Ratiometric fluorescent probe with AIE characteristics for hypochlorite detection and biological imaging.

It is very important and highly valuable to detect ClO- in samples and living cells with accuracy and speed. In this work, a novel fluorescent probe NA was prepared from 4-bromo-1,8-naphthalic anhydride by acylation reaction and Suzuki coupling reaction and used for the detection of ClO-. Thiomethyl serves as the recognition group for probe NA, while naphthalimide serves as fluorescent chromophore. The probe exhibited an extremely pronounced blue shift from yellow to blue fluorescence within 1 min after the addition of hypochlorite (ClO-). The probe demonstrates high sensitivity to ClO- with a limit of detection (LOD) of 1.22 µM. Also, probe NA demonstrates excellent selectivity and immunity to interference. Additionally, simple fluorescent test strips containing probe NA were prepared in this study, enabling rapid detection of ClO- in water samples. And NA had been effectively used to image endogenous and exogenous ClO-fluorescence in living cells. The results suggest that probe NA has significant potential for portable detection and biological applications.

Protocol for machine-learning-based 3D image analysis of nuclear envelope tubules in cultured cells.

The nuclear envelope can form complex structures in physiological and pathological contexts. Current approaches to quantify nuclear envelope structures can be time-consuming or inaccurate. Here, we present a protocol to measure nuclear envelope tubules induced by DNA double-strand breaks using a mid-throughput approach. We describe steps for the induction of these nuclear envelope structures and 3D image analysis using machine-learning-based image segmentation. This protocol can be applied to analyze various nuclear envelope structures in contexts beyond DNA repair. For complete details on the use and execution of this protocol, please refer to Shokrollahi et al.1.

Recent advances in fluorescent probes for ATP imaging.

Adenosine-5'-triphosphate (ATP) is a critical biological molecule that functions as the primary energy currency within cells. ATP synthesis occurs in the mitochondria, and variations in its concentration can significantly influence mitochondrial and cellular performance. Prior studies have established a link between ATP levels and a variety of diseases, such as cancer, neurodegenerative conditions, ischemia, and hypoglycemia. Consequently, researchers have developed many fluorescent probes for ATP detection, recognizing the importance of monitoring intracellular ATP levels to understand cellular processes. These probes have been effectively utilized for visualizing ATP in living cells and biological samples. In this comprehensive review, we categorize fluorescent sensors developed in the last five years for ATP detection. We base our classification on fluorophores, structure, multi-response channels, and application. We also evaluate the challenges and potential for advancing new generations of fluorescence imaging probes for monitoring ATP in living cells. We hope this summary motivates researchers to design innovative and effective probes tailored to ATP sensing. We foresee imminent progress in the development of highly sophisticated ATP probes.

Retinoids Molecular Probes by Late-stage Azide Insertion - Functional Tools to Decrypt Retinoid Metabolism.

Studying the complex and intricate retinoids metabolic pathways by chemical biology approaches requires design and synthesis of biologically functional molecular probes. Only few of such molecular retinoid probes could be found in literature, most of them bearing a molecular structure quite different from natural retinoids. To provide close-to-native retinoid probes, we have developed a versatile late-stage method for the insertion of azide function at the C4 position of several retinoids. This one-step process opens straightforward access to different retinoid and carotenoid probes from commercially available precursors. We have further demonstrated that the different molecular probes retain ability of the original compound to activate genes' transcription, despite azide insertion, highlighting biological activities that were further validated in zebrafish in vivo model. The present work paves the way to future studies on vitamin A's metabolism.

Emerging technologies toward the integration of multiple functionalities on non-planar implantable neurophotonics probes.

The continuous exchange between the neuroscience and neuroengineering communities that took place over the past decades has uncovered a multitude of technological solutions to interface with the brain. In this framework, a fascinating approach relies on the integration of multiple activation and monitoring capabilities in the same implantable neural probe to better study the multifaceted nature of neural signaling and related functions in the deep brain regions. We highlight current challenges and perspectives on technological developments that could potentially enable the integration of multiple functionalities on optical fiber-based non-planar implantable neurophotonics probes.

In Situ PL Probes the Effect of 2D SnSe Nanosheets on the Crystallization Process of CsPbI2Br Perovskite Solar Cells.

Reducing defects in the active layer is important for improving the crystalline quality of all-inorganic perovskite solar cells (PSCs). Exploring novel additives is one of the most promising approaches to minimize active layer defects. In this work, two-dimensional (2D) SnSe nanosheets with excellent optoelectronic properties are prepared using an ultrasonic exfoliation method. The prepared 2D SnSe nanosheets are introduced into a CsPbI2Br precursor, which reduces the defect formation at grain boundaries and enhances the crystallinity of CsPbI2Br perovskites. We use the in situ photoluminescence (PL) technique to investigate the role of 2D materials in the crystallization process. The results show that SnSe nanosheets primarily shorten the grain boundary merging time and reduce the defect generation during the grain boundary merging stage, thereby regulating the crystallization of perovskite. In addition, SnSe nanosheets passivate uncoordinated Pb atoms at grain boundaries by Se atoms, further reducing the defect density in perovskite. As a result, PSCs exhibit a higher power conversion efficiency (PCE) of 14.24% and a Voc of 1.22 V. This study highlights the role of 2D materials in enhancing the crystalline quality and PCE of PSCs.

A simulated microgravity-oriented AIE probe-ECM hydrogel-integrated chip for cell culture and superoxide anion radical detection.

Human space activities have been continuously increasing. Astronauts experiencing spaceflight are faced with health problems caused by special space environments such as microgravity, and the investigation of cell injury is fundamental. The development of a platform capable of cell culture and injury detection is the prerequisite for the investigation. Constructing a platform suitable for special conditions in space life science research is the key issue. The ground-based investigation is an indispensable part of the research. Accordingly, a simulated microgravity (SMG)-oriented integrated chip platform capable of 3D cell culture and in situ visual detection of superoxide anion radical (O2•-) is developed. SMG can cause oxidative stress in human cells, and O2•- is one of the signaling molecules. Thus, a O2•--responsive aggregation-induced emission (AIE) probe is designed, which shows high selectivity and sensitivity to O2•-. Moreover, the probe exhibits abilities of long-term and wash-free staining to cells due to the AIE behavior, which is precious for space cell imaging. Meanwhile, a chip with a high-aspect-ratio chamber for adequate medium storage for the lack of the perfusion system during the SMG experiment and a cell culture chamber which can integrate the extracellular matrix (ECM) hydrogel for the bioinspired 3D cell culture is fabricated. In addition, a porous membrane is introduced between the chambers to prevent the hydrogel from separating during the SMG experiment. The afforded AIE probe-ECM hydrogel-integrated chip can achieve 3D culturing of U87-MG cells and in situ fluorescent detection of endogenous O2•- in the cells after long-term staining under SMG. The chip provides a powerful and potential platform for ground-based investigation in space life science and biomedical research.

Development and performance evaluation of a novel scintillation-based active shielding gamma probe.

The gamma probe is a commonly used detector for localizing sentinel lymph nodes after the injection of radiopharmaceuticals. In recent years, studies have focused on improving the features of gamma probes to achieve more consistent localization of the radiotracer uptake. As part of this effort, a novel gamma probe prototype based on an active shielding was developed, and its characteristics, including sensitivity, resolution and shielding effectiveness, were determined. The prototype integrates trapezoidal-shaped bismuth germanate (BGO) array coupled with a silicon photomultiplier (SiPM) array, accompanied by dedicated electronics and software for stand alone usage. We conducted a thorough characterization, validating experimental observations through Monte Carlo simulations using the GEANT4 simulation package. In scattering environment, with a probe-source distance of 30 mm, the experimental results show that the detector sensitivity is 120 ± 5 cps/MBq, and the spatial and angular resolutions, in terms of full width at half maximum (FWHM), are 44.8 ± 1.3 mm and 87.3 ± 1 . 5 ∘ , respectively. The shielding effectiveness of the probe was determined to be greater than 95 % . The prototype with active shielding was found to have comparable performance to conventional gamma probes.

Mechanistically Different Mechanochromophores Enable Calibration and Validation of Molecular Forces in Glassy Polymers and Elastomeric Networks.

Sterically distorted donor-acceptor p­systems, termed DA springs, can be progressively planarized under mechanical load causing a bathochromic shift of the photoluminescence (PL) spectrum. By combining theory and experiment, we here use a simple linear force calibration for two different conformational mechanochromophores to determine molecular forces in polymers from the mechanochromic shift in PL wavelength during multiple uniaxial tensile tests. Two systems are used, i) a highly entangled linear glassy polyphenylene and ii) a covalent elastomeric polydimethylsiloxane network. The mean forces estimated by this method are validated using known threshold forces for the mechanochemical ring-opening reactions of two different spiropyran force probes. The agreement between both approaches underlines that these DA springs provide the unique opportunity for the online monitoring of local molecular forces present in diverse polymer matrices.

Preparation of Fluorescent "on-off-on" Carbon Quantum Dots and Their Application to the Detection of Fe3+ and Ascorbic Acid and Information Encryption.

Carbon quantum dots are a new type of fluorescent carbon-based nanomaterials, and their excellent properties have provoked a strong research interest. Herein, blue-fluorescent carbon quantum dots (k-CQDs) were successfully synthesized by a simple one-step hydrothermal method using chitosan and ethylenediaminetetraacetic acid as precursors. It was found that Fe3+ could quench the fluorescence of k-CQDs by a dynamic quenching mechanism that increased the positive charge in solution. Due to ascorbic acid (AA) can reduce Fe3+ to Fe2+, the positive charge in solution was reduced and the fluorescence of k-CQDs was restored. Based on the mechanism of the fluorescence "on-off-on", k-CQDs were used for the detection of Fe3+ and AA with strong antijamming capability. The LOD for Fe3+ concentrations in the ranges of 0 to 30 µM and 30 to 100 µM were 0.3 µM and 0.76 µM, respectively. The LOD for AA concentrations in the ranges of 0 to 82.5 µM and 82.5 to 172.5 µM were 3.93 µM and 1.63 µM, respectively. Spiking recoveries of Fe3+ in tap water, AA in orange juice and tomato juice were 87.93 ∼ 101.13%, 86.77 ∼ 105.15% and 86.43 ∼ 103.80%, respectively. Meanwhile, k-CQDs also showed good potential for anti-counterfeiting encryption.

Primer exchange reaction assisted CRISPR/Cas9 cleavage for detection of dual microRNAs with electrochemistry method.

An electrochemical sensor assisted by primer exchange reaction (PER) and CRISPR/Cas9 system (PER-CRISPR/Cas9-E) was established for the sensitive detection of dual microRNAs (miRNAs). Two PER hairpin (HP) were designed to produce a lot of extended PER products, which could hybridize with two kinds of hairpin probes modified on the electrode and initiate the cleavage of two CRISPR/Cas9 systems guided by single guide RNAs (sgRNAs) with different recognition sequences. The decrease of the two electrochemical redox signals indicated the presence of dual-target miRNAs. With the robustness and high specificity of PER amplification and CRISPR/Cas9 cleavage system, simultaneous detection of two targets was achieved and the detection limits for miRNA-21 and miRNA-155 were 0.43 fM and 0.12 fM, respectively. The developed biosensor has the advantages of low cost, easy operation, and in-situ detection, providing a promising platform for point-of-care detection of multiple miRNAs.

Development of caspase-3-selective activity-based probes for PET imaging of apoptosis.

BACKGROUND: The cysteine-aspartic acid protease caspase-3 is recognized as the main executioner of apoptosis in cells responding to specific extrinsic and intrinsic stimuli. Caspase-3 represents an interesting biomarker to evaluate treatment response, as many cancer therapies exert their effect by inducing tumour cell death. Previously developed caspase-3 PET tracers were unable to reach routine clinical use due to low tumour uptake or lack of target selectivity, which are two important requirements for effective treatment response evaluation in cancer patients. Therefore, the goal of this study was to develop and preclinically evaluate novel caspase-3-selective activity-based probes (ABPs) for apoptosis imaging. RESULTS: A library of caspase-3-selective ABPs was developed for tumour apoptosis detection. In a first attempt, the inhibitor Ac-DW3-KE (Ac-3Pal-Asp-ßhLeu-Phe-Asp-KE) was 18F-labelled on the N-terminus to generate a radiotracer that was incapable of adequately detecting an increase in apoptosis in vivo. The inability to effectively detect active caspase-3 in vivo was likely attributable to slow binding, as demonstrated with in vitro inhibition kinetics. Hence, a second generation of caspase-3 selective ABPs was developed based on the Ac-ATS010-KE (Ac-3Pal-Asp-Phe(F5)-Phe-Asp-KE) with greatly improved binding kinetics over Ac-DW3-KE. Our probes based on Ac-ATS010-KE were made by modifying the N-terminus with 6 different linkers. All the linker modifications had limited effect on the binding kinetics, target selectivity, and pharmacokinetic profile in healthy mice. In an in vitro apoptosis model, the least hydrophilic tracer [18F]MICA-316 showed an increased uptake in apoptotic cells in comparison to the control group. Finally, [18F]MICA-316 was tested in an in vivo colorectal cancer model, where it showed a limited tumour uptake and was unable to discriminate treated tumours from the untreated group, despite demonstrating that the radiotracer was able to bind caspase-3 in complex mixtures in vitro. In contrast, the phosphatidylethanolamine (PE)-binding radiotracer [99mTc]Tc-duramycin was able to recognize the increased cell death in the disease model, making it the best performing treatment response assessment tracer developed thus far. CONCLUSIONS: In conclusion, a novel library of caspase-3-binding PET tracers retaining similar binding kinetics as the original inhibitor was developed. The most promising tracer, [18F]MICA-316, showed an increase uptake in an in vitro apoptosis model and was able to selectively bind caspase-3 in apoptotic tumour cells. In order to distinguish therapy-responsive from non-responsive tumours, the next generation of caspase-3-selective ABPs will be developed with higher tumour accumulation and in vivo stability.

Human NQO1 as a Selective Target for Anticancer Therapeutics and Tumor Imaging.

Human NAD(P)H-quinone oxidoreductase1 (HNQO1) is a two-electron reductase antioxidant enzyme whose expression is driven by the NRF2 transcription factor highly active in the prooxidant milieu found in human malignancies. The resulting abundance of NQO1 expression (up to 200-fold) in cancers and a barely detectable expression in body tissues makes it a selective marker of neoplasms. NQO1 can catalyze the repeated futile redox cycling of certain natural and synthetic quinones to their hydroxyquinones, consuming NADPH and generating rapid bursts of cytotoxic reactive oxygen species (ROS) and H2O2. A greater level of this quinone bioactivation due to elevated NQO1 content has been recognized as a tumor-specific therapeutic strategy, which, however, has not been clinically exploited. We review here the natural and new quinones activated by NQO1, the catalytic inhibitors, and the ensuing cell death mechanisms. Further, the cancer-selective expression of NQO1 has opened excellent opportunities for distinguishing cancer cells/tissues from their normal counterparts. Given this diagnostic, prognostic, and therapeutic importance, we and others have engineered a large number of specific NQO1 turn-on small molecule probes that remain latent but release intense fluorescence groups at near-infrared and other wavelengths, following enzymatic cleavage in cancer cells and tumor masses. This sensitive visualization/quantitation and powerful imaging technology based on NQO1 expression offers promise for guided cancer surgery, and the reagents suggest a theranostic potential for NQO1-targeted chemotherapy.

Labeling selectivity of lipid droplets fluorescent probes: Twisted intramolecular charge transfer (TICT) vs intramolecular charge transfer (ICT).

Fluorescence imaging technology is a versatile and essential tool in the field of biomedical research. To obtain excellent imaging results, the precise labeling of fluorescent probes is an important prerequisite. Nevertheless, the labeling selectivity of most fluorescent probes is not satisfactory, new design concepts are desperately needed. In this context, two isomeric lipid droplets (LDs) fluorescent probes Lipi-Cz-1 and Lipi-Cz-2 have been sophisticatedly developed with TICT and ICT-emitting characteristic, respectively. The more environmentally sensitive TICT-emitting Lipi-Cz-1 exhibits a significantly enhanced labeling selectivity in LDs imaging compared to the ICT-emitting Lipi-Cz-2, sufficiently illustrating the effectiveness of TICT-emitting characteristic in improving labeling selectivity. Additionally, Lipi-Cz-1 displays high photostability and biocompatibility. These advantages enable Lipi-Cz-1 to be finely applied in multimode fluorescence imaging, e.g. time-lapse 3D confocal imaging to monitor changes of the number and size of LDs during starvation, two-photon 3D imaging to compare the variations of LDs in various liver tissues, and STED super-resolution imaging to visualize the nanoscale LDs with the resolution of 65 nm. Overall, these imaging findings validate the effectiveness of the new strategy for improving the labeling selectivity.

Real-Time RT-PCR Assays for Typing of Epizootic Hemorrhagic Disease Virus.

Real-time RT-PCR for the detection of epizootic hemorrhagic disease virus (EHDV) in clinical samples is a fast and sensitive tool for the diagnosis and confirmation of disease. Several real-time RT-PCR methods have been reported over the last 10 years. In this chapter, we describe seven duplex real-time RT-PCR assays to amplify part of genome segment 2 of EHDV to enable serotype identification. The assay includes the detection of an endogenous control gene-beta-actin.

Contactless integrated photonic probes: fundamentals, characteristics, and applications.

On-chip optical power monitors are indispensable for functional implementation and stabilization of large-scale and complex photonic integrated circuits (PICs). Traditional on-chip optical monitoring is implemented by tapping a small portion of optical power from the waveguide, which leads to significant loss. Due to its advantages like non-invasive nature, miniaturization, and complementary metal-oxide-semiconductor (CMOS) process compatibility, a transparent monitor named the contactless integrated photonic probe (CLIPP), has been attracting great attention in recent years. The CLIPP indirectly monitors the optical power in the waveguide by detecting the conductance variation of the local optical waveguide caused by the surface state absorption (SSA) effect. In this review, we first introduce the fundamentals of the CLIPP including the concept, the equivalent electric model and the impedance read-out method, and then summarize some characteristics of the CLIPP. Finally, the functional applications of the CLIPP on the identification and feedback control of optical signal are discussed, followed by a brief outlook on the prospects of the CLIPP.

Open-source, high-throughput targeted in-situ transcriptomics for developmental and tissue biology.

Multiplexed spatial profiling of mRNAs has recently gained traction as a tool to explore the cellular diversity and the architecture of tissues. We propose a sensitive, open-source, simple and flexible method for the generation of in-situ expression maps of hundreds of genes. We exploit direct ligation of padlock probes on mRNAs, coupled with rolling circle amplification and hybridization-based in situ combinatorial barcoding, to achieve high detection efficiency, high throughput and large multiplexing. We validate the method across a number of species, and show its use in combination with orthogonal methods such as antibody staining, highlighting its potential value for developmental and tissue biology studies. Finally, we provide an end-to-end computational workflow that covers the steps of probe design, image processing, data extraction, cell segmentation, clustering and annotation of cell types. By enabling easier access to high-throughput spatially resolved transcriptomics, we hope to encourage a diversity of applications and the exploration of a wide range of biological questions.

Charge Transfer Mechanisms of Adaptive Multicomponent Solutions at Solid-Liquid Interfaces for Real-Time Coolant State Monitoring.

Charge-transfer mechanisms in adaptive multicomponent solutions at liquid-solid interfaces with triboelectric probes are crucial for understanding chemistry dynamics. However, liquid-solid charge transfer becomes unpredictable, due to the components or interactions in solutions, restricting its potential application for precise monitoring of liquid environments. This study utilizes triboelectric probes to investigate the charge transfer of chemicals, applying this approach to real-time coolant state monitoring. Analysis of electrical signal dynamics induced by ethylene glycol and its oxidation byproduct, oxalic acid, in ethylene glycol solutions reveals that hydrogen bond and ion adsorption diminishes the efficiency of electron transfer at the liquid-solid interface. These findings promote the engineering of the triboelectric probe that enhances coolant quality with remarkable sensitivity (detection limit: 0.0001%) and a broad freezing point operational range (0 to -49 °C). This work advances the precise control of the charge dynamics and demonstrates the potential of triboelectric probes for interdisciplinary applications.

A Pyridinium fluorophore for the detection of zinc ions under autophagy conditions.

Molecular probes for sensing and imaging of various analytes and biological specimens are of great importance in clinical diagnostics, therapy, and disease management. Since the cellular concentration of free Zn2+ varies from nanomolar to micromolar range during cellular processes and the high affinity Zn2+ imaging probes tend to saturate at lower concentrations of free Zn2+, fluorescence based probes with moderate binding affinity are desirable in distinguishing the occurrence of higher zinc concentrations in the cells. Herein, we report a new, pentacyclic pyridinium based probe, PYD-PA, having a pendant N,N-di(pyridin-2-ylmethyl)amine (DPA) for Zn2+ detection in the cellular environment. The designed probe is soluble in water and serves as a mitochondria targeting unit, whereas the pendent DPA acts as the coordination site for Zn2+. PYD-PA displayed a threefold enhancement in fluorescence intensity upon Zn2+ binding with a 1:1 binding stoichiometry. Further, the probe showed a selective response to Zn2+ over other biologically relevant metal ions with a moderate binding affinity (Ka = 6.29 × 104 M-1), good photostability, pH insensitivity, and low cytotoxicity. The demonstration of bioimaging in SK-BR-3 breast cancer cell lines confirmed the intracellular Zn ion sensing ability of the probe. The probe was successfully applied for real time monitoring of the fluctuation of intracellular free zinc ions during autophagy conditions, demonstrating its potential for cellular imaging of Zn2+ at higher intracellular concentrations.

Capturing Legionella pneumophila effector enzymes using a ubiquitin derived photo-activatable probe.

Upon infection of host cells the Legionella pneumophila bacterium releases a multitude of effector enzymes into the host's cytoplasm that manipulate cellular host pathways, including the host-ubiquitination pathways. The effectors belonging to the SidE-family are involved in non-canonical phosphoribosyl serine ubiquitination (PR-ubiquitination) of host substrate proteins. This results in the recruitment of ER-remodeling proteins and the formation of a Legionella-containing vacuole which is crucial in the onset of legionnaires disease. PR-ubiquitination is a dynamic process reversed by other Legionella effectors called Dups. During PR-Ubiquitin phosphodiester hydrolysis Dups form a covalent intermediate with the phosphoribosyl ubiquitylated protein using its active site His67 residue. We envisioned that covalent probes to target Legionella effectors could be of value to study these effectors and contribute to deciphering the complex biology of Legionella infection. Hence we effectively installed a photo-activatable pyridinium warhead on the 5'-OH of triazole-linked ribosylated ubiquitin allowing crosslinking of the probe to the catalytic histidine residues in Legionella SidE or Dup enzymes. In vitro tests on recombinantly expressed DupA and SdeAPDE revealed that the probe was able to capture the enzymes covalently upon photo-activation.