Acid In Living Cells Research Articles

Development of an activatable far-red fluorescent probe for rapid visualization of hypochlorous acid in live cells and mice with neuroinflammation.

Recent investigations have suggested that abnormally elevated levels of HOCl may be tightly related to the severity of neuroinflammation. Although some successes have been achieved, fluorescent probes with far-red fluorescence emission and capable of detecting HOCl with high specificity in pure aqueous solution are still urgently needed. Herein, a responsive far-red fluorescent probe, DCI-H, has been constructed to monitor HOCl activity in vivo and in vitro. DCI-H could rapidly respond to HOCl within 120s and had a low detection limit for HOCl of 1.5nM. Importantly, physiologically common interfering species, except for HOCl, did not cause a change in the fluorescence intensity of DCI-HOCl at 655nm. The results of confocal imaging demonstrated the ability of DCI-H to visualize endogenous HOCl produced by MPO-catalyzed H2O2/Cl- and LPS stimulation. With the assistance of DCI-H, upregulation of HOCl levels was observed in the mice model of LPS-induced neuroinflammation. Thus, we believed that DCI-H provided a valuable tool for HOCl detection and diagnosis of inflammation-related diseases.

Structural details of a Class B GPCR-arrestin complex revealed by genetically encoded crosslinkers in living cells

Understanding the molecular basis of arrestin-mediated regulation of GPCRs is critical for deciphering signaling mechanisms and designing functional selectivity. However, structural studies of GPCR-arrestin complexes are hampered by their highly dynamic nature. Here, we dissect the interaction of arrestin-2 (arr2) with the secretin-like parathyroid hormone 1 receptor PTH1R using genetically encoded crosslinking amino acids in live cells. We identify 136 intermolecular proximity points that guide the construction of energy-optimized molecular models for the PTH1R-arr2 complex. Our data reveal flexible receptor elements missing in existing structures, including intracellular loop 3 and the proximal C-tail, and suggest a functional role of a hitherto overlooked positively charged region at the arrestin N-edge. Unbiased MD simulations highlight the stability and dynamic nature of the complex. Our integrative approach yields structural insights into protein-protein complexes in a biologically relevant live-cell environment and provides information inaccessible to classical structural methods, while also revealing the dynamics of the system.

A big blue-shift phenanthroline fluorescent probe with for detecting hypochlorous acid in live cells

A small-molecule reactive oxygen species (ROS) called hypochlorous acid or hypochlorite (HClO/ClO−) plays important roles in both healthy physiological functions and the development of numerous illnesses. On the basis of the imidazo-1,10-phenanthroline skeleton, we have created a new probe for the detection of ClO−. The novel probe demonstrated high selectivity and sensitivity for the detection of ClO−. While other investigated analytes failed to elicit the fluorescence augmentation, the emission spectra after treatment with ClO− showed a dramatic alteration with an approximately 4-fold increase in fluorescence. The emission spectrum is also blue-shifted with a sizable Stokes shift (from 520 nm to 418 nm). In particular, the probe had a fast response time to ClO− (2 min) and a detection limit of up to 0.011 M. The probe could also capture images of HClO in the living cells. The above extraordinary properties illustrated that the probe was able to determine HClO in the living cells.

Color-Switchable Nanosilicon Fluorescent Probes.

Fluorescent probes are vital to cell imaging by allowing specific parts of cells to be visualized and quantified. Color-switchable probes (CSPs), with tunable emission wavelength upon contact with specific targets, are particularly powerful because they not only eliminate the need to wash away all unbound probe but also allow for internal controls of probe concentrations, thereby facilitating quantification. Several such CSPs exist and have proven very useful, but not for all key cellular targets. Here we report a pioneering CSP for in situ cell imaging using aldehyde-functionalized silicon nanocrystals (SiNCs) that switch their intrinsic photoluminescence from red to blue quickly when interacting with amino acids in live cells. Though conventional probes often work better in cell-free extracts than in live cells, the SiNCs display the opposite behavior and function well and fast in universal cell lines at 37 °C while requiring much higher temperature in extracts. Furthermore, the SiNCs only disperse in cytoplasm not nucleus, and their fluorescence intensity correlated linearly with the concentration of fed amino acids. We believe these nanosilicon probes will be promising tools to visualize distribution of amino acids and potentially quantify amino acid related processes in live cells.

Live-Cell Imaging of Sterculic Acid-a Naturally Occurring 1,2-Cyclopropene Fatty Acid-by Bioorthogonal Reaction with Turn-On Tetrazine-Fluorophore Conjugates.

In the field of lipid research, bioorthogonal chemistry has made the study of lipid uptake and processing in living systems possible, whilst minimising biological properties arising from detectable pendant groups. To allow the study of unsaturated free fatty acids in live cells, we here report the use of sterculic acid, a 1,2‐cyclopropene‐containing oleic acid analogue, as a bioorthogonal probe. We show that this lipid can be readily taken up by dendritic cells without toxic side effects, and that it can subsequently be visualised using an inverse electron‐demand Diels–Alder reaction with quenched tetrazine‐fluorophore conjugates. In addition, the lipid can be used to identify changes in protein oleoylation after immune cell activation. Finally, this reaction can be integrated into a multiplexed bioorthogonal reaction workflow by combining it with two sequential copper‐catalysed Huisgen ligation reactions. This allows for the study of multiple biomolecules in the cell simultaneously by multimodal confocal imaging.

Live‐Cell Imaging of Sterculic Acid—a Naturally Occurring 1,2‐Cyclopropene Fatty Acid—by Bioorthogonal Reaction with Turn‐On Tetrazine‐Fluorophore Conjugates**

AbstractIn the field of lipid research, bioorthogonal chemistry has made the study of lipid uptake and processing in living systems possible, whilst minimising biological properties arising from detectable pendant groups. To allow the study of unsaturated free fatty acids in live cells, we here report the use of sterculic acid, a 1,2‐cyclopropene‐containing oleic acid analogue, as a bioorthogonal probe. We show that this lipid can be readily taken up by dendritic cells without toxic side effects, and that it can subsequently be visualised using an inverse electron‐demand Diels–Alder reaction with quenched tetrazine‐fluorophore conjugates. In addition, the lipid can be used to identify changes in protein oleoylation after immune cell activation. Finally, this reaction can be integrated into a multiplexed bioorthogonal reaction workflow by combining it with two sequential copper‐catalysed Huisgen ligation reactions. This allows for the study of multiple biomolecules in the cell simultaneously by multimodal confocal imaging.

Graphene quantum dots-based nanocomposite for electrocatalytic application of L-cysteine in whole blood and live cells

Driven by the urgent need to detect amino acids in live cells at trace levels, as they are an important biomarker for various diseases. For this purpose, we have developed a nanomolar level detection of l-cysteine (L-Cys) and real-time cellular quantification from live cells based on graphene quantum dots (GQDs)-AuNPs-metal-free organic framework (MFOF). Different analytical and microscopic techniques were used to confirm the synthesized nanocomposite. The sensitive electrochemical sensing system of l-Cys-was detected synergistically with GQDs-AuNPs-MFOF/GCE by voltammetry and amperometric i-t curve. The electrocatalytic oxidation signal of l-Cys-at Epa = none, +0.45, +0.36 and +0.21 V (vs. Ag/AgCl) with enhanced current responses were observed at control GCE, MFOF, GQDs-MFOF, and GQDs-AuNPs-MFOF/GCEs by DPV. The nanocomposite modified sensor probe showed a wide linear range concentration from 1 nM to 0.5 mM with LOD of 0.124 nM (S/N = 3), LOQ of 0.863 nM (S/N = 10) and sensitivity of 2.364 μA μM−1 cm−2. Subsequently, the selective detection of l-Cys-in the presence of possible other interfering biothiols. amino acids, and metal ions were achieved. The practical application was demonstrated in human whole blood and bio-fluid samples. Finally, the endogenous real-time detection of cellular cysteine in live cells was demonstrated.

A mitochondria-tracing fluorescent probe for real-time detection of mitochondrial dynamics and hypochlorous acid in live cells

Mitochondrial dynamics and hypochlorous acid (HOCl) are involved in many physiological and pathological processes. Therefore, the development of real-time analysis tools for biological studies involving mitochondrial dynamics and HOCl is highly significant. Herein, we report a fluorescent probe, RIC, consisting of rhodamine, imidazolium salt, and coumarin, which has high selectivity and sensitivity toward HOCl. RIC has an inherently blue fluorescence signal and can be anchored in mitochondria, which enables it to reveal changes in mitochondrial dynamics. This mitochondria-anchoring characteristic also facilitates the real-time detection of HOCl in mitochondria. To our knowledge, RIC is the first probe to detect mitochondrial dynamics and HOCl in real time. The detection limit of HOCl is as low as 3.8 nM, and RIC analyzes endogenous and exogenous HOCl in live cells. The detection capacity is pH-insensitive in the physiological pH range of mitochondria, and the HOCl-sensing process is completed within seconds in cuvettes. Furthermore, we prove that the molecular design strategy provides a general synthetic method for constructing new multifunctional probes for HOCl.

Hypocrates is a genetically encoded fluorescent biosensor for (pseudo)hypohalous acids and their derivatives

The lack of tools to monitor the dynamics of (pseudo)hypohalous acids in live cells and tissues hinders a better understanding of inflammatory processes. Here we present a fluorescent genetically encoded biosensor, Hypocrates, for the visualization of (pseudo)hypohalous acids and their derivatives. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from Escherichia coli. We show that Hypocrates is ratiometric, reversible, and responds to its analytes in the 106 M−1s−1 range. Solving the Hypocrates X-ray structure provided insights into its sensing mechanism, allowing determination of the spatial organization in this circularly permuted fluorescent protein-based redox probe. We exemplify its applicability by imaging hypohalous stress in bacteria phagocytosed by primary neutrophils. Finally, we demonstrate that Hypocrates can be utilized in combination with HyPerRed for the simultaneous visualization of (pseudo)hypohalous acids and hydrogen peroxide dynamics in a zebrafish tail fin injury model.

A simple p-methylaminophenylether-based fluorescent probe for detecting native hypochlorous acid in live cells and zebrafish

As one of the reactive oxygen species (ROS), hypochlorous acid (HOCl) plays a key role in the pathogenesis of a range of diseases. The detection of HOCl has been a hot research topic. Herein, this paper proposes a new conjugate of 4-hydroxy-1,8-naphthalimide and p-methylaminophenylether as fluorescent probe HNPE with simple synthesis and excellent performances to detect HOCl. The probe has a good linear relationship with HOCl (0–1 μM), and its detection limit is 6.56 nM. Moreover, this probe has good selectivity, which can distinguish HOCl from other common analytes. Importantly, the probe HNPE has some achievements in HOCl imaging of living cells and zebrafish. Hence, the probe HNPE is competent as a tool to visualize HOCl in biosystems.

Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor.

Isoleucine is one of the branched chain amino acids that plays a major role in the energy metabolism of human beings and animals. However, detailed investigation of specific receptors for isoleucine has not been carried out because of the non-availability of a tool that can monitor the metabolic flux of this amino acid in live cells. This study presents a novel genetically-encoded nanosensor for real-time monitoring of isoleucine in living cells. This nanosensor was developed by sandwiching a periplasmic binding protein (LivJ) of E. coli between a fluorescent protein pair, ECFP (Enhanced Cyan Fluorescent Protein), and Venus. The sensor, named GEII (Genetically Encoded Isoleucine Indicator), was pH stable, isoleucine-specific, and had a binding affinity (Kd) of 63 ± 6 μM. The GEII successfully performed real-time monitoring of isoleucine in bacterial and yeast cells, thereby, establishing its bio-compatibility in monitoring isoleucine in living cells. As a further enhancement, in silico random mutagenesis was carried out to identify a set of viable mutations, which were subsequently experimentally verified to create a library of affinity mutants with a significantly expanded operating range (96 nM–1493 μM). In addition to its applicability in understanding the underlying functions of receptors of isoleucine in metabolic regulation, the GEII can also be used for metabolic engineering of bacteria for enhanced production of isoleucine in animal feed industries.

Enzymatic Labeling of Bacterial Proteins for Super-resolution Imaging in Live Cells

Methods that enable the super-resolution imaging of intracellular proteins in live bacterial cells provide powerful tools for the study of prokaryotic cell biology. Photoswitchable organic dyes exhibit many of the photophysical properties needed for super-resolution imaging, including high brightness, photostability, and photon output, but most such dyes require organisms to be fixed and permeabilized if intracellular targets are to be labeled. We recently reported a general strategy for the chemoenzymatic labeling of bacterial proteins with azide-bearing fatty acids in live cells using the eukaryotic enzyme N-myristoyltransferase. Here we demonstrate the labeling of proteins in live Escherichia coli using cell-permeant bicyclononyne-functionalized photoswitchable rhodamine spirolactams. Single-molecule fluorescence measurements on model rhodamine spirolactam salts show that these dyes emit hundreds of photons per switching event. Super-resolution imaging was performed on bacterial chemotaxis proteins Tar and CheA and cell division proteins FtsZ and FtsA. High-resolution imaging of Tar revealed a helical pattern; imaging of FtsZ yielded banded patterns dispersed throughout the cell. The precision of radial and axial localization in reconstructed images approaches 15 and 30 nm, respectively. The simplicity of the method, which does not require redox imaging buffers, should make this approach broadly useful for imaging intracellular bacterial proteins in live cells with nanometer resolution.

Quantum Dot Nanobeacons for Single RNA Labeling and Imaging.

Detection and imaging RNAs in live cells is in high demand. Methodology for such a purpose is still a challenge, particularly for single RNA detection and imaging in live cells. In this study, a type of quantum dot (QD) nanobeacon with controllable valencies was constructed by precisely conjugating the black hole quencher (BHQ1) and phosphorothioate comodified DNA onto CdTe:Zn2+ QDs via a one-pot hydrothermal method. The nanobeacon with only one conjugated DNA was used to label and detect low-abundance nucleic acids in live cells, and single HIV-1 RNAs were detected and imaged in live HIV-1 integrated cells. Additionally, QD nanobeacon-labeled HIV-1 genomic RNAs were encapsulated in progeny viral particles, which can be used to track the uncoating process of single viruses. The current study provides a platform for nucleic acid labeling and imaging with high sensitivity, being especially meaningful for tracking of individual RNAs in live cells.

Dual-Reactivity trans-Cyclooctenol Probes for Sulfenylation in Live Cells Enable Temporal Control via Bioorthogonal Quenching.

Sulfenylation (RSH → RSOH) is a post-translational protein modification associated with cellular mechanisms for signal transduction and the regulation of reactive oxygen species. Protein sulfenic acids are challenging to identify and study due to their electrophilic and transient nature. Described here are sulfenic acid modifying trans-cycloocten-5-ol (SAM-TCO) probes for labeling sulfenic acid functionality in live cells. These probes enable a new mode of capturing sulfenic acids via transannular thioetherification, whereas "ordinary" trans-cyclooctenes react only slowly with sulfenic acids. SAM-TCOs combine with sulfenic acid forms of a model peptide and proteins to form stable adducts. Analogously, SAM-TCO with the selenenic acid form of a model protein leads to a selenoetherification product. Control experiments illustrate the need for the transannulation process coupled with the activated trans-cycloalkene functionality. Bioorthogonal quenching of excess unreacted SAM-TCOs with tetrazines in live cells provides both temporal control and a means of preventing artifacts caused by cellular-lysis. A SAM-TCO biotin conjugate was used to label protein sulfenic acids in live cells, and subsequent quenching by tetrazine prevented further labeling even under harshly oxidizing conditions. A cell-based proteomic study validates the ability of SAM-TCO probes to identify and quantify known sulfenic acid redox proteins as well as targets not captured by dimedone-based probes.

Novel fluorescent probe with a bridged Si-O-Si bond for the reversible detection of hypochlorous acid and biothiol amino acids in live cells and zebrafish.

Herein, we report the design of a novel fluorescent probe consisting of a naphthalimide fluorophore and a silicone small molecule for the reversible detection of hypochlorous acid and biothiol amino acids. The response mechanism of BSi-1 is based on the concept of the S-based oxidation/reduction. The probe was found to be suitable for imaging HOCl in HeLa, RAW 264.7 cells and zebrafish, demonstrating its utility in biological applications.

Front Cover: Responsive Fluorescence Probe for Selective and Sensitive Detection of Hypochlorous Acid in Live Cells and Animals (Chem. Asian J. 18/2018)

Front Cover: Responsive Fluorescence Probe for Selective and Sensitive Detection of Hypochlorous Acid in Live Cells and Animals (Chem. Asian J. 18/2018)

Responsive Fluorescence Probe for Selective and Sensitive Detection of Hypochlorous Acid in Live Cells and Animals.

The development of effective bioanalytical methods for rapid, sensitive and specific detection of HOCl in vitro and in vivo plays a key role for better understanding the roles of this molecule in normal and diseased conditions, but remains challenging due to the highly reactive nature of HOCl and the complicated biological conditions. In this work, a new fluorescence probe, PQI, was developed for monitoring of the HOCl level in biological samples. PQI was easily synthesized by a one-step condensation reaction. Upon addition of HOCl, significant changes in the absorption spectra and the color of the solution were noticed, facilitating the "naked eye" detection of HOCl in PBS buffer. The fluorescence of PQI was found to be significantly increased within a few seconds, leading to "OFF-ON" fluorescence response towards HOCl. The sensing mechanism, oxidation of thioether by HOCl, was confirmed by HRMS titration analysis. PQI features a large Stokes shift, high sensitivity and selectivity, and rapid fluorescence response towards HOCl. Quantitative detection of HOCl in single live cells was demonstrated through fluorescence imaging and flow cytometry analysis. PQI was then successfully used in visualisation of HOCl in live zebrafish and nude mice.

Boosting the turn-on fluorescent signaling ability of thiazole orange dyes: The effectiveness of structural modification site and its unusual interaction behavior with nucleic acids

New fluorescent dyes derivatized on a classical thiazole orange framework are able to show unexpectedly strong interaction signal and unusual binding selectivity with different structures of nucleic acid, particularly when bound with double-stranded DNA or G-quadruplex DNA. The present study reveals that these small binding ligands simply bearing an additional amino side group on its parent molecule of thiazole orange have almost no background fluorescence in solution. Conversely, they are able to produce an extremely strong yellow emission signal upon interaction with targeting nucleic acids in live cells. The induced fluorescence intensity was approximately 10–15 times stronger than that of thiazole orange. The dyes are therefore excellent fluorescent stains for bio-sensing and bio-imaging applications. It is particularly suitable for fluorescence microscopy experiments requiring very low working concentration (0.25 μM or less) targeting nucleic acids. This was demonstrated using pu27 G-quadruplex DNA, which has a low limit of detection (LOD = 3–4 nM) while thiazole orange was much higher (LOD = 48.7 nM) under the same conditions. In addition, it was found that structural modification on the quinolinium scaffold of thiazole orange was less effective than modification on the benzothiazole moiety. The findings of the present study provide important information for structural advancement of small molecules based on the widely used thiazole orange skeleton, resulting in analogues that are able to achieve higher sensitivity and selectivity for targeting at a specific class of nucleic acids. Computational docking studies were also conducted to illustrate the interaction behaviors of the dyes with different DNA structures.

A two-photon fluorescent probe for ratiometric visualization of hypochlorous acid in live cells and animals based on a selenide oxidation/elimination tandem reaction.

Utilizing the oxidation/elimination tandem reaction of the α-phenylseleno carbonyl moiety, a two-photon fluorescent probe for ratiometric visualization of hypochlorous acid was developed. Its superior sensing performance and practical applications were well demonstrated.

Water solubility is essential for fluorescent probes to image hypochlorous acid in live cells.

We synthesised four probes and compared their HClO-detecting ability in different solvents. The data showed that only hydrophilic probes could sensitively and accurately detect HClO in live cells. Meanwhile, the addition of organic solvents, as is commonly practised, weakens the oxidising capacity of HClO and thus generates inaccurate outcomes.