Real-time imaging of cell-surface proteins with antibody-based fluorogenic probes

Cell-surface proteins, working as key agents in various diseases, are the targets for around 66% of approved human drugs. A general strategy to selectively detect these proteins in a real-time manner is expected to facilitate the development of new drugs and medical diagnoses. Although brilliant successes were attained using small-molecule probes, they could cover a narrow range of targets due to the lack of suitable ligands and some of them suffer from selectivity issues. We report herein an antibody-based fluorogenic probe prepared via a two-step chemical modification under physiological conditions, to fulfill the selective recognition and wash-free imaging of membrane proteins, establishing a modular strategy with broad implications for biochemical research and for therapeutics.

A modular strategy to convert commercially available antibodies into fluorogenic probes has been developed, enabling selective recognition and wash-free imaging of endogenous membrane proteins.      

Near-infrared fluorescence activation probes based on disassembly-induced emission cyanine dye

Currently most of the fluorogenic probes are designed for the detection of enzymes which work by converting the non-fluorescence substrate into the fluorescence product via an enzymatic reaction. On the other hand, the design of fluorogenic probes for non-enzymatic proteins remains a great challenge. Herein, we report a general strategy to create near-IR fluorogenic probes, where a small molecule ligand is conjugated to a novel γ-phenyl-substituted Cy5 fluorophore, for the selective detection of proteins through a non-enzymatic process. Detail mechanistic studies reveal that the probes self-assemble to form fluorescence-quenched J-type aggregate. In the presence of target analyte, bright fluorescence in the near-IR region is emitted through the recognition-induced disassembly of the probe aggregate. This Cy5 fluorophore is a unique self-assembly/disassembly dye as it gives remarkable fluorescence enhancement. Based on the same design, three different fluorogenic probes were constructed and one of them was applied for the no-wash imaging of tumor cells for the detection of hypoxia-induced cancer-specific biomarker, transmembrane-type carbonic anhydrase IX.     

Real-time quantification of nuclear RNA export using an intracellular relocation probe

Nuclear RNA export into the cytoplasm is one of the key steps in protein expression to realize biological functions. Despite the broad availability of nucleic acid dyes, tracking and quantifying the highly dynamic process of RNA export in live cells is challenging. When dye-labeled RNA enters the cytoplasm, the dye molecules are released upon degradation of the RNA, allowing them to re-enter the cell nucleus. As a result, the ratio between the dye exported with RNA into the cytoplasm and the portion staying inside the nucleus cannot be determined. To address this common limitation, we report the design of a smart probe that can only check into the nucleus once. When adding to cells, this probe rapidly binds with nuclear RNAs in live cells and reacts with intrinsic H₂S. This reaction not only activates the fluorescence for RNA tracking but also changes the structure of probe and consequently its intracellular localization. After disassociating from exported RNAs in cytoplasm, the probe preferentially enters lysosomes rather than cell nucleus, enabling real-time quantitative measurement of nuclear RNA exports. Using this probe, we successfully evaluated the effects of hormones and cancer drugs on nuclear RNA export in live cells. Interestingly, we found that hormones inhibiting RNA exports can partially offset the effect of chemotherapy.     

Development of a water-soluble 3-formylBODIPY dye for fluorogenic sensing and cell imaging of sulfur dioxide derivatives

A new water-soluble, highly fluorogenic 3-formylBODIPY dye that enables the sensing of SO₂ derivatives in aqueous buffers and cancer cells is reported. The quaternary ammonium group appended through the meso-position of the BODIPY dye ensures water solubility. The probe exhibits high specificity for cytosolic (bi)sulfites and fluoresces brightly in human lung adenocarcinoma cells (A549).     

SNAP-tag蛋白标签技术与荧光分析法在蛋白原位分析中的联合应用

为将生物体内微观的蛋白行为可视化并以宏观信号呈现出来对蛋白进行实时、动态分析，借助SNAP-tag蛋白标签技术与有机小分子荧光染料，构建了一系列用于活细胞内实时监测目标蛋白的免洗荧光探针。标签蛋白SNAP-tag能够特异性识别探针中的苄基鸟嘌呤，从而使目标蛋白共价连接上荧光团（萘酰亚胺），携带上荧光信使。此外，由于萘酰亚胺从水环境中被牵引至SNAP-tag蛋白的疏水空腔，其荧光信号呈现出2~13倍的增强。通过SNAP-tag标签蛋白与目标蛋白的融合，该荧光探针实现了对活细胞内线粒体蛋白CoX8A及核内蛋白H2B特异性识别，在免洗条件下完成了对目标蛋白的实时追踪及原位分析。     

An NIR Fluorescence Turn-on and MRl Bimodal Probe for Concurrent Real-time in vivo Sensing and Labeling of β-Galactosidase

To realize sensing and labeling biomarkers is quite challenging in terms of designing multimodal imaging probes. In this study, we developed a novel β-galactosidase (β-gal) activated bimodal imaging probe that combines near-infrared (NIR) fluorescence and magnetic resonance imaging (MRI) to enable real-time visualization of activity in living organisms. Upon β-gal activation, Gal-Cy-Gd-1 exhibits a remarkable 42-fold increase in NIR fluorescence intensity at 717 nm, allowing covalent labeling of adjacent target enzymes or proteins and avoiding molecular escape to promote probe accumulation at the tumor site. This fluorescence reaction enhances the longitudinal relaxivity by approximately 1.9 times, facilitating high-resolution MRI. The unique features of Gal-Cy-Gd-1 enable real-time and precise visualization of β-gal activity in live tumor cells and mice. The probe's utilization aids in identifying in situ ovarian tumors, offering valuable assistance in the precise removal of tumor tissue during surgical procedures in mice. The fusion of NIR fluorescence and MRI activation through self-immobilizing target enzymes or proteins provides a robust approach for visualizing β-gal activity. Moreover, this approach sets the groundwork for developing other activatable bimodal probes, allowing real-time in vivo imaging of enzyme activity and localization.     

A general strategy for in situ assembly of light-up fluorophores via bioorthogonal Suzuki-Miyaura cross-coupling

Herein we presented a general strategy for in situ assembly of intramolecular charge-transfer (ICT)-based light-up fluorophores via bioorthogonal Suzuki-Miyaura cross-coupling reaction. By introducing iodo group at the appropriate position, five fluorophores with different scaffolds including naphthalimide, coumarin, naphthalene sulfonate, nitrobenzoxadiazole, and acetonaphthone, were designed as bioorthogonal multicolor fluorogenic probes, which could produce significant fluorescence enhancement and high fluorescence quantum yield after Suzuki-Miyaura reaction with aryl boronic acid or boronate. Manipulating the substituents and π scaffold in the fluorophores allows fine-tuning of their photophysical properties. With this strategy, we succeeded in peptide conjugation, no-wash fluorogenic protein labeling, and mitochondria-selective bioorthogonal imaging in live cells.     

In Situ Localization of Enzyme Activity in Live Cells by a Molecular Probe Releasing a Precipitating Fluorochrome

Current enzyme‐responsive, fluorogenic probes fail to provide in situ information because the released fluorophores tend to diffuse away from the reaction sites. The problem of diffusive signal dilution can be addressed by designing a probe that upon enzyme conversion releases a fluorophore that precipitates. An excited‐state intramolecular proton transfer (ESIPT)‐based solid‐state fluorophore HTPQ was developed that is strictly insoluble in water and emits intense fluorescence in the solid state, with λ _ex/em=410/550 nm, thus making it far better suited to use with a commercial confocal microscope. HTPQ was further utilized in the design of an enzyme‐responsive, fluorogenic probe (HTPQA), targeting alkaline phosphatase (ALP) as a model enzyme. HTPQA makes possible diffusion‐resistant in situ detection of endogenous ALP in live cells. It was also employed in the visualizing of different levels of ALP in osteosarcoma cells and tissue, thus demonstrating its interest for the diagnosis of this type of cancer.     

Quantitative interrogation of protein co-aggregation using multi-color fluorogenic protein aggregation sensors

Co-aggregation of multiple pathogenic proteins is common in neurodegenerative diseases but deconvolution of such biochemical process is challenging. Herein, we developed a dual-color fluorogenic thermal shift assay to simultaneously report on the aggregation of two different proteins and quantitatively study their thermodynamic stability during co-aggregation. Expansion of spectral coverage was first achieved by developing multi-color fluorogenic protein aggregation sensors. Orthogonal detection was enabled by conjugating sensors of minimal fluorescence crosstalk to two different proteins via sortase-tag technology. Using this assay, we quantified shifts in melting temperatures in a heterozygous model protein system, revealing that the thermodynamic stability of wild-type proteins was significantly compromised by the mutant ones but not vice versa. We also examined how small molecule ligands selectively and differentially interfere with such interplay. Finally, we demonstrated these sensors are suited to visualize how different proteins exert influence on each other upon their co-aggregation in live cells.

A little leak will sink a great ship! We prepared a series of multi-color protein aggregation sensors and developed a dual-color thermal shift assay to simultaneously and quantitatively report on protein co-aggregation of two different proteins.     

HSA-Lys-161 covalent bound fluorescent dye for in vivo blood drug dynamic imaging and tumor mapping

The specific combination of human serum albumin and fluorescent dye will endow superior performance to a coupled fluorescent platform for in vivo fluorescence labeling. In this study, we found that lysine-161 in human serum albumin is a covalent binding site and could spontaneously bind a ketone skeleton quinoxaline–coumarin fluorescent dye with a specific turn-on fluorescence signal for the first time. Supported by the abundant drug binding domains in human serum albumin, drugs such as ibuprofen, warfarin and clopidogrel could interact with the fluorescent dye labeled human serum albumin to feature a substantial enhancement in fluorescence intensity (6.6-fold for ibuprofen, 4.5-fold for warfarin and 5-fold for clopidogrel). The drug concentration dependent fluorescence intensity amplification realized real-time, in situ blood drug concentration monitoring in mice, utilizing ibuprofen as a model drug. The non-invasive method avoided continuous blood sample collection, which fundamentally causes suffering and consumption of experimental animals in the study of pharmacokinetics. At the same time, the coupled fluorescent probe can be efficiently enriched in tumors in mice which could map a tumor with a high-contrast red fluorescence signal and could hold great potential in clinical tumor marking and surgical resection.

HSA lysine-161 covalent bound quinoxaline–coumarin based fluorescent dye realized in situ blood drug concentration monitoring and tumor visualization.     

Detection of oncoprotein platelet-derived growth factor using a fluorescent signaling complex of an aptamer and TOTO

There have recently been advances in the application of aptamers, a new class of nucleic acids that bind specifically with target proteins, as protein recognition probes for biomedical study. The development of a signaling aptamer with the capability of simple and rapid real-time detection of disease-related proteins has attracted increasing interest. We have recently reported a new protein-detection strategy using a signaling aptamer based on a DNA molecular light-switching complex, [Ru(phen)₂(dppz)]²⁺. In this work we have used the commercially available DNA-intercalating dye, TOTO, to replace [Ru(phen)₂(dppz)]²⁺ for detection of oncoprotein platelet-derived growth factor BB (PDGF-BB), a potential cancer marker. Taking advantage of the high affinity of the aptamer to PDGF-BB and the sensitive fluorescence change of the aptamer–TOTO signaling complex on protein binding, PDGF-BB was detected in physiological buffer with high selectivity and sensitivity. The detection limit was 0.1 nmol L⁻¹, which was better than that of other reported aptamer-based methods for PDGF-BB, including that using [Ru(phen)₂(dppz)]²⁺. The method is very simple with no need for covalent labeling of the aptamer or probe synthesis. It facilitates wide application of the signaling mechanism to the analysis and study of cancer markers and other proteins.      

Detection of LacZ‐Positive Cells in Living Tissue with Single‐Cell Resolution

The LacZ gene, which encodes Escherichia coli β‐galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ‐positive cells in living organisms or tissues at single‐cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic β‐galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single‐cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms.     

Intracellular protein labeling with prodrug-like probes using a mutant β-lactamase tag

Intracellular protein labeling with small molecular probes that do not require a washing step for the removal of excess probe is greatly desired for real-time investigation of protein dynamics in living cells. Successful labeling of proteins on the cell membrane has been performed using mutant β-lactamase tag (BL-tag) technology. In the present study, intracellular protein labeling with novel cell membrane permeable probes based on β-lactam prodrugs is described. The prodrug-based probes quickly permeated the plasma membranes of living mammalian cells, and efficiently labeled intracellular proteins at low probe concentrations. Because these cell-permeable probes were activated only inside cells, simultaneous discriminative labeling of intracellular and cell surface BL-tag fusion proteins was attained by using cell-permeable and impermeable probes. Thus, this technology enables adequate discrimination of the location of proteins labeled with the same protein tag, in conjunction with different color probes, by dual-color fluorescence. Moreover, the combination of BL-tag technology and the prodrug-based probes enabled the labeling of target proteins without requiring a washing step, owing to the efficient entry of probes into cells and the fast covalent labeling achieved with BL-tag technology after bioactivation. This prodrug-based probe design strategy for BL-tags provides a simple experimental procedure with application to cellular studies with the additional advantage of reduced stress to living cells.     

Constructing real-time,wash-free,and reiterative sensors for cell surface proteins using binding-induced dynamic DNA assembly

Cell surface proteins are an important class of biomarkers for fundamental biological research and for disease diagnostics and treatment. In this communication, we report a universal strategy to construct sensors that can achieve rapid imaging of cell surface proteins without any separation by using binding-induced dynamic DNA assembly. As a proof-of-principle, we developed a real-time and wash-free sensor for an important breast cancer biomarker, human epidermal growth factor receptor-2 (HER2). We then demonstrated that this sensor could be used for imaging and sensing HER2 on both fixed and live breast cancer cells. Additionally, we have also incorporated toehold-mediated DNA strand displacement reactions into the HER2 sensor, which allows for reiterating (switching on/off) fluorescence signals for HER2 from breast cancer cells in real-time.     

A Far-Red Molecular Rotor Fluorogenic Trehalose Probe for Live Mycobacteria Detection and Drug-Susceptibility Testing

Increasing the speed, specificity, sensitivity, and accessibility of mycobacteria detection tools are important challenges for tuberculosis (TB) research and diagnosis. In this regard, previously reported fluorogenic trehalose analogues have shown potential, but their green-emitting dyes may limit sensitivity and applications in complex settings. Here, we describe a trehalose-based fluorogenic probe featuring a molecular rotor turn-on fluorophore with bright far-red emission (RMR-Tre). RMR-Tre, which exploits the unique biosynthetic enzymes and environment of the mycobacterial outer membrane to achieve fluorescence activation, enables fast, no-wash, low-background fluorescence detection of live mycobacteria. Aided by the red-shifted molecular rotor fluorophore, RMR-Tre exhibited up to a 100-fold enhancement in M. tuberculosis labeling compared to existing fluorogenic trehalose probes. We show that RMR-Tre reports on M. tuberculosis drug resistance in a facile assay, demonstrating its potential as a TB diagnostic tool.     

SNAP-tag fluorogenic probes for wash free protein labeling

In this review, we described the design strategies of SNAP-tag fl uorogenic probes with turn-on fl uorescence responses, which minimized the fl uorescence background and allowed for direct imaging in living cells without wash-out steps. These probes can apply in real-time analysis of protein localization, dynamics, and protein– protein interactions in living cells. Furthermore, the excellent fl uorescent properties made it possible to apply some of the probes in super-resolution fl uorescence imaging.     

Red-Shifted Substrates for FAST Fluorogen-Activating Protein Based on the GFP-Like Chromophores

A genetically encoded fluorescent tag for live cell microscopy is presented. This tag is composed of previously published fluorogen-activating protein FAST and a novel fluorogenic derivative of green fluorescent protein (GFP)-like chromophore with red fluorescence. The reversible binding of the novel fluorogen and FAST is accompanied by three orders of magnitude increase in red fluorescence (580–650 nm). The proposed dye instantly stains target cellular proteins fused with FAST, washes out in a minute timescale, and exhibits higher photostability of the fluorescence signal in confocal and widefield microscopy, in contrast with previously published fluorogen:FAST complexes.     

Rapid differentiation between bacterial infections and cancer using a near-infrared fluorogenic probe

The reliable differentiation between bacterial infections and other pathologies is crucial for both diagnostics and therapeutic approaches. To accommodate such needs, we herein report the development of an activatable near-infrared fluorescent probe 1 that could be applied in the ultrafast, ultrasensitive and specific detection of nitroreductase (NTR) activity in bacterial pathogens both in vitro and in vivo. Upon reaction with NTR, the nitro-group of the para-nitro phenyl sulfonic moiety present in probe 1 was reduced to an amino-group, resulting in a near-infrared fluorescence turn-on of the latent cyanine 7 fluorophore. Probe 1 was capable of rapid and real-time quantitative detection of 0–150 ng mL⁻¹ NTR with a limit of detection as low as 0.67 ng mL⁻¹in vitro. In addition, probe 1 exhibited an outstanding performance of ultrafast measurements and suitable selectivity toward NTR to accurately sense intracellular basal NTR in ESKAPE bacterial pathogens. Most remarkably, probe 1 was capable of noninvasively identifying bacterial infection sites without showing any significantly increased signal of tumour sites in the same animal within 30 min.

A new nitroreductase-responsive near-infrared fluorogenic probe can specifically image live bacteria in mouse models and does not accumulate at sites of inflammation or tumor.     

Fluorogenic Protein Probes with Red and Near-Infrared Emission for Genetically Targeted Imaging**

Fluorogenic probes are important tools to image proteins with high contrast and no wash protocols. In this work, we rationally designed and synthesized a small set of four protein fluorogens with red or near-infrared emission. The fluorophores were characterized in the presence of albumin as a model protein environment and exhibited good fluorogenicity and brightness (fluorescence quantum yield up to 36 %). Once conjugated to a haloalkane ligand, the probes reacted with the protein self-labeling tag HaloTag with a high fluorescence enhancement (up to 156-fold). The spectroscopic properties of the fluorogens and their reaction with HaloTag were investigated experimentally in vitro and with the help of molecular dynamics. The two most promising probes, one in the red and one in the near-infrared range, were finally applied to image the nucleus or actin in live-cell and in wash-free conditions using fluorogenic and chemogenetic targeting of HaloTag fusion proteins.     

Nile-red and Nile-blue-based near-infrared fluorescent probes for in-cellulo imaging of hydrogen sulfide

Hydrogen sulfide has recently been identified as a biologically responsive species. The design and synthesis of fluorescence probes, which are constructed with Nile-red or Nile-blue fluorophores and a fluorescence-controllable dinitrophenyl group, for hydrogen sulfide are reported in this paper. The Nile-red–dinitrophenyl-ether-group-based probe (1a) is essentially non-fluorescent because of the inhibition of the photo-induced electron-transfer process; when the dinitrobenzene moiety is removed by nucleophilic substitution with the hydrosulfide anion, probe 1a is converted into hydroxy Nile red, eliciting a H₂S-induced fluorescence turn-on signal. Furthermore, probe 1a has high selectivity and sensitivity for the hydrosulfide anion, and its potential for biological applications was confirmed by using it for real-time fluorescence imaging of hydrogen sulfide in live HeLa cells. The Nile-blue–dinitrobenzene-based probe (1b) has gradually diminishing brightness in the red-emission channel with increased hydrogen-sulfide concentration. Thus, this paper reports a comparative study of Nile-red and Nile-blue-based hydrogen-sulfide probes. Graphical Abstract

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