uptake being the critical organ for radiation therapy. This suggests that using a shorter 9-mer primary probe, Z-HP9, in combination with 9-mer HP16 or 8-mer HP20 secondary probes effectively targets tumors while minimizing the dose-limiting kidney uptake of radionuclide. In conclusion, the Z-HP9:HP16 and Z-HP9:HP20 probe combinations offer good prospects for both cost-effective production and efficient in vivo pretargeting of HER2-expressing tumors.

Dual-Probe SERS Nanosensor: A Promising Approach for Sensitive and Ratiometric Detection of Glucose in Clinical Settings.

Diabetes is a major global health concern, with millions of annual deaths. Monitoring glucose levels is vital for clinical management, and urine samples offer a noninvasive alternative to blood samples. Optical techniques for urine glucose sensing have gained notable traction due to their cost-effectiveness and portability. Among these methods, surface-enhanced Raman spectroscopy (SERS) has attracted considerable attention thanks to its remarkable sensitivity and multiplexing capabilities. However, challenges remain in achieving reliable quantification through SERS. In this study, an alternative approach is proposed to enhance quantification involving the use of dual probes. Each probe is encoded with unique SERS signatures strategically positioned in the biologically silent region. One probe indicates the glucose presence, while the other acts as an internal reference for calibration. This setup enables ratiometric analysis of the SERS signal, directly correlating it with the glucose concentration. The fabrication of the sensor relies on the prefunctionalization of Fe sheets using an aryl diazonium salt bearing a -C≡CH group (internal reference), followed by the immobilization of Ag nanoparticles modified with an aryl diazonium salt bearing a -B(OH)2 group (for glucose capture). A secondary probe bearing a -B(OH)2 group on one side and a -C≡N group on the other side enables the ratiometric analysis by forming a sandwich-like structure in the presence of glucose (glucose indicator). Validation studies in aqueous solutions and artificial urine demonstrated the high spectral stability and the potential of this dual-probe nanosensor for sensitive glucose monitoring in clinical settings.

Abstract 5188: Defining PD-1/PD-L1 receptor-ligand interactions in the head and neck cancer tumor microenvironment identifies unique ‘immune cell rivers’ of cellular interactions

Abstract Head and neck squamous cell carcinoma (HNSCC) is the 7th most common cancer globally with a poor 5-year survival rate of ~50%. With the emerging successes with immune checkpoint blockade (ICB) in the metastatic and recurrent setting, there is a need for predictive biomarkers to identify patients likely to achieve clinical benefit. Whilst PD-L1 expression on tumour cells and PD-1 on immune cells have been investigated, these remain inconclusive biomarkers. To better understand and map the PD-L1/PD-1 receptor-ligand interactions, we evaluated a novel in situ proximity ligation assay (is PLA) across whole tissue sections of pre-treatment HNSCC patients to received ICB therapy. Pre-treatment FFPE whole tissue samples were collected from n=25 advanced stage HNSCC patients across two major Queensland Hospitals (Royal Brisbane and Women’s Hospital and the Princess Alexandra Hospital). Tissues were stained with multiplex immunofluorescence and the Navinci Diagnostics fluorescent is PLA assay for PD-1/PD-L1. Tissues were incubated with the PD1/PD-L1 antibodies and subsequent incubation with oligonucleotide-tagged secondary probes enabled PLA, which highlights PD1 and PD-L1 interactions. Following the application of secondary probes, the sections underwent a ligation step, then a post-blocking, and an amplification process to enhance the detection of protein interactions. Next, the sections were treated with a detection solution to visualize the amplification product. The PLA assay findings were measured against clinical endpoints (PFS/OS criteria). In n=5/25 HNSCC tissues, positive PD-1/PD-L1 interactions were measured across the tumour microenvironment (TME), specifically in the immune rivers on the immediate tumour periphery. These cells showed highly specific binding of the PLA product. These areas positive by the PLA assay were in stark contrast to the tumour bulk/nests which were absent in PD-1/PD-L1 interactions. Notably, the PLA PD-1/PD-L1 do not correlate with tumour PD-L1 expression or tumour proportion scores (TPS). Rather, our study highlighted the difference in spatially mapping PD-1/PD-L1 interactions which may better recapitulate the tumour-immune recognition and activity in the TME. Taken together, these data provide a novel approach for determining clinically relevant receptor/ligand interactions in the HNSCC TME. Citation Format: Habib Sadeghirad, Vahid Yaghoubi Naei, James Monkman, Subham Basu, Agata Wicher, Rahul Ladwa, Brett GM Hughes, Arutha Kulasinghe. Defining PD-1/PD-L1 receptor-ligand interactions in the head and neck cancer tumor microenvironment identifies unique ‘immune cell rivers’ of cellular interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5188.

Abstract 5354: High-resolution analysis of immune checkpoint activation utilizing a combined PD1/PD-L1 in situ proximity ligation assay (isPLA) and multiplex immunofluorescence (mIF) imaging approach

Abstract The immune checkpoint involving PD-1 and PD-L1 generates suppressive signals in T cells, which help to prevent autoimmunity by inducing a state of immune exhaustion. In the context of the tumor microenvironment (TME), however, cancer cells can manipulate these pathways to camouflage themselves from an immune attack. Blocking these checkpoints has thus emerged as a key immunotherapeutic tactic. Nonetheless, the success rate of immune checkpoint inhibitors (ICIs) has been mixed, even for patients who test positive for relevant diagnostic biomarkers. Patient stratification currently depends on immunohistochemical staining for checkpoint proteins like PD-L1, but these tests do not provide adequate stratification. A more comprehensive stratification, one that includes immune profiling of the TME plus an evaluation of immune checkpoint interactions, might provide better patient stratification and ICI responsiveness. Here we describe mIF that is augmented with a PD-1 and PD-L1 protein-protein interaction assay. Human formalin-fixed, paraffin-embedded (FFPE) tissue sections were subjected to standard histological processing and then incubated with primary antibodies against PD1 and PD-L1. Sections were then treated with oligonucleotide-modified secondary probes suitable for in situ Proximity Ligation Assay (isPLA), enabling the detection of PD1 and PD-L1 interactions. The PLA protocol utilized a fluorescent probe complementary to amplified nucleic acid to enhance the visibility of the protein interactions. The sample was then mounted in a closed microfluidic chamber and imaged on the CellScape™ platform for Precise Spatial Multiplexing. After imaging of the isPLA signal, the CellScape cyclic multiplex mIF staining approach was used to iteratively stain immune and structural markers on the tissue sample utilizing the VistaPlex™ Spatial Immune Profiling Assay Kit. This proof-of-concept study demonstrates that high-plex mIF can be augmented with an isPLA on human FFPE samples. The combined application of both methods allows visualization of PD1/PD-L1 protein-protein interactions and integrates this interaction within the spatial context of the surrounding cell populations. This approach allows for a more comprehensive insight into the interplay of different immune cell and non-immune cell populations during checkpoint activation processes in normal and neoplastic tissues. Citation Format: Arne Christians, Jannik Boog, Charles E. Jackson, Matthew H. Ingalls, J Spencer Schwarz, Sara Bodbin, Agata Zieba Wicher, Subham Basu, Oliver Braubach. High-resolution analysis of immune checkpoint activation utilizing a combined PD1/PD-L1 in situ proximity ligation assay (isPLA) and multiplex immunofluorescence (mIF) imaging approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5354.

Distribution of Boron in 9.5Cr–1.5MoCoVNbNB Martensitic Heat-Resistant Steel Studied by Secondary Ion Mass Spectroscopy and atom Probe Tomography

Distribution of Boron in 9.5Cr–1.5MoCoVNbNB Martensitic Heat-Resistant Steel Studied by Secondary Ion Mass Spectroscopy and atom Probe Tomography

On the Metallicities and Kinematics of the Circumgalactic Media of Damped Lyα Systems at z ∼ 2.5* * The data presented herein were obtained at the W. M. Keck Observatory, which is operated as a scientific partnership among the California Institute of Technology, the University of California and the National Aeronautics and Space Administration. The Observatory was made possible by

We use medium- and high-resolution spectroscopy of close pairs of quasars to analyze the circumgalactic medium (CGM) surrounding 32 damped Lyα absorption systems (DLAs). The primary quasar sightline in each pair probes an intervening DLA in the redshift range 1.6 < z abs < 3.5, such that the secondary sightline probes absorption from Lyα and a large suite of metal-line transitions (including O i, C ii, C iv, Si ii, and Si iv) in the DLA host galaxy’s CGM at transverse distances 24 kpc ≤ R ⊥ ≤ 284 kpc. Analysis of Lyα in the CGM sightlines shows an anticorrelation between R ⊥ and H i column density (N HI) with 99.8% confidence, similar to that observed around luminous galaxies. The incidences of C ii and Si ii with N > 1013 cm−2 within 100 kpc of DLAs are larger by 2σ than those measured in the CGM of Lyman break galaxies (C f (N CII ) > 0.89 and ). Metallicity constraints derived from ionic ratios for nine CGM systems with negligible ionization corrections and N HI > 1018.5 cm−2 show a significant degree of scatter (with metallicities/limits across the range ), suggesting inhomogeneity in the metal distribution in these environments. Velocity widths of C iv λ1548 and low-ionization metal species in the DLA versus CGM sightlines are strongly (>2σ) correlated, suggesting that they trace the potential well of the host halo over R ⊥ ≲ 300 kpc scales. At the same time, velocity centroids for C iv λ1548 differ in DLA versus CGM sightlines by >100 km s−1 for ∼50% of velocity components, but few components have velocities that would exceed the escape velocity assuming dark matter host halos of ≥1012 M ⊙.

Proximity Ligation Assay (PLA) for Fillets of Drosophila Larvae.

The ability to detect protein-protein interactions is critical for understanding the mechanisms underlying protein and cell function. Current methods to assay protein-protein interactions, such as co-immunoprecipitation (Co-IP) and fluorescence resonance energy transfer (FRET), have limitations; for example, Co-IP is an in vitro technique and may not reflect the situation in vivo, and FRET typically suffers from low signal-to-noise ratio. The proximity ligation assay (PLA) is an in situ method for inferring protein-protein interaction with a high signal-to-noise ratio. The PLA technique can indicate that two different proteins are closely associated by the ability of two secondary antibody oligonucleotide probes to hybridize when they are close to each other. This interaction generates a signal with rolling-circle amplification using fluorescent nucleotides. Although a positive result does not indicate that two proteins directly interact, it implies a potential in vivo interaction that can then be verified in vitro. PLA uses primary antibodies against the two proteins (or epitopes) of interest, one raised in mouse and the other raised in rabbit. When these antibodies bind to proteins that lie within 40 nm of each other in the tissue, complementary oligonucleotides conjugated individually to mouse and rabbit secondary antibodies can anneal to form a template for rolling-circle amplification. Using fluorescently labeled nucleotides, rolling circle amplification generates a strong fluorescent signal in areas of the tissue where the two proteins are found together that is detected using conventional fluorescence microscopy. This protocol describes how to perform PLA in vivo on the central nervous system and peripheral nervous system of third-instar larvae of the fruit fly Drosophila melanogaster.

Paper-based biosensors relying on core biological immune scaffolds for the detection of procymidone in vegetables

In order to achieve a highly sensitive detection of procymidone in vegetables, three paper-based biosensors based on a core biological immune scaffold (CBIS) were developed, which were time-resolved fluorescence immunochromatography strips with Europium (III) oxide (Eu-TRFICS). Goat anti-mouse IgG and europium oxide time-resolved fluorescent microspheres formed secondary fluorescent probes. CBIS was formed by secondary fluorescent probes and procymidone monoclonal antibody (PCM-Ab). The first type of Eu-TRFICS (Eu-TRFICS-(1)) fixed secondary fluorescent probes on a conjugate pad, and PCM-Ab was mixed with a sample solution. The second type of Eu-TRFICS (Eu-TRFICS-(2)) fixed CBIS on the conjugate pad. The third type of Eu-TRFICS (Eu-TRFICS-(3)) was directly mixed CBIS with the sample solution. They solved the problems of steric hindrance of antibody labeling, insufficient exposure of antigen recognition region and easy loss of activity in traditional methods. They realized multi-dimensional labeling and directional coupling. They replaced the loss of antibody activity. And the three types of Eu-TRFICS were compared, among which Eu-TRFICS-(1) was the best detection choice. Antibody usage was reduced by 25% and sensitivity was increased by 3 times. Its detection range was 1–800 ng/mL, the limit of detection (LOD) was 0.12 ng/mL with the visible LOD (vLOD) of 5 ng/mL.

Integrated effect of aging and heavy ion radiation on FeNiCrAl duplex alloy for accident-tolerant fuel cladding

Alumina-forming duplex FeNiCrAl alloy with superior mechanical properties and high-temperature oxidation resistance has been regarded as an accident-tolerant fuel cladding material. This work investigated the effect of aging and the radiation resistance of FeNiCrAl duplex alloy after up to 2000 hours thermal aging and 3.5 MeV Fe13+ ion radiation. The ferrite phase containing B2 phase shows much fewer radiation defects and lower radiation hardening rate than conventional FeCrAl alloy. The coherent B2 phase precipitate is critical in hindering dislocation and inhibiting the formation of radiation induced defects which is proved via experiment and molecular dynamics simulation. Besides, the Al depletion of B2 phase and nano-sized precipitation are characterized by using Time-of-Flight Secondary Ion Mass Spectrometry and Atom Probe Tomography. These findings effectively illuminate microstructure evolution of FeNiCrAl duplex alloy after long-term aging and radiation and lead to new insight for designing advanced accident-tolerant fuel material.

Disrupting bacterial cytoskeletal protein interaction partners modulates geometric localization and alters cell shape.

The shape a bacterial cell assumes is an essential component in its ability to interact with its physical environment. For many bacterial species, shape is genetically encoded in the action of a small number of cell shape determining proteins. These proteins guide the building of a rigid peptidoglycan cell wall of a given geometry. We use a computational imaging framework to reconstruct the 3D shape of individual cells from fluorescence microscopy images and determine where a secondary fluorescent probe is enriched. In the straight-rod bacterium Escherichia coli, the bacterial actin MreB is enriched in areas with low Gaussian curvature, avoiding the poles. Disrupting MreB's interactions with the transmembrane protein RodZ alters MreB's geometric localization and cells lose their characteristic rod-like morphology. In the helical-rod bacterium Helicobacter pylori, the bactofilin CcmA is preferentially enriched at regions of small positive Gaussian curvature along the outer or major helical axis. Disrupting CcmA interaction partners results in less CcmA on the membrane, altered geometric localization of CcmA, and loss of the characteristic helical morphology of cells. These results are consistent with a model of cell shape determination that includes proper geometric localization of bacterial cytoskeletal proteins.

Evaluation of cold atmospheric pressure plasma irradiation of water as a method of singlet oxygen generation.

We used cold atmospheric pressure plasma jet to examine in detail 1O2 generation in water. ESR with 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide, a secondary amine probe, was used for the detection of 1O2. Nitroxide radical formation was detected after cold atmospheric pressure plasma jet irradiation of a 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide solution. An 1O2 scavenger/quencher inhibited the ESR signal intensity induced by cold atmospheric pressure plasma jet irradiation, but this inhibition was not 100%. As 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide reacts with oxidizing species other than 1O2, it was assumed that the signal intensity inhibited by NaN3 corresponds to only the nitroxide radical generated by 1O2. The concentration of 1O2 produced by cold atmospheric pressure plasma jet irradiation for 60 s was estimated at 8 μM. When this 1O2 generation was compared to methods of 1O2 generation like rose bengal photoirradiation and 4-methyl-1,4-etheno-2,3-benzodioxin-1(4H)-propanoic acid (endoperoxide) thermal decomposition, 1O2 generation was found to be, in decreasing order, rose bengal photoirradiation ≥ cold atmospheric pressure plasma jet > endoperoxide thermal decomposition. Cold atmospheric pressure plasma jet is presumed to not specifically generate 1O2, but can be used to mimic states of oxidative stress involving multiple ROS.

Exploration for oxygen diffusion during deoxidation process of terbium with 18O as isotope tracer

Oxygen (O) is the most difficult kind of impurity to be removed from rare earth terbium (Tb), which seriously restricts the promotion of high purity rare earth metal quality. Study the law of oxygen removal during the purification process and clarify the influence and synergism of active substance are of great significance. Herein, isotopic tracer technique (using 18O) has been used to explore the oxygen diffusion process. Secondary ion probe mass spectrometer (SIMS) and Scanning Electron Microscope (SEM) investigated the occurrence state, migration trajectory and relative concentration gradient of oxygen in Tb. The diffusion coefficient as a function of depth and temperature can be simulated based on the experimental data, which is of great significance for the purification and corrosion prevention of Tb. X-ray photoelectron spectroscopy (XPS) clarified the variation of metal valence, indicating the bonding characteristics between O and Tb.

Introducing gallium in silicon and thin film polysilicon using self assembled monolayer doping

• Gallium compound synthesized to form Ga molecular monolayers on silicon surface. • Ga monolayer doping in silicon and thin film polysilicon demonstrated. • Junction depth of 200 nm with a dose 1.6*10 15 Ga atoms/cm 2 demonstrated in n -Si. • Near uniform doping in polysilicon films is achieved with 20% electrical activation. Monolayer Doping (MLD) is a technique involving the formation of a self-assembled dopant-containing layer on the substrate. The dopant is subsequently incorporated into the substrate by annealing, forming a diffused region. Following MLD, samples were capped with silicon dioxide and rapid thermal annealed (RTA). In this work, gallium doping using MLD has been demonstrated. Gallium containing compound Tris (2,4 pentanedionato) gallium(III) was synthesized, and shown to be suitable for monolayer doping silicon substrates and deposited thin film polysilicon. Secondary ion mass spectroscopy (SIMS) and spreading resistance probe (SRP) measurements were performed to determine the dopant profiles and dopant electrical activation. These results showed that a dose of 1.6*10 15 atoms/cm 2 was received, and the gallium dopant produced a 0.2 µm junction in n -type silicon. For polysilicon, the entire 0.4 μm film was evenly doped, with a concentration greater than 10 19 atoms/cm 3 throughout.

A novel peptide-based relay fluorescent probe with a large Stokes shift for detection of Hg2+ and S2− in 100 % aqueous medium and living cells: Visual detection via test strips and smartphone

Herein, a novel relay peptide-based fluorescent probe DGRK was synthesized via solid phase peptide synthesis (SPPS) technology. DGRK exhibited excellent water-solubility, good stability, remarkably large Stokes shift (230 nm) and high selectivity response to Hg2+ with a non-fluorescence complex DGRK-Hg2+ formation via a 1:1 binding mode. Further studies indicated that the DGRK-Hg2+ complex could act as a secondary probe for rapidly and sequentially detecting S2− based on fluorescent “off–on” response, and without interference from a range of anions. The limit of detection (LOD) for Hg2+ and S2− were calculated to be 33.6 nM and 60.9 nM, respectively. In addition, The reversibility of interaction of confirmed that the continuous and reversible recognition behavior of Hg2+ and S2− by the probe DGRK, and could be cycled more than 5 times. In addition, DGRK could be successfully applied to the fluorescence imaging of Hg2+ and S2− in two living cells based on excellent cells permeability and low cytotoxicity. Meanwhile, DGRK was successfully used to create the low-cost and portable test strips for visual detection and rapid analysis under 365 nm UV lamp, and the test strips combined with a smartphone (RGB color) was successfully applied to the semi-quantitative analysis and monitoring of dynamic changes of Hg2+ levels.

Fluorescent “on–off–on” probe based on copper peptide backbone for specific detection of Cu(II) and hydrogen sulfide and its applications in cell imaging, real water samples and test strips

Copper ions (Cu2+) and hydrogen sulfide (H2S) detection is of great significance in environmental monitoring and disease screening. Herein, a novel fluorescent probe (DAHK) was rationally designed and easily synthesized based on a copper peptide backbone labelled a dansyl fluorophore. DAHK exhibited large Stokes shift (210 nm), high sensitivity (38.9 nM) and high selectivity response to Cu2+ ions based on obvious fluorescence quenching in HEPES buffer (10.0 mM, pH 7.4) solutions. In addition, the in situ formed DAHK-Cu2+ ensemble was successfully used as a secondary probe for rapidly and sequentially detected H2S via fluorescence “off–on” response, and without interference by a range of other anions. The limit of detection (LOD) for 47.3 nM was determined, which was a much lower value than WHO and EPA guidelines (H2S) for drinking water. The reversibility of interaction of DAHK with Cu2+ and H2S was monitored using fluorescence emission spectra. Cells imaging experiments exhibited that DAHK had good membrane permeability and excellent low toxicity. Moreover, DAHK was successfully made into fluorescent test strips for visual detection and rapid analysis based on the exciting stability and excellent water solubility.

Response preparation of a secondary reaction time task is influenced by movement phase within a continuous visuomotor tracking task.

The simultaneous performance of two motor tasks is challenging. Currently, it is unclear how response preparation of a secondary task is impacted by the performance of a continuous primary task. The purpose of the present experiment was to investigate whether the position of the limb performing the primary cyclical tracking task impacts response preparation of a secondary reaction time task. Participants (n = 20) performed a continuous tracking task with their left hand that involved cyclical and targeted wrist flexion and extension. Occasionally, a probe reaction time task requiring isometric wrist extension was performed with the right hand in response to an auditory stimulus (80 or 120 dB) that was triggered when the left hand passed through one of 10 locations identified within the movement cycle. On separate trials, transcranial magnetic stimulation was applied over the left primary motor cortex and triggered at the same 10 stimulus locations to assess corticospinal excitability associated with the probe reaction time task. Results revealed that probe reaction times were significantly longer and motor-evoked potential amplitudes were significantly larger when the left hand was in the middle of a movement cycle compared with an endpoint, suggesting that response preparation of a secondary probe reaction time task was modulated by the phase of movement within the continuous primary task. These results indicate that primary motor task requirements can impact preparation of a secondary task, reinforcing the importance of considering primary task characteristics in dual-task experimental design.

Detection of sub-micrometer thermomechanical and thermochemical failure mechanisms in titanium with a laser-based thermoreflectance technique

This work reports on a pump–probe laser-based heating and sensing metrology to study the failure mechanisms of materials during extreme heat fluxes localized near surfaces, the localization of which is controlled by the focus of the laser beam and sensed by the reflection of a secondary probe laser. We focus the demonstration of these power density at failure tests on the damage mechanisms of commercially pure titanium metal during and after high heat fluxes induced from the absorbed laser energy. Using this steady-state thermoreflectance pump–probe metrology, a localized region of the material was irradiated at a low modulated frequency, while the average change in the thermoreflectance signal was monitored. We observe surface and cross-sectional oxidation of the titanium, revealing correlations between microstructural evolution events and shifts in thermoreflectance trends as a function of absorbed power density. Furthermore, the damage morphology was shown to be heavily influenced by the size of the heater (dictated by the radius of the pump laser beam), which controlled the relative degree of thermomechanical, melting, and oxidative decohesion failure mechanisms in the samples. The analysis of the temperature distribution coupled with the observed microstructural damage gives rise to a high-throughput experimental technique to induce desired deformation modes through cyclic thermal testing.

Adding a diazo-transfer reagent to culture to generate secondary metabolite probes for click chemistry.

Converting discrete microbial metabolites into chemical probes for chemical biology and medicinal chemistry studies is typically preceded by lengthy purification and chemical derivatization processes. Standard practice involves purifying the target microbial metabolite from culture, followed by derivatization and/or conjugation chemistry to convert the pure metabolite into a tagged species. This multistep approach can pose difficulties in generating useful yields of chemical probes, particularly in the case of low-abundant metabolites, as common in metabolomes. This chapter describes a methodological approach to simplify the steps towards generating chemical probes from complex mixtures, that combines: (a) tailored purification processes; (b) compound identification using state-of-the-art tandem mass spectrometry and data-dependent fragmentation; and (c) in situ bioorthogonal bioconjugation chemistries. The combination of these methods, as illustrated by the conversion of a set of amine-bearing metabolites to the cognate azide analogs suitable for biotinylation through azide-alkyne cycloaddition, describes a powerful approach to access new chemical probes of low-abundant metabolites that might otherwise be inaccessible using traditional methods.