Bioimaging In Living Cells Research Articles

Rapid discovery and evolution of nanosensors containing fluorogenic amino acids

Binding-activated optical sensors are powerful tools for imaging, diagnostics, and biomolecular sensing. However, biosensor discovery is slow and requires tedious steps in rational design, screening, and characterization. Here we report on a platform that streamlines biosensor discovery and unlocks directed nanosensor evolution through genetically encodable fluorogenic amino acids (FgAAs). Building on the classical knowledge-based semisynthetic approach, we engineer ~15 kDa nanosensors that recognize specific proteins, peptides, and small molecules with up to 100-fold fluorescence increases and subsecond kinetics, allowing real-time and wash-free target sensing and live-cell bioimaging. An optimized genetic code expansion chemistry with FgAAs further enables rapid (~3 h) ribosomal nanosensor discovery via the cell-free translation of hundreds of candidates in parallel and directed nanosensor evolution with improved variant-specific sensitivities (up to ~250-fold) for SARS-CoV-2 antigens. Altogether, this platform could accelerate the discovery of fluorogenic nanosensors and pave the way to modify proteins with other non-standard functionalities for diverse applications.

Rational Design of Cyanine-Based Fluorogenic Dimers to Reduce Nonspecific Interactions with Albumin and Lipid Bilayers: Application to Highly Sensitive Imaging of GPCRs in Living Cells.

Fluorogenic dimers with polarity-sensitive folding are powerful probes for live-cell bioimaging. They switch on their fluorescence only after interacting with their targets, thus leading to a high signal-to-noise ratio in wash-free bioimaging. We previously reported the first near-infrared fluorogenic dimers derived from cyanine 5.5 dyes for the optical detection of G protein-coupled receptors. Owing to their hydrophobic character, these dimers are prone to form nonspecific interactions with proteins such as albumin and with the lipid bilayer of the cell membrane resulting in a residual background fluorescence in complex biological media. Herein, we report the rational design of new fluorogenic dimers derived from cyanine 5. By modulating the chemical structure of the cyanine units, we discovered that the two asymmetric cyanine 5.25 dyes were able to form intramolecular H-aggregates and self-quenched in aqueous media. Moreover, the resulting original dimeric probes enabled a significant reduction of the nonspecific interactions with bovine serum albumin and lipid bilayers compared with the first generation of cyanine 5.5 dimers. Finally, the optimized asymmetric fluorogenic dimer was grafted to carbetocin for the specific imaging of the oxytocin receptor under no-wash conditions directly in cell culture media, notably improving the signal-to-background ratio compared with the previous generation of cyanine 5.5 dimers.

Advances in Organic Fluorescent Probes for Intracellular Zn2+ Detection and Bioimaging.

Zinc ions (Zn2+) play a key role in maintaining and regulating protein structures and functions. To better understand the intracellular Zn2+ homeostasis and signaling role, various fluorescent sensors have been developed that allow the monitoring of Zn2+ concentrations and bioimaging in live cells in real time. This review highlights the recent development of organic fluorescent probes for the detection and imaging of intracellular Zn2+, including the design and construction of the probes, fluorescent response mechanisms, and their applications to intracellular Zn2+ detection and imaging on-site. Finally, the current challenges and prospects are discussed.

Pyridine appended pyrimidine bis hydrazone: Zn2+/ATP detection, bioimaging and functional properties of its dinuclear Zn(II) complex

A pyridine functionalized pyrimidine-based system, H2P was successfully synthesized, characterized, and evaluated for its remarkable selective characteristics towards Zn2+ and ATP ions. The chemical sensing capabilities of H2P were demonstrated through absorption, fluorescence, and NMR spectroscopic techniques. The probe exhibited outstanding sensitivity when interacting with the ions, demonstrating relatively strong association constants and impressively low detection limits. The comprehensive binding mechanism of H2P with respect to Zn2+ and ATP ions was investigated using a combination of analytical methods, including Job's plot, NMR spectroscopy, mass spectrometry, and density functional theory (DFT) experiments. The interesting sensing ability of H2P for Zn2+/ATP ions was harnessed for live cell bioimaging and other diverse on-site detection purposes, including paper strips, cotton swabs, and applications involving mung bean sprouts. Further, the fluorescent probe demonstrated its effectiveness in detecting Zn2+ and ATP within live cells, indicating its significant potential in the realm of biological imaging applications. Moreover, the molecular configuration of the zinc complex (H2P–Zn2Cl4), derived from H2P, was elucidated using X-ray crystallography. This complex exhibited intriguing multifunctional attributes, encompassing its capability for detecting picric acid and for reversible acid/base sensing responses. The enhanced conducting behavior of the complex as well as its resistance properties were investigated by performing I–V characteristics and electrochemical impedance spectroscopic (EIS) experiments respectively.

Photoresponsive CHA-Integrated Self-Propelling 3D DNA Walking Amplifier within the Concentration Localization Effect of DNA Molecular Framework Enables Highly Efficient Fluorescence Bioimaging.

While three-dimensional (3D) DNA walking amplifiers hold considerable promise in the construction of advanced DNA-based fluorescent biosensors for bioimaging, they encounter certain difficulties such as inadequate sensitivity, premature activation, the need for exogenous propelling forces, and low reaction rates. In this contribution, a variety of profitable solutions have been explored. First, a catalytic hairpin assembly (CHA)-achieved nonenzymatic isothermal nucleic acid amplification is integrated to enhance sensitivity. Subsequently, one DNA component is simply functionalized with a photocleavage-bond to conduct a photoresponsive manner, whereby the target recognition occurs only when the biosensor is exposed to an external ultraviolet light source, overcoming premature activation during biodelivery. Furthermore, a special self-propelling walking mechanism is implemented by reducing biothiols to MnO2 nanosheets, thereby propelling forces that are self-supplied to a Mn2+-reliant DNAzyme. By carrying the biosensing system with a DNA molecular framework to induce a unique concentration localization effect, the nucleic acid contact reaction rate is notably elevated by 6 times. Following these, an ultrasensitive in vitro detection performance with a limit of detection down to 2.89 fM is verified for a cancer-correlated microRNA biomarker (miRNA-21). Of particular importance, our multiple concepts combined 3D DNA walking amplifier that enables highly efficient fluorescence bioimaging in live cells and even bodies, exhibiting a favorable application prospect in disease analysis.

Modulation of the binding sites for an adaptable DNA interactive probe: efficient chromo-fluorogenic recognition of Al3+ and live cell bioimaging

Herein, a simple chromone-based biocompatible reversible fluorescent “turn-on” probe, HMCP, is successfully utilized to detect Al3+ over a group of other coexisting metal ions.

A Rhodamine-coumarin Triazole Conjugate as a Fluorescent Chemodosimeter for Cu(II) Detection and its Application in Live Cell Bioimaging.

A rhodamine-triazole fluorescent probe bearing a coumarin moiety RTC was synthesized using the Cu(I)-catalyzed click reaction. The rhodamine-triazole conjugate was highly selective to Cu2+ among other metal ions, including Ca2+, Co2+, Cu2+, Cd2+, Mg2+, Fe2+, Fe3+, Hg2+, Zn2+, Ni2+, Pd2+ and Pb2+ in physiological conditions. Upon the addition of Cu2+, the colorless RTC solution turned pink and exhibited a significant fluorescence emission centered at 578 nm. The binding of Cu2+ induced a hydrolysis reaction, leading to a release of the coumarin unit from the rhodamine probe, as confirmed by mass spectrometric data. From the fluorescence titration, the detection limit of RTC for Cu2+ was determined to be 21 nM (1.3 ppb). The sensor was responsive to Cu2+ in a wide pH range and successfully applied to monitor Cu2+ in HEK293T cells by confocal fluorescence imaging.

Quinazoli-4-one ionic liquid as a fluorescent sensor for NH3 detection: Interaction with ctDNA, theoretical investigation and live cell bioimaging

A novel quinazoli-4-one based ionic liquid, 1-(3-aminopropyl)-3-methyl-4-oxo-3,4-dihydroquinazolin-1-ium bromide (QIL) for fluorometric determination of dissolved ammonia has been successfully synthesized and characterized by spectroscopic techniques such as 1H and 13C NMR, FTIR and HRMS spectrometry. In the proposed method, QIL is converted to a fluorescent derivative by the reaction with ammonia in aqueous medium. The excitation and emission wavelengths were 250 and 436 nm, respectively. Remarkably with the reaction time of >1 s, the binding constant and detection limit was found to be 6.43 × 108 M−1 and 0.73 × 10−8 M, respectively. QIL is found to be highly selective as no interference is observed from various cations, anions, organic molecules and amino acids. The sensing mechanism was further validated by the density functional theory studies. The fluorophore exhibited great sensing property in 3.0–14.0 pH range, hence, it can be employed in diverse matrices. In addition, the fluoro-sensor is highly reversible and reusable in the presence of ctDNA molecule. Moreover, a live-cell imaging study of QIL in Drosophila larval gut tissue has also been carried out to investigate the cell permeability of QIL and its efficiency for selective detection of NH3 in cellular micro environment. To show practical applicability of the fluoro-sensor, test strip kit has been constructed. A detailed comparison table has been shown to evaluate the efficiency of this method.

Self-Stacking Autocatalytic Molecular Circuit with Minimal Catalytic DNA Assembly.

Isothermal autocatalytic DNA circuits have been proven to be versatile and powerful biocomputing platforms by virtue of their self-sustainable and self-accelerating reaction profiles, yet they are currently constrained by their complicated designs, severe signal leakages, and unclear reaction mechanisms. Herein, we developed a simpler-yet-efficient autocatalytic assembly circuit (AAC) for highly robust bioimaging in live cells and mice. The scalable and sustainable AAC system was composed of a mere catalytic DNA assembly reaction with minimal strand complexity and, upon specific stimulation, could reproduce numerous new triggers to expedite the whole reaction. Through in-depth theoretical simulations and systematic experimental demonstrations, the catalytic efficiency of these reproduced triggers was found to play a vital role in the autocatalytic profile and thus could be facilely improved to achieve more efficient and characteristic autocatalytic signal amplification. Due to its exponentially high signal amplification and minimal reaction components, our self-stacking AAC facilitated the efficient detection of trace biomolecules with low signal leakage, thus providing great clinical diagnosis and therapeutic assessment potential.

Visual and quantitative monitoring of thiophenol by a novel deep-red emitting fluorescent probe in environmental and biological systems

Detection of highly toxic thiophenols in biological or environmental systems is of great importance. Therefore, fast, reliable, and sensitive probes are needed to detect thiophenols. Herein, a novel triphenylamine conjugated dicyanoisophorone-based near infrared fluorescence probe is reported to determine trace thiophenol (PhSH) levels. The probe demonstrates a distinct “turn-on” fluorescence response to thiophenol among the tested analytes and its quantum yield (Φ) increases from 0.011 to 0.142. It has low cytotoxicity with cell viability of 90–100% up to 10.0 μM of the probe, a strong anti-interference capability, a large Stokes shift (150 nm), and a fast response time (<1 min). In addition, the probe exhibits a good linear response to PhSH over the range from 0 to 15.0 μM with a detection limit of 32.3 nM (R2 = 0.9978). The detection process is also confirmed through HPLC. The practical applicability of the probe is proved by a smartphone platform, TLC kit, plant tissue imaging, soil assay, tap, and lake water analysis with good recovery values (92.3–117%), and concentration-dependent live cell bioimaging PhSH from 5.0 to 15.0 μM. Therefore, the present probe is a robust candidate for monitoring PhSH levels in biological and environmental systems.

Highly sensitive and rapid detection of hypochlorous acid in aqueous media and its bioimaging in live cells and zebrafish using an ESIPT-driven mycophenolic acid-based fluorescent probe.

Excessive production of potent biological oxidants such as HOCl has been implicated in numerous diseases. Thus, it is crucial to develop highly specific and precise methods to detect HOCl in living systems, preferably with molecules that can show a distinct therapeutic effect. Our study introduces the synthesis and application of a highly sensitive fluorescence "turn-on" probe, Myco-OCl, based on the mycophenolic acid scaffold with exceptional water solubility. The ESIPT-driven mechanism enables Myco-OCl to specifically and rapidly detect (<5 s) HOCl with an impressive Stokes shift of 105 nm (λex = 417 nm, λem = 522 nm) and a sub-nanomolar (97.3 nM) detection limit with the detection range of 0 to 50 μM. The potential of Myco-OCl as an excellent biosensor is evident from its successful application for live cell imaging of exogenous and endogenous HOCl. In addition, Myco-OCl enabled us to detect HOCl in a zebrafish inflammatory animal model. These underscore the great potential of Myco-OCl for detecting HOCl in diverse physiological systems. Our findings thus offer a highly promising tool for detecting HOCl in living organisms.

A ratiometric fluorescent dye for detection of Lys and Arg and its bioimaging in live cells and zebrafish larvae.

A ratiometric and pH-sensitive fluorescent dye named IDE was applied to the detection of argine and lysine from common amino acids and exploited to monitor the Lys and Arg levels in living cells and zebrafish larvae successfully. IDE will be a useful fluorescence indicator of pH changes by Lys and Arg.

Photocontrollable Fluorescence Imaging of Mitochondrial Peroxynitrite during Ferroptosis with High Fidelity.

Ferroptosis, a new regulatory cell death modality, underlies the pathogenesis of a broad range of disorders. Although much efforts have been made to uncover the molecular mechanisms, some mechanistic details of ferroptosis still remain poorly understood. Particularly, the functional relevance of mitochondrial reactive oxygen species (ROS) in ferroptosis is still highly controversial, which is partially due to the fact that it still remains puzzled how the mitochondrial ROS level varies during ferroptosis. The conventional mitochondria-targeted probes may react with cytosolic ROS and show fluorescence variation before entering mitochondria, thus probably giving a false result on the mitochondrial ROS level and leading to the misjudgment on its biofunction. To circumvent this issue, we rationally designed a photocontrollable and mitochondria-targeted fluorescent probe to in situ visualize the mitochondrial peroxynitrite (ONOO-), which is the ROS member and mediator of ferroptosis. The photoactivated probe was endowed with a highly specific and sensitive fluorescence response to ONOO-. Notably, the response activity could be artificially regulated with light irradiation, which ensured that all the probe molecules passed through the cytosol in the locked status and were then photoactivated after reaching mitochondria. This photocontrolled fluorescence imaging strategy eliminated the interference of ONOO- outside the mitochondria, thus potentially afforded improved fidelity for mitochondrial ONOO- bioimaging in live cells and animal models. With this probe, for the first time, we revealed the mitochondrial ONOO- flux and its probable biological source during erastin-induced ferroptosis. These results suggest a tight correlation between mitochondrial ONOO-/ROS and ferroptotic progression, which will further facilitate the comprehensive exploration and manipulation of ferroptosis.

Amphiphilicity-Controlled Localization of Red Emitting Bicationic Fluorophores in Tumor Cells Acting as Bio-Probes and Anticancer Drugs.

Small organic molecules arouse lively interest for their plethora of possible biological applications, such as anticancer therapy, for their ability to interact with nucleic acids, or bioimaging, thanks to their fluorescence emission. Here, a panchromatic series of styryl-azinium bicationic dyes, which have already proved to exhibit high water-solubility and significant red fluorescence in water, were investigated through spectrofluorimetric titrations to assess the extent of their association constants with DNA and RNA. Femtosecond-resolved transient absorption spectroscopy was also employed to characterize the changes in the photophysical properties of these fluorophores upon interaction with their biological targets. Finally, in vitro experiments conducted on tumor cell lines revealed that some of the bicationic fluorophores had a peculiar localization within cell nuclei exerting important antiproliferative effects, others were instead found to localize in the cytoplasm without leading to cell death, being useful to mark specific organelles in light of live cell bioimaging. Interestingly, this molecule-dependent behavior matched the different amphiphilicity featured by these bioactive compounds, which are thus expected to be caught in a tug-of-war between lipophilicity, ensured by the presence of aromatic rings and needed to pass cell membranes, and hydrophilicity, granted by charged groups and necessary for stability in aqueous media.

Silicon-Vacancy Nanodiamonds as High Performance Near-Infrared Emitters for Live-Cell Dual-Color Imaging and Thermometry.

Nanodiamonds (NDs) with color centers are excellent emitters for various bioimaging and quantum biosensing applications. In our work, we explore new applications of NDs with silicon-vacancy centers (SiV) obtained by high-pressure high-temperature (HPHT) synthesis based on metal-catalyst-free growth. They are coated with a polypeptide biopolymer, which is essential for efficient cellular uptake. The unique optical properties of NDs with SiV are their high photostability and narrow emission in the near-infrared region. Our results demonstrate for the first time that NDs with SiV allow live-cell dual-color imaging and intracellular tracking. Also, intracellular thermometry and challenges associated with SiV atomic defects in NDs are investigated and discussed for the first time. NDs with SiV nanoemitters provide new avenues for live-cell bioimaging, diagnostic (SiV as a nanosized thermometer), and theranostic (nanodiamonds as drug carrier) applications.

Phosphorogenic Iridium(III) bis‐Tetrazine Complexes for Bioorthogonal Peptide Stapling, Bioimaging, Photocytotoxic Applications, and the Construction of Nanosized Hydrogels

AbstractThe dual functionality of 1,2,4,5‐tetrazine as a bioorthogonal reactive unit and a luminescence quencher has shaped tetrazine‐based probes as attractive candidates for luminogenic labeling of biomolecules in living systems. In this work, three cyclometalated iridium(III) complexes featuring two tetrazine units were synthesized and characterized. Upon photoexcitation, the complexes were non‐emissive but displayed up to 3900‐fold emission enhancement upon the inverse electron‐demand Diels–Alder (IEDDA) [4+2] cycloaddition with (1R,8S,9s)‐bicyclo[6.1.0]non‐4‐yne (BCN) substrates. The rapid reaction kinetics (k2 up to 1.47×104 M−1 s−1) of the complexes toward BCN substrates allowed effective peptide labeling. The complexes were also applied as live cell bioimaging reagents and photocytotoxic agents. One of the complexes was utilized in the preparation of luminescent nanosized hydrogels that exhibited interesting cargo delivery properties.

Phosphorogenic Iridium(III) bis-Tetrazine Complexes for Bioorthogonal Peptide Stapling, Bioimaging, Photocytotoxic Applications, and the Construction of Nanosized Hydrogels.

The dual functionality of 1,2,4,5-tetrazine as a bioorthogonal reactive unit and a luminescence quencher has shaped tetrazine-based probes as attractive candidates for luminogenic labeling of biomolecules in living systems. In this work, three cyclometalated iridium(III) complexes featuring two tetrazine units were synthesized and characterized. Upon photoexcitation, the complexes were non-emissive but displayed up to 3900-fold emission enhancement upon the inverse electron-demand Diels-Alder (IEDDA) [4+2] cycloaddition with (1R,8S,9s)-bicyclo[6.1.0]non-4-yne (BCN) substrates. The rapid reaction kinetics (k2 up to 1.47×104 M-1 s-1 ) of the complexes toward BCN substrates allowed effective peptide labeling. The complexes were also applied as live cell bioimaging reagents and photocytotoxic agents. One of the complexes was utilized in the preparation of luminescent nanosized hydrogels that exhibited interesting cargo delivery properties.

Multiple heteroatom-doped photoluminescent carbon dots for ratiometric detection of Hg2+ ions in cell imaging and environmental applications.

Photoluminescence detection and imaging of Hg2+ ions in the biochemical living system are of great importance. In this study, a new photoluminescent probe based on nitrogen (N), sulfur (S), and boron (B) multiple heteroatom co-doped carbon dots (NSB-CDs) is synthesized for the ratiometric detection of Hg2+ ions. The prepared NSB-CDs possess good aqueous solubility, excellent pH and ionic stability, excitation dependency, and high quantum yield (QY = 17.6%). The ratiometric photoluminescent sensor NSB-CDs exhibit high selectivity, sensitivity, and interference towards Hg2+ ions over other metal ions. After adding Hg2+ ions, the emission intensity of the NSB-CDs exhibits a large redshift from 452 to 496 nm (up to 44 nm), corresponding to a notable change from blue to green emission in aqueous solutions. The association constant (Ka), the limit of detection (LOD), and the limit of quantification (LOQ) for NSB-CDs/Hg2+ complex are calculated to be 3.6 × 104 M-1, 3.1 × 10-9 M, and 10.4 × 10-9 M, respectively, in the range of 0-30 × 10-6 M. The live cell bioimaging of HCT-116 cells with NSB-CDs validates the application of multicolor imaging for the detection of Hg2+ ions in aqueous media and biological systems. Moreover, the potential use of the NSB-CDs/Hg2+ complex for real sample analysis is demonstrated.

Dual-Locked Near-Infrared Fluorescent Probes for Precise Detection of Melanoma via Hydrogen Peroxide-Tyrosinase Cascade Activation.

Activity-based near-infrared (NIR) fluorescent probes provide powerful tools for diagnosis of diseases. However, most of these probes suffer from low specificity due to "off-target" reaction. The dual-locked strategy, which utilizes two biomarkers as triggers, can increase the specificity and precision of diagnosis. Here, we report a dual-locked NIR probe, MB-m-borate, which releases fluorophore methylene blue (MB) after hydrogen peroxide-tyrosinase (H2O2-TYR) cascade activation. Both MB-m-borate and its intermediate MB-m-phenol (the product after H2O2 activation) show almost nondetectable fluorescence. MB-m-borate exhibits "turn on" fluorescence upon H2O2-TYR cascade activation. The further live cell bioimaging results indicate that MB-m-borate only responds to melanoma cells, providing it as a robust probe for precise detection of melanoma. Finally, the probe is applied for the diagnosis of melanoma in vivo with a xenogeneic mouse model.

"Covalent-Assembly"-Triggered Striking Far-Red to near-Infrared Emitting Fluorescent Probe for Abrupt Detection of Nerve-Agent Mimic (DCP): Real Time Application in Monitoring the Presence of Trace Amounts in Soil and Live Cells.

Detection of chemical warfare agents (CWA) by simple and rapid methods with real-sample applications are quite inevitable in order to ease the threats to living systems caused by uncertain terror attacks and wars. Herein we have developed the first far-red to near infra-red (NIR) probe based on a covalent assembly approach for the detection of trace amounts of nerve agent mimic diethyl chloro phosphate (DCP) in soil and their fluorescent bio imaging in live cells. The probe features abrupt fluorescence turn on sensing of DCP with fluorescence quantum yield Φ = 0.622. It senses DCP selectively over other analytes in excellent sensitivity with a detection limit of 6.9 nM. In real time, the probe treated strips were employed to detect the DCP vapor effectively with eye catching fluorescence response. The presence of trace amounts of these acute warfare agents in the environment were monitored by soil analysis. Further fluorescent bio imaging was carried out to monitor trace level DCP in living cells using the HeLa cell line.