Search

Visualization of the spatiotemporal organization and dynamics of the genome in the nucleus is essential to elucidate how chromatin structure and nuclear function are intertwined. In the past, such studies were limited by the lack of appropriate tools. However, various imaging techniques now allow direct visualization of genomic loci in living cells. One of the main drivers behind this progress is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) technology. Initially used as an efficient and specific genome editing tool, the versatility of the CRISPR/Cas system has rapidly expanded its application range to include diagnostics, therapeutics, and, more recently, live cell imaging. In contrast to traditional fluorescent in situ hybridization, CRISPR/Cas-based imaging systems enable real-time dynamic tracking of genomic sequences down to the level of single cells and even a single locus. Recognizing the vital role played by CRISPR/Cas-based imaging systems in advancing our understanding of genome function in relation to its spatiotemporal organization, this review aimed to depict the current landscape of CRISPR/Cas-based technologies in genomic imaging. It discusses the principles, notable features, advantages, and limitations of these CRISPR/Cas-based imaging systems and concludes with a brief outlook into the future. [ABSTRACT FROM AUTHOR]

The viscosity within cells is a crucial microenvironmental factor, and sulfur dioxide (SO2) has essential functions in regulating cellular apoptosis and inflammation. Some evidence has been confirmed that changes in viscosity and overexposure of SO2 within the cell may cause detrimental effects including, but not limited to, respiratory and cardiovascular illnesses, inflammation, fatty liver, and various types of cancer. Therefore, precise monitoring of SO2 and viscosity in biological entities holds immense practical importance. Therefore, in this research, we developed a versatile fluorescent TCF‐Cou that enables the dual detection of SO2 and viscosity in the living system. Probe TCF‐Cou possessed a response to viscosity and SO2 through red and green emissions. The alteration of SO2 and viscosity levels in live cells and zebrafish were also monitored using probe TCF‐Cou. We hope that this fluorescent probe could be a potential tool for revealing the related pathological and physiological processes through monitoring the changes in SO2 and viscosity. [ABSTRACT FROM AUTHOR]

Cytotoxicity assays are crucial for assessing the efficacy of drugs in killing cancer cells and determining their potential therapeutic value. Measurement of the effect of drug concentration, which is an influence factor on cytotoxicity, is of great importance. This paper proposes a cytotoxicity assay using microwave sensors in an end-point approach based on the detection of the number of live cells for the first time. In contrast to optical methods like fluorescent labeling, this research uses a resonator-type microwave biosensor to evaluate the effects of drug concentrations on cytotoxicity by monitoring electrical parameter changes due to varying cell densities. Initially, the feasibility of treating cells with ultrapure water for cell counting by a microwave biosensor is confirmed. Subsequently, inhibition curves generated by both the CCK-8 method and the new microwave biosensor for various drug concentrations were compared and found to be congruent. This agreement supports the potential of microwave-based methods to quantify cell growth inhibition by drug concentrations.

Multi‐color two‐photon microscopy imaging of live cells is essential in biology. However, the limited diffraction resolution of conventional two‐photon microscopy restricts its application to subcellular organelle imaging. Recently, we developed a laser scanning two‐photon non‐linear structured illumination microscope (2P‐NLSIM), whose resolution improved three‐fold. However, its ability to image polychromatic live cells under low excitation power has not been verified. Here, to improve the reconstruction super‐resolution image quality under low excitation power, we increased the image modulation depth by multiplying the raw images with the reference fringe patterns in the reconstruction process. Simultaneously, we optimized the 2P‐NLSIM system to image live cells, including the excitation power, imaging speed, and field of view. The proposed system could provide a new imaging tool for live cells. [ABSTRACT FROM AUTHOR]

In this report, we presented our results on a recognition of multiple analytes using trisubstituted PDI‐based chemosensor DNP in 50 % HEPES buffered‐CH3CN solution. Upon addition of Pd2+ or Pd0, DNP showed near IR ratiometric changes (A759nm/A560nm) with detection limit of 283 nM and 190 nM, respectively. These spectroscopic changes are associated with color change from violet to light yellow (de‐propargylation). The de‐propargylation reaction of DNP is faster in the presence of Pd2+ than Pd0. Upon addition of PPh3, DNP resulted in appearance of new bands at 615 and 698 nm with decrease of absorbance intensity at 560 nm with detection limit of 349 nM. Two isosbestic points at 504 nm and 600 nm was observed. This change is associated with color changes from violet to blue. DNP coated TLC‐strips can be used for the detection of 4×10−4 M Pd0 and PPh3. We have demonstrated the practical application of DNP (i) for the detection of Pd0 in live A549 cells and (ii) for detection of Pd2+/Pd0 in simulated urine (10 %) samples with recovery of (>98±5 %). [ABSTRACT FROM AUTHOR]

This study employs TLD1433, a RuII‐based photodynamic therapy (PDT) agent in human clinical trials, as a benchmark to establish protocols for studying the excited‐state dynamics of photosensitizers (PSs) in cellulo, in the local environment provided by human cancer cells. Very little is known about the excited‐state properties of any PS in live cells, and for TLD1433, it is terra incognita. This contribution targets a general problem in phototherapy, which is how to interrogate the light‐triggered, function‐determining processes of the PSs in the relevant biological environment, and establishes methodological advances to study the ultrafast photoinduced processes for TLD1433 when taken up by MCF7 cells. We generalize the methodological developments and results in terms of molecular physics by applying them to TLD1433's analogue TLD1633, making this study a benchmark to investigate the excited‐state dynamics of phototoxic compounds in the complex biological environment. [ABSTRACT FROM AUTHOR]

Point accumulation for imaging in nanoscale topography (PAINT) is a single‐molecule technique for super‐resolution microscopy, which uses exchangeable single stranded DNA oligos or peptide‐pairs to create blinking phenomenon and achieves ≈5–25 nanometer resolution. Here, it is shown that by transfecting the protein‐of‐interest with a docker‐coil, rather than by adding the docker externally—as is the norm when using DNA tethers or antibodies as dockers—similar localization can be achieved, ≈10 nm. However, using a transfected docker has several experimental advances and simplifications. Most importantly, it allows Peptide‐PAINT to be applied to transfected live cells for imaging surface proteins in mammalian cells and neurons under physiological conditions. The enhanced resolution of Peptide‐PAINT is also shown for organelles in fixed cells to unravel structural details including ≈40‐nm and ≈60‐nm axial repeats in vimentin filaments in the cytoplasm, and fiber shapes of sub‐100‐nm histone‐rich regions in the nucleus. [ABSTRACT FROM AUTHOR]

Two four-coordinate organoboron N,C-chelate complexes with different functional terminals on the PEG chains are studied with respect to their photophysical properties within human MCF-7 cells. Their excited-state properties are characterized by time-resolved pump-probe spectroscopy and fluorescence lifetime microscopy. The excited-state relaxation dynamics of the two complexes are similar when studied in DMSO. Aggregation of the complexes with the carboxylate terminal group is observed in water. When studying the light-driven excited-state dynamics of both complexes in cellulo, i. e., after being taken up into human MCF-7 cells, both complexes show different features depending on the nature of the anchoring PEG chains. The lifetime of a characteristic intramolecular charge-transfer state is significantly shorter when studied in cellulo (360±170 ps) as compared to in DMSO (-960 ps) at 600 nm for the complexes with an amino group. However, the kinetics of the complexes with the carboxylate group are in line with those recorded in DMSO. On the other hand, the lifetimes of the fluorescent state are almost identical for both complexes in cellulo. These findings underline the importance to evaluate the excited-state properties of fluorophores in a complex biological environment in order to fully account for intra- and intermolecular effects governing the light-induced processes in functional dyes. [ABSTRACT FROM AUTHOR]

Protein–protein interactions (PPIs) have great importance for intracellular signal transduction and sustaining the homeostasis of an organism. Thus, the identification of PPIs is necessary to better understand the downstream signaling functions of the proteins in healthy and pathological conditions. Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool for detecting PPIs in living cells. In literature, FRET analysis methods such as donor photobleaching (FLIM), acceptor photobleaching, spectral imaging, and the three-filter cube method (sensitized emission) are abundantly applied to investigate PPIs; however, they require various expensive instrumentations, and their calculation methods are very time consuming. Since confocal microscopy applications and live cell-based techniques of FRET are very costly, scientists sometimes prefer plate readers for FRET experiments. However, plate reader applications also have many disadvantages and considerations compared to confocal fluorescence microscopy, and complex calculation procedures should be performed. To overcome these problems, we propose a FRET-based high-throughput assay method with a standard monochromator-based microplate reader, which is generally available in most biochemistry laboratories, and an alternative calculation procedure. This rapid, low cost, and effective analysis method enables the scientists to prescreen PPIs in living cells as a preliminary study and quick glance at the experiment before preparing the whole experimental setup with the expensive instrumentations. Additionally, the alternative calculation procedure provides the FRET area comparison without complex bleed-through calculations in a non-conventional manner by shortening the analysis processes with this quick and uncomplicated spectral representation. Graphical Abstract [ABSTRACT FROM AUTHOR]

Biological pathways rely on the formation of intricate protein interaction networks called interactomes. Getting a comprehensive map of interactomes implies the development of tools that allow one to capture transient and low-affinity protein–protein interactions (PPIs) in live conditions. Here we presented an experimental strategy: the Cell-PCA (cell-based protein complementation assay), which was based on bimolecular fluorescence complementation (BiFC) for ORFeome-wide screening of proteins that interact with different bait proteins in the same live cell context, by combining high-throughput sequencing method. The specificity and sensitivity of the Cell-PCA was established by using a wild-type and a single-amino-acid-mutated HOXA9 protein, and the approach was subsequently applied to seven additional human HOX proteins. These proof-of-concept experiments revealed novel molecular properties of HOX interactomes and led to the identification of a novel cofactor of HOXB13 that promoted its proliferative activity in a cancer cell context. Taken together, our work demonstrated that the Cell-PCA was pertinent for revealing and, importantly, comparing the interactomes of different or highly related bait proteins in the same cell context. [ABSTRACT FROM AUTHOR]

Graphene (GR) has engrossed immense research attention as an emerging carbon material owing to its enthralling electrochemical (EC) and physical properties. Herein, we debate the role of GR-based nanomaterials (NMs) in refining EC sensing performance toward bioanalytes detection. Following the introduction, we briefly discuss the GR fabrication, properties, application as electrode materials, the principle of EC sensing system, and the importance of bioanalytes detection in early disease diagnosis. Along with the brief description of GR-derivatives, simulation, and doping, classification of GR-based EC sensors such as cancer biomarkers, neurotransmitters, DNA sensors, immunosensors, and various other bioanalytes detection is provided. The working mechanism of topical GR-based EC sensors, advantages, and real-time analysis of these along with details of analytical merit of figures for EC sensors are discussed. Last, we have concluded the review by providing some suggestions to overcome the existing downsides of GR-based sensors and future outlook. The advancement of electrochemistry, nanotechnology, and point-of-care (POC) devices could offer the next generation of precise, sensitive, and reliable EC sensors. [ABSTRACT FROM AUTHOR]

Nitrogen rich carbon nanoparticles are known to provide higher fluorescence stokes shift, and thereby are potential candidates for fluorescent sensors. Herein, a facile one-step hydrothermal synthesis is reported for N-rich carbon nanospheres (G-CNS) from caffeine and o -phenylenediamine as precursors. The as-synthesized G-CNS showed high fluorescence with λ em at 509 nm, with a highly selective fluorescence turn-off response towards Fe2+/Fe3+, rendering these carbon nanospheres as potential candidates to detect intracellular labile iron pool in live cells. The intracellular labile iron pool in iron-overloaded cells was sensed using the synthesized G-CNS. Mechanistically, the fluorescence quenching via dynamic pathway involves the formation of an excited state charge transfer process, which undergoes non-radiative decay. [Display omitted] • An N-doped carbon nanosphere with a green fluorescence has been developed. • The nanosphere shows highly selective fluorescence turn-off response towards iron with low limit of detection. • The probe is effective in sensing intracellular labile iron pool in live cells. [ABSTRACT FROM AUTHOR]

Here, in the present work, a new hydroxybenzothiazole derivative (HBT 2) with AIE+ESIPT features was synthesized by Suzuki–Miyora coupling of HBT 1 with 4-formylphenylboronic acid. The AIE and ESIPT features were confirmed by optical, microscopic (AFM) and dynamic light scattering (DLS) techniques. The yellow fluorescent aggregates of HBT 2 can specifically detect Cu2+/Cu+ ions with limits of detection as low as 250 nM and 69 nM. The Job's plot revealed the formation of a 1:1 complex. The Cu2+ complexation was further confirmed by optical, NMR, AFM and DLS techniques. HBT 2 was also used for the detection of Cu2+ ions in real water samples collected from different regions of Punjab. HBT 2 was successfully used for the bio-imaging of Cu2+ ions in live A549 and its anticancer activity was checked on different cancer cell lines, such as MG63, and HeLa, and normal cell lines such as L929. We successfully utilized HBT 2 to develop security labels for anticounterfeiting applications. [ABSTRACT FROM AUTHOR]

A series of three-dimensional copper oxide (CuO) inverse opals anchored with carboxylated graphene quantum dots (CuO/cGQDs) have been fabricated for non-enzymatic tracking of dopamine (DA). Heterostructures composed of various building blocks are promising to construct versatile biosensing platforms. The optimal CuO/cGQDs modified electrode demonstrates sensitivities of 243.45 μA mM−1 cm−2 (50 nM–1888.5 μM) with the practical detection limit as low as 0.5 nM in mimic physiological environment (at + 0.45 V vs. Ag/AgCl). The extraordinary tolerance to various interferents enables the practical detection of intracellular DA amount in human neural cells. On this basis, the proposed biosensor attains precise evaluation of antipsychotic drug effects on stimulated DA release. Particularly, it successfully spots fluctuation of DA in plasma and cerebrospinal fluid in murine model of Parkinson's disease, which serves as a crucial tool to understand neuropathology and symptomatology of DA-related diseases. This study developed a reliable sensing platform and is expected to be applied to physiological and pathological studies. [ABSTRACT FROM AUTHOR]

Pantetheinase is a key biomarker for the diagnosis of acute kidney injury and the monitoring of malaria progression. Currently, existing methods for sensing pantetheinase, also known as Vanin-1, show considerable potential but come with certain limitations, including their inability to directly sense analytes in turbid biofluid samples without tedious sample pretreatment. Here, we describe the first activity-based electrochemical probe, termed VaninLP, for convenient and specific direct targeting of pantetheinase activity in turbid liquid biopsy samples. The probe was designed such that cleavage of the pantetheinase amide linkage, triggered by a self-immolative reaction, simultaneously ejects an amino ferrocene reporter. Among the distinctive properties of the VaninLP probe for sensing pantetheinase are its high selectivity, sensitivity, and enzyme affinity, a wide linear concentration range (8-300 ng/mL), and low limit of detection (2.47 ng/mL). The designed probe precisely targeted pantetheinase and was free of interference by other electroactive biological species. We further successfully applied the VaninLP probe to monitor and quantify the activity of pantetheinase on the surfaces of HepG2 tumor cells, blood, and urine samples. Collectively, our findings indicate that VaninLP holds significant promise as a point-of-care tool for diagnosing early-stage kidney injury, as well as monitoring the progression of malaria.

Fourier transform infrared (FTIR) spectroscopy is widely used for molecular analysis. However, for the materials situated in an aqueous environment, a precondition for live biological objects such as cells, transmission-based FTIR is prevented by strong water absorption of mid-infrared (MIR) light. Reflection-based cellular assays using internal reflection elements (IREs) such as high-index prisms or flat plasmonic metasurfaces mitigate these issues but suffer from a shallow probing volume localized near the plasma membrane. Inspired by the recent introduction of high-aspect-ratio nanostructures as a novel platform for manipulating cellular behavior, we demonstrate that the integration of plasmonic metasurfaces with tall dielectric nanostructures dramatically enhances the sensing capabilities of FTIR spectroscopy. We also demonstrate the ability of a metal-on-dielectric metasurface to transduce intracellular processes, such as protein translocation to high-curvature membrane regions during cell adhesion, into interpretable spectral signatures of the reflected light.

Abstract Chemically manipulating bacterial surface structures, a cutting‐edge research direction in the biomedical field, predominantly relies on metabolic labeling by now. However, this method may involve daunting precursor synthesis and only labels nascent surface structures. Here, we report a facile and rapid modification strategy based on a tyrosinase‐catalyzed oxidative coupling reaction (TyOCR) for bacterial surface engineering. This strategy employs phenol‐tagged small molecules and tyrosinase to initiate direct chemical modification of Gram‐positive bacterial cell walls with high labeling efficiency, while Gram‐negative bacteria are inert to this modification due to the hindrance of an outer membrane. By using the biotin‒avidin system, we further present the selective deposition of various materials, including photosensitizer, magnetic nanoparticle, and horseradish peroxidase, on Gram‐positive bacterial surfaces, and realize the purification/isolation/enrichment and naked‐eye detection of bacterial strains. This work demonstrates that TyOCR is a promising strategy for engineering live bacterial cells.

Toxic hydrazine poses a huge threat to living organisms and our environment. Extremely acidic pH is also harmful to various ecosystems. Here, a coumarin-based dye called CMM has been synthesized for the ratiometric detection of hydrazine (F 504 nm /F 560 nm). CMM could detect gaseous hydrazine in the environment and endogenous hydrazine in live cells. Furthermore, the fluorescence at 563 nm of CMM was stable from pH 10.0 to 2.0, whereas increased greatly from pH 2.0 to 1.2. Thus, the probe could also detect extremely acidic pH with great fluorescence enhancement. The p K a value was determined to be as low as 1.41. We anticipate that the probe could provide a good candidate for detection of toxic hydrazine and extremely acidic pH. [Display omitted] • A "two-in-one" fluorescent probe was synthesized for detection of hydrazine and extremely acidic pH. • The probe could respond to hydrazine and extremely acidic pH through different signals. • The probe could detect hydrazine in real environment samples and in live cells. [ABSTRACT FROM AUTHOR]

A long-standing desire in biological and biomedical sciences is to be able to probe cellular chemistry as biological processes are happening inside living cells. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectral microscopy is a label-free and nondestructive analytical technique that can provide spatiotemporal distributions and relative abundances of biomolecules of a specimen by their characteristic vibrational modes. Despite great progress in recent years, SR-FTIR imaging of living biological systems remains challenging because of the demanding requirements on environmental control and strong infrared absorption of water. To meet this challenge, microfluidic devices have emerged as a method to control the water thickness while providing a hospitable environment to measure cellular processes and responses over many hours or days. This paper will provide an overview of microfluidic device development for SR-FTIR imaging of living biological systems, provide contrast between the various techniques including closed and open-channel designs, and discuss future directions of development within this area. Even as the fundamental science and technological demonstrations develop, other ongoing issues must be addressed; for example, choosing applications whose experimental requirements closely match device capabilities, and developing strategies to efficiently complete the cycle of development. These will require imagination, ingenuity and collaboration.

Biological pathways rely on the formation of intricate protein interaction networks called interactomes. Getting a comprehensive map of interactomes implies the development of tools that allow one to capture transient and low-affinity protein–protein interactions (PPIs) in live conditions. Here we presented an experimental strategy: the Cell-PCA (cell-based protein complementation assay), which was based on bimolecular fluorescence complementation (BiFC) for ORFeome-wide screening of proteins that interact with different bait proteins in the same live cell context, by combining high-throughput sequencing method. The specificity and sensitivity of the Cell-PCA was established by using a wild-type and a single-amino-acid-mutated HOXA9 protein, and the approach was subsequently applied to seven additional human HOX proteins. These proof-of-concept experiments revealed novel molecular properties of HOX interactomes and led to the identification of a novel cofactor of HOXB13 that promoted its proliferative activity in a cancer cell context. Taken together, our work demonstrated that the Cell-PCA was pertinent for revealing and, importantly, comparing the interactomes of different or highly related bait proteins in the same cell context.

Graphene (GR) has engrossed immense research attention as an emerging carbon material owing to its enthralling electrochemical (EC) and physical properties. Herein, we debate the role of GR-based nanomaterials (NMs) in refining EC sensing performance toward bioanalytes detection. Following the introduction, we briefly discuss the GR fabrication, properties, application as electrode materials, the principle of EC sensing system, and the importance of bioanalytes detection in early disease diagnosis. Along with the brief description of GR-derivatives, simulation, and doping, classification of GR-based EC sensors such as cancer biomarkers, neurotransmitters, DNA sensors, immunosensors, and various other bioanalytes detection is provided. The working mechanism of topical GR-based EC sensors, advantages, and real-time analysis of these along with details of analytical merit of figures for EC sensors are discussed. Last, we have concluded the review by providing some suggestions to overcome the existing downsides of GR-based sensors and future outlook. The advancement of electrochemistry, nanotechnology, and point-of-care (POC) devices could offer the next generation of precise, sensitive, and reliable EC sensors.

Live cells are implicated in diverse biological processes, including nutrient transport and removal of foreign substances. Their intelligent biofunctions with complex mechanisms cannot be replicated at all in man‐made materials despite the significant advance in material design. Taking advantages of their biocompatibility and biotropic capability, various live cells have been developed as a kind of special carriers for the on‐demand delivery of therapeutic and diagnostic agents in recent years. Furthermore, synthetic materials can be integrated with live cells to provide bio‐hybrid systems that inherit advantages from both the synthetic particles and the natural cells. Herein, recent strategies and advances in cell‐based bio‐hybrid delivery systems for disease treatment are summarized. Challenges and opportunities in this field are also discussed.

Here, in the present work, a new hydroxybenzothiazole derivative (HBT 2) with AIE+ESIPT features was synthesized by Suzuki–Miyora coupling of HBT 1 with 4-formylphenylboronic acid. The AIE and ESIPT features were confirmed by optical, microscopic (AFM) and dynamic light scattering (DLS) techniques. The yellow fluorescent aggregates of HBT 2 can specifically detect Cu2+/Cu+ ions with limits of detection as low as 250 nM and 69 nM. The Job’s plot revealed the formation of a 1:1 complex. The Cu2+ complexation was further confirmed by optical, NMR, AFM and DLS techniques. HBT 2 was also used for the detection of Cu2+ ions in real water samples collected from different regions of Punjab. HBT 2 was successfully used for the bio-imaging of Cu2+ ions in live A549 and its anticancer activity was checked on different cancer cell lines, such as MG63, and HeLa, and normal cell lines such as L929. We successfully utilized HBT 2 to develop security labels for anticounterfeiting applications.

[Display omitted] • A new pH-sensitive fluorescent probe 1 based on hemicyanine fluorophore is developed. • The probe can respond to pH with high selectivity and sensitivity. • The probe can solely illumine the lysosomes and detect the exogenous pH difference in live cells. • The probe can successfully monitor the pH changes during food spoilage. pH as an important parameter is closely related to many pathological processes and food freshness. In this paper, a new pH-sensitive fluorescent probe 1 based on hemicyanine dye was synthesized and used for the detection of pH in live cells and food spoilage. Under neutral and alkaline conditions, the phenolic hydroxyl group of the probe deprotonates, forming a closed spirocyclic structure and resulting in fluorescence quenching. Under acidic conditions, the probe maintains an open-loop structure and has intense fluorescence. The probe possessing good selectivity and stability can target lysosomes and visualize the exogenous pH in live cells. Furthermore, by using this probe, we successfully achieved the monitoring of pH in metamorphic process of shrimp and pork. This probe is expected to provide a good imaging tool for real-time detection of pH in live cells and food monitoring. [ABSTRACT FROM AUTHOR]

Abstract Background GPCRs are considered essential for various physiological processes and have been the most productive drug targets. Therefore, development of the methods of GPCR ligands screening is a high priority for pharmaceutical industries and research institutions. Methods We developed a potential method (piggyBac-TANGO) based on the TANGO and PRESTO-TANGO assays. The system was optimized with a piggyBac transposon as a transgene vehicle, and eGFP was used as a reporter instead of luciferase. The assay was validated in the HEK 293T and U87-MG cell lines and antagonist activities of the compounds were assessed. The transgene copy number and long-term stability were evaluated by qPCR. Then, we performed a DRD2-targeted screening for natural products using the piggyBac-TANGO assay. Results The validation assay showed that using the piggyBac transposon as a transgene vehicle produced high signal-to-background ratio and stable readout confirmed by investigation of the transgene copy number and long-term stability. Use of eGFP instead of luciferase as a reporter enabled to create a high throughput system suitable for live cells. Moreover, the piggyBac-TANGO assay permitted versatile detection of antagonist activity of compounds and was not limited to a particular cell type. With the use of the piggyBac-TANGO assay, we have successfully identified a novel agonist of DRD2. Conclusion Thus, the results indicate that the piggyBac-TANGO method is a user-friendly, robust and imaging-based assay that provides a novel approach to high throughput GPCR-targeted ligand screening and drug development.

Cytotoxicity assays are crucial for assessing the efficacy of drugs in killing cancer cells and determining their potential therapeutic value. Measurement of the effect of drug concentration, which is an influence factor on cytotoxicity, is of great importance. This paper proposes a cytotoxicity assay using microwave sensors in an end-point approach based on the detection of the number of live cells for the first time. In contrast to optical methods like fluorescent labeling, this research uses a resonator-type microwave biosensor to evaluate the effects of drug concentrations on cytotoxicity by monitoring electrical parameter changes due to varying cell densities. Initially, the feasibility of treating cells with ultrapure water for cell counting by a microwave biosensor is confirmed. Subsequently, inhibition curves generated by both the CCK-8 method and the new microwave biosensor for various drug concentrations were compared and found to be congruent. This agreement supports the potential of microwave-based methods to quantify cell growth inhibition by drug concentrations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhao, Wang, Shi, Wang, Jiang, Yang, Gao and Qiang.)

Water absorption of mid-infrared (MIR) radiation severely limits the options for vibrational spectroscopy of the analytes-including live biological cells-that must be probed in aqueous environments. While internal reflection elements, such as attenuated total reflection prisms and metasurfaces, partially overcome this limitation, such devices have their own limitations: ATR prisms are difficult to integrate with multiwell cell culture workflows, while metasurfaces suffer from a limited spectral range and small penetration depth into analytes. In this work, we introduce an alternative live cell biosensing platform based on metallic nanogratings fabricated on top of elevated dielectric pillars. For the MIR wavelengths that are significantly longer than the grating period, reflection-based spectroscopy enables broadband sensing of the analytes inside the trenches separating the dielectric pillars. Because the depth of the analyte twice-traversed by the MIR light excludes the highly absorbing thick water layer above the grating, we refer to the technique as inverted transflection spectroscopy (ITS). The analytic power of ITS is established by measuring a wide range of protein concentrations in solution, with the limit of detection in the single-digit mg mL -1 . The ability of ITS to interrogate live cells that naturally wrap themselves around the grating is used to characterize their adhesion kinetic.

Hydrogen sulfide (H2S), an endogenous gasotransmitter, is produced in mammalian systems and is closely associated with pathological and physiological functions. Nevertheless, the complete conversion of H2S is still unpredictable owing to the limited number of sensors for accurate and quantitative detection of H2S in biological samples. In this study, we constructed a disposable electrochemical sensor based on PtNi alloy nanoparticles (PtNi NPs) for sensitive and specific in situ monitoring of H2S released by human breast cancer cells. PtNi alloy NPs with an average size of 5.6 nm were prepared by a simple hydrothermal approach. The conversion of different forms of sulfides (e.g., H2S, HS−, and S2−) under various physiological conditions hindered the direct detection of H2S in live cells. PtNi NPs catalyze the electrochemical oxidation of H2S in a neutral phosphate buffer (PB, pH 7.0). The PtNi-based sensing platform demonstrated a linear detection range of 0.013–1031 µM and the limit of detection was 0.004 µM (S/N = 3). Moreover, the PtNi sensor exhibited a sensitivity of 0.323 μA μM−1 cm−2. In addition, the stability, repeatability, reproducibility, and anti-interference ability of the PtNi sensor exhibited satisfactory results. The PtNi sensor was able to successfully quantify H2S in pond water, urine, and saliva samples. Finally, the biocompatible PtNi electrode was effectively employed for the real-time quantification of H2S released from breast cancer cells and mouse fibroblasts.

Acoustics have a wide range of uses, from noise-cancelling to ultrasonic imaging. There has been a surge in interest in developing acoustic-based approaches for biological and biomedical applications in the last decade. This review focused on the application of surface acoustic waves (SAW) based on interdigital transducers (IDT) for live-cell investigations, such as cell manipulation, cell separation, cell seeding, cell migration, cell characteristics, and cell behaviours. The approach is also known as acoustofluidic, because the SAW device is coupled with a microfluidic system that contains live cells. This article provides an overview of several forms of IDT of SAW devices on recently used cells. Conclusively, a brief viewpoint and overview of the future application of SAW techniques in live-cell investigations were presented.

β‐arrestins (βarr1 and βarr2) are ubiquitous regulators of G protein‐coupled receptor (GPCR) signaling. Available data suggest that β‐arrestins dock to different receptors in different ways. However, the structural characterization of GPCR‐arrestin complexes is challenging and alternative approaches to study GPCR‐arrestin complexes are needed. Here, starting from the finger loop as a major site for the interaction of arrestins with GPCRs, we genetically incorporate non‐canonical amino acids for photo‐ and chemical crosslinking into βarr1 and βarr2 and explore binding topologies to GPCRs forming either stable or transient complexes with arrestins: the vasopressin receptor 2 (rhodopsin‐like), the corticotropin‐releasing factor receptor 1, and the parathyroid hormone receptor 1 (both secretin‐like). We show that each receptor leaves a unique footprint on arrestins, whereas the two β‐arrestins yield quite similar crosslinking patterns. Furthermore, we show that the method allows defining the orientation of arrestin with respect to the GPCR. Finally, we provide direct evidence for the formation of arrestin oligomers in the cell. Synopsis: Chemical and photo‐crosslinkers genetically incorporated into β‐arrestins allow the mapping of footprints of different receptors on the arrestin surface in living cells, and to identify intermolecular pairs of proximal amino acids in GPCR‐arrestin complexes. Genetically encoded crosslinkers can capture arrestin‐GPCR interactions with high spatial precision.Each G protein‐coupled receptor leaves a unique footprint on arrestins.Arrestins βarr1 and βarr2 may bind to a GPCR in a similar way. [ABSTRACT FROM AUTHOR]

A simple ultrasonic assisted chemical technique was used to synthesise cadmium oxide (CdO) nanoparticles (NPs) and CdO NPs/c‐Multiwalled carbon nanotube (c‐MWCNT) nanocomposite fibres.To confirm the physio‐chemico properties and to analyse surface morphology of the obtained nanomaterials X‐Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM) were performed. To evaluate the anti‐cancer property of CdO NPs, c‐MWCNT NPs and CdO NPs/c‐MWCNT nanocomposite fibres, an anti‐proliferative assay test (Methylthiazolyl diphenyl‐ tetrazolium bromide ‐ MTT assay) were performed on HeLa cells which further estimated IC50 value (Least concentration of sample in which nearly 50% of cells remain alive) under in‐vitro conditions. On comparison, CdONPs/c‐MWCNT based system was found to be superior by achieving 52.3% cell viability with its minimal IC50 value of 31.2 μg/ml. Lastly, the CdO NPs based system was taken up for an apoptotic study using DNA fragmentation assay for estimating its ability to cleave the DNA of the HeLa cells into internucleosomal fragments using the agarose gel electrophoresis method. In conclusion, based on our observations, CdO NPs/c‐MWCNT hybrid based system can be further used for the development of efficient drug delivery and therapeutic systems. [ABSTRACT FROM AUTHOR]

The production of livestock and poultry faces major challenges to meet the global demand for meat and dairy products and eggs due to a steady increase in the world's population and the ban of antibiotics in animal production. This ban has forced animal nutritionists to seek for natural alternatives to antibiotics. In this context, the yeast Saccharomyces cerevisiae has received considerable attention in the last decade. It has been reported that feed supplementation with live yeast cells improve feed efficiency, enhance feed digestibility, increase animal performance, reduce the number of pathogenic bacteria, improve animal health and reduce the negative environmental impacts of livestock production. The current review sheds light on the effects of the use of live S. cerevisiae cells in the diets of nonruminant and pseudo‐ruminant's animals and the mechanisms by which they exert its effects. This review work revealed that the addition of S. cerevisiae in poultry feed causes a phenomenon called competitive exclusion of pathogenic bacteria capable of causing disease adhere to the yeast surface, and so removing a large amount of harmful micro‐organisms and allowing the Animal defend more effectively, the production of antimicrobial agents, the balancing the gut microbiota and stimulation of host adaptive immune system and improving gut morphological structure, thus these benefits are reflected on the overall poultry health. In addition, in the presence of live S. cerevisiae cells, the immunity of rabbits was improved due to the high number of white blood cell. In addition, apparent digestibility of acid and neutral detergent fibre was improved in horses and rabbits. Saccharomyces cerevisiae in pig diets augment mucosal immunity by increasing IgM and IgA activity against pathogens, enhance intestinal development and function, adsorb mycotoxins, modulate gut microbiota and reduce postweaning diarrhoea. [ABSTRACT FROM AUTHOR]

[Display omitted] • Probe LTA has a low detection limit (4.5 μM) and good selectivity for H 2 O 2. • Compared with probe LTQ, probe LTA has a faster response to H 2 O 2. • LTA can effectively monitor the changes in endogenous and exogenous H 2 O 2 levels in cells and zebrafish. • It was successfully used to detect the elevated H 2 O 2 level caused by APAP-induced liver injury. H 2 O 2 is an important signaling molecule in the body, and its levels fluctuate in many pathological sites, therefore, it can be used as a biomarker for early diagnosis of disease. Since the environment in vivo is extremely complex, it is of great significance to develop a probe that can accurately monitor the fluctuation of H 2 O 2 level without interference from other physiological processes. Based on this, we designed and synthesized two new near-infrared H 2 O 2 fluorescent probes, LTA and LTQ, based on the ICT mechanism. Both of them have good responses to H 2 O 2 , but LTA has a faster response speed. In addition, the probe LTA has good biocompatibility, good water solubility, and a large Stokes shift (95 nm). The detection limit is 4.525 μM. The probe was successfully used to visually detect H 2 O 2 in living cells and zebrafish and was successfully used to monitor the changes in H 2 O 2 levels in zebrafish due to APAP-induced liver injury. [ABSTRACT FROM AUTHOR]

Endogenous sulfur dioxide (SO 2) and cysteine (Cys) are important species and interacted with each other. However, due to the reactivity of these species, it is difficult to differentiated detection them. Thus, it is necessary to develop new fluorescent probe for discriminative detect these reactive sulfur species in biological environment. Herein, A new hemicyanine derivative Mito-AE with dual recognition sites for SO 2 derivatives and Cys detection was constructed and applied in biological detection. Mito-AE exhibited ratiometric fluorescence change (F 470 /F 610) of over 90-fold when interacted with SO 2 , and a dramatic fluorescence turn-on at 610 nm when interacted with Cys. The probe can monitor both SO 2 derivatives and Cys with fast response rate (within 1 min), high sensitivity and selectivity. As proved by biological experiments, Mito-AE was able to successfully detect exogenous and endogenous SO 2 in living cells as well as in mitochondria area. More importantly, the detection of SO 2 derivatives and Cys in zebrafish model suggesting a potential application for studying the multiple interaction of biological sulfurous species in live organism. [Display omitted] • A new fluorescent probe for discriminative detection of SO 2 and Cys was constructed. • The probe shows ratiometric response toward SO 2 and fluorescence turn-on toward Cys. • The probe was successfully applied to detect SO 2 and Cys in live cells and zebrafish. • These results shown potential application for studying the interaction of SO 2 and Cys. [ABSTRACT FROM AUTHOR]

The spectroscopic properties, ultrafast kinetics and utilization of a photochromic molecule as a bi-stable fluorescing sensor of polarity in live cells are described. This molecule is a photochromic fulgimide, 2,3-dialkylidenesuccinimide, which emits fluorescence that can be switched optically on and off. The fluorescence intensity is a function of the polarity of the molecular environment, namely it fluoresces strongly when the molecule is in its polar isomeric structure form. We demonstrate that this molecule enters live cells without inducing damage, it binds primarily to internal membranous organelles (mitochondria) and its fluorescence can be switched optically "on" and "off" repeatedly while inside the living cell. A possible use as a bi-stable, on/off sensor is discussed.

The viscosity within cells is a crucial microenvironmental factor, and sulfur dioxide (SO 2 ) has essential functions in regulating cellular apoptosis and inflammation. Some evidence has been confirmed that changes in viscosity and overexposure of SO 2 within the cell may cause detrimental effects including, but not limited to, respiratory and cardiovascular illnesses, inflammation, fatty liver, and various types of cancer. Therefore, precise monitoring of SO 2 and viscosity in biological entities holds immense practical importance. Therefore, in this research, we developed a versatile fluorescent TCF-Cou that enables the dual detection of SO 2 and viscosity in the living system. Probe TCF-Cou possessed a response to viscosity and SO 2 through red and green emissions. The alteration of SO 2 and viscosity levels in live cells and zebrafish were also monitored using probe TCF-Cou. We hope that this fluorescent probe could be a potential tool for revealing the related pathological and physiological processes through monitoring the changes in SO 2 and viscosity., (© 2023 John Wiley & Sons Ltd.)

The impacts of biofouling on the retention of pharmaceutically active compounds (PhACs) by a commercially available nanofiltration membrane (NF 270) were systematically studied. Biofouling was achieved through inoculating live and dead Pseudomonas aeruginosa into artificial wastewater. In comparison to a clean membrane, an increase in PhAC rejection during biofouling with live cells was observed. However, the rejection behaviors presented more complex changes during biofouling with dead cells: PhAC rejection was below the clean membrane in the early biofouling stage; however, in the later stage, PhAC rejection was above the clean membrane. In addition, PhAC rejection behaviors present the similar tendency as salt rejection under both biofouling conditions. In addition, solute rejections were much lower for biofouling with dead cells than those for biofouling with live cells. Combined with biofilm characterization under both biofouling conditions, we could conclude that biofilm enhanced osmotic pressure (BEOP) due to higher cell counts and biofilm thickness led to a decrease in PhAC retention, especially for the dead cells. In addition, more dominant steric exclusion in the later stage of biofouling due to higher extracellular polymeric substances (EPS) concentration on the membrane surface resulted in an increase in PhAC retention. [ABSTRACT FROM AUTHOR]

A novel coumarin-derived Schiff base fluorescence probe (CTB) has been successfully designed and synthesized through exploiting tris-(2-aminothyl)-amine moiety as a recognition unit for the highly selective and sensitive detection of Cd2+. It is based on C N isomerization and the photo-induced electron transfer (PET) mechanism. The investigation into the sensing processes showed that CTB exhibited an excellent selectivity for Cd2+. The sensitivity exceeded that of other competing metal ions, and had a high sensitivity, a detection limit of 1.16 × 10−7 M with the association constants of 1.37 × 1011 M−2. The experiments including Job's plot, UV–Vis titration, 1H NMR titration and ESI-MS spectrum established that the probe CTB binds to Cd2+ in a 1:2 ratio. Further studies also demonstrated that probe CTB can be successfully applied to the fluorescence imaging of Cd2+ in HepG-2 cells. Unlabelled Image • A tripodal coumarin-derived Schiff base fluorescence probe for Cd2+ was designed and synthesized. • This fluorescence probe has good selectivity and sensitivity for Cd2+. • This fluorescence probe could discriminate between Zn2+ and Cd2+ by using EDTA as an auxiliary reagent. • This fluorescence probe can be applied in live cells. [ABSTRACT FROM AUTHOR]

Intracellular Ca2+ signaling controls numerous cellular functions. Mitochondria respond to cytosolic Ca2+ changes by adapting mitochondrial functions and, in some cell types, shaping the spatiotemporal properties of the cytosolic Ca2+ signal. Numerous methods have been developed to specifically and quantitatively measure the mitochondrial‐free Ca2+ concentrations ([Ca2+]m), but there are still significant discrepancies in the calculated absolute values of [Ca2+]m in stimulated live cells. These discrepancies may be due to the distinct properties of the methods used to measure [Ca2+]m, the calcium‐free/bound ratio, and the cell‐type and stimulus‐dependent Ca2+ dynamics. Critical processes happening in the mitochondria, such as ATP generation, ROS homeostasis, and mitochondrial permeability transition opening, depend directly on the [Ca2+]m values. Thus, precise determination of absolute [Ca2+]m values is imperative for understanding Ca2+ signaling. This review summarizes the reported calibrated [Ca2+]m values in many cell types and discusses the discrepancies among these values. Areas for future research are also proposed. [ABSTRACT FROM AUTHOR]

BACKGROUND The recently developed super‐resolution microscopy has been applied to characterize the nanometer‐scale structure of actin filaments in live cells, owing to its live‐cell imaging ability at sub‐diffraction resolution. However, there are still some limitations to existing live‐cell labeling methods for super‐resolution imaging of F‐actin. RESULTS: A series of cell‐permeable fluorescent probes was developed for super‐resolution imaging of actin filaments with the introduction of a cell‐penetrating peptide, (rR)3R2. These probes are highly specific, multicolor available and suitable for different cell lines. With these probes, we resolve actin filaments at spatial resolution of ca 60 nm in live‐cell super‐resolution imaging. CONCLUSIONS: Our results here extensively expand the types of probes for live‐cell super‐resolution imaging of F‐actin and open up a new avenue in the design of cell‐permeable fluorescent probes. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Abstract A nitrogen and sulfur dual-doped carbon nanodots (N,S-CNDs) sample was fabricated by an one-step hydrothermal treatment of acid fuchsin. The obtained N,S-CNDs possess yellow fluorescent, excellent water solubility, high fluorescence stability and religious biocompatibility, which can be used as a di-functional and label-free chem-nanosensor for the determination of pH and curcumin (Cur). When the pH is decreased from 8.0 to 4.4, the N,S-CNDs reveals an extraordinary emission escalation at emission wavelength (λ em) of 543 nm, and the p K a value of the N,S-CNDs was calculated as 6.06. The N,S-CNDs displays a favourable linear relationship in the physiological pH range of 5.0–7.4, which is meaningful for the research of near-neutral cytosolic pH. Furthermore, the laser scanning confocal microscopic images of intracellular distribution and the determination of pH in HeLa cells were successfully carried out, implying that the N,S-CNDs possess excellent cell membrane permeability and are capable of being further applied to real-time pH fluctuations monitoring in live cells with negligible autofluorescence. More importantly, the fluorescence of N,S-CNDs may be dramatically quenched by Cur via a combination of electrostatic and hydrogen-bond interaction and fluorescence inner filter effect (FIFE). The limit of detection is as low as 81 nmol/L Cur, and the linearity range is 0.5–20 μmol/L Cur. Ultimately, a satisfactory result was obtained when applying the as-constructed fluorescent chem-nanosensing system for the analyses of Cur in practical samples. Graphical abstract The as-fabricated nitrogen and sulfur dual-doped carbon nanodots (N,S-CNDs) could serve as a di-functional and label-free chem-nanosensor for real-time monitoring of pH fluctuation in live cells and quantitative determining of Curcumin in real samples. Image 1 Highlights • Nitrogen and sulfur dual-doped carbon nanodots (N,S-CNDs) with yellow fluorescent was fabricated by one-step hydrothermal treatment of acid fuchsin. • "Mostly-Off.→ Partially-On → Entirely-On" fluorescent chem-nanosensor for pH detection by N,S-CNDs. • "Turn-off" fluorescence chem-nanosensor for Cur determination by N,S-CNDs. • Real-time monitoring of pH fluctuation in live cells and quantitative determining of Cur in real samples. [ABSTRACT FROM AUTHOR]

Abstract As an efficient turn-on fluorescent chemosensor for Al3+, a new coumarin derivative (CND) has been designed and synthesized by the condensation of 8-formyl-7-hydroxycoumarin with niacin hydrazide. The spectroscopic studies revealed that the sensor CND exhibited a remarkable fluorescence enhancement towards Al3+ with high selectivity and sensitivity in EtOH-HEPES (95:5, v/v, pH = 7.40), which was attributed to the photoinduced electron transfer (PET) and C N isomerization mechanism. Fluorescence titration calculations data showed that the detection limit and the association constants of CND for Al3+ were found to be 2.51 × 10−7 M and 9.64 × 104 M−1, respectively. The results of experiments, including Job's plot, 1H NMR titration and ESI-MS, revealed that the stoichiometric binding between CND and Al3+ was 1:1. The investigations of the pH dependency of CND for Al3+ detection, and the cell imaging suggested the sensor CND could be promisingly applied for the recognition of Al3+ in biological cells. Graphical Abstract Unlabelled Image Highlights • A novel coumarin-based fluorescence sensor for Al3+ was designed and synthesized. • This sensor has good selectivity and sensitivity for Al3+. • This sensor can be applied in live cells. [ABSTRACT FROM AUTHOR]

Hypochlorous acid (HClO) is a reactive oxygen species (ROS) that plays a critical role in numerous pathological and physiological processes. Abnormal concentrations of HClO in vivo have been closely linked to many diseases, including inflammatory disorders and cancer. In this work, an on-off fluorescent probe PTZ based on phenothiazine was synthesized for specific detection of HClO. PTZ demonstrated excellent selectivity, biocompatibility, rapid response, pH stability, and low limits of detection (5.2 nM). Furthermore, PTZ can detect both exogenous and endogenous HClO rapidly in living cells and zebrafish, which provides it with broad potential applications in life sciences. Rapid and sensitive phenothiazine-based fluorescent probe for detecting endogenous HClO in living cells and zebrafish. [Display omitted] [ABSTRACT FROM AUTHOR]

Sensitive and specific sensing of intracellular telomerase is of significance for early cancer diagnosis and treatment. And, efficient signal amplification technology is a powerful tool to achieve low level biomarker detection. However, the delivery and release of amplification reagents in live cells is currently a major challenge to achieve sensitive intracellular detection of biomarkers. Here, we describe the development of a powerful in-cell amplification approach for label-free and highly sensitive detection of telomerase in live cells via zeolitic imidazolate framework-8 (ZIF-8)-mediated DNAzyme/light-up RNA aptamer transcription amplification cascades. The amplification reagents can be readily transported into and released in the target cells by ZIF-8 under intracellular acidic environment. Telomerase then activates DNAzyme to trigger the DNAzyme-aided cleavage reaction and T7 RNA polymerase-initiated light-up RNA aptamer transcription cycles for the yield of many light-up RNA aptamers, which associate with the non-fluorescent dye to recover significant fluorescence for achieving intracellular telomerase imaging with high sensitivity in a label-free approach. Moreover, the proposed strategy can discriminate telomerase level variations in different cells and shows promising potentials for anticancer drug screening, which makes such method a robust bioassay for early tumor-related diagnosis and therapeutic monitoring. • An in-cell amplification method is established for sensitive label-free imaging of telomerase activity. • The DNAzyme, RNA transcription probes and enzymes are readily delivered into the target cells via biodegradable MOFs. • Telomerase level variation against different drug concentrations is interrogated for potential drug screening. [ABSTRACT FROM AUTHOR]