Small-molecule Fluorescent Probes Research Articles

How to Target Small-Molecule Fluorescent Imaging Probes to the Plasma Membrane-The Influence and QSAR Modelling of Amphiphilicity, Lipophilicity, and Flip-Flop.

Many new fluorescent probes targeting the plasma membrane (PM) of living cells are currently being described. Such probes are carefully designed to report on relevant membrane features, but oddly, the structural features required for effective and selective targeting of PM often receive less attention, constituting a lacuna in the molecular design process. We aim to rectify this by clarifying how the amphiphilicity and lipophilicity of a probe, together with the tendency to flip-flop across the membrane, contribute to selective PM accumulation. A simplistic decision-rule QSAR model has been devised that predicts the accumulation/non-accumulation of small-molecule fluorescent probes in the PM. The model was based on probe log P plus various derived measures, allowing the roles of amphiphilicity, lipophilicity, and flip-flop to be taken into account. The validity and wide applicability of the model were demonstrated by evaluating its ability to predict amphiphilicity or PM accumulation patterns in surfactants, drugs, saponins, and PM probes. It is hoped that the model will aid in the more efficient design of effective PM probes.

A novel near-infrared fluorescent probe for specific imaging of telomerase reverse transcriptase in vivo

Identifying the expression levels of human telomerase reverse transcriptase (hTERT) is important for early cancer diagnosis and cancer-related drug screening. However, a small-molecule fluorescent probe has rarely been used for rapidly determining the hTERT level with high sensitivity has been scarce. Herein, we designed and synthesized a novel near-infrared fluorescent probe (NB-BIBRA) by conjugating an inhibitor analog (BIBRA) to the fluorophore Nile blue (NB) for specific imaging of hTERT in living cells and in vivo. In the absence of hTERT, NB-BIBRA mainly exists in aggregates, exhibiting self-quenching photoactivity. However, once NB-BIBRA interacts with hTERT, NB-BIBRA aggregates are disrupted and their fluorescence intensity is drastically enhanced. Notably, the probe exhibited a low detection limit, high selectivity and rapid response to hTERT, allowing rapid imaging of hTERT in living cells and solid tumor tissue in vivo. To the best of our knowledge, NB-BIBRA is the first near-infrared fluorescent probe for the detection and imaging of hTERT in living cells and in vivo, thus providing a powerful tool for early cancer diagnosis and cancer-related drug screening.

Recent Development of Lysosome-Targeted Organic Fluorescent Probes for Reactive Oxygen Species.

Reactive oxygen species (ROS) are extremely important for various biological functions. Lysosome plays key roles in cellular metabolism and has been known as the stomach of cells. The abnormalities and malfunctioning of lysosomal function are associated with many diseases. Accordingly, the quantitative monitoring and real-time imaging of ROS in lysosomes are of great interest. In recent years, with the advancement of fluorescence imaging, fluorescent ROS probes have received considerable interest in the biomedical field. Thus far, considerable efforts have been undertaken to create synthetic fluorescent probes for sensing ROS in lysosomes; however, specific review articles on this topic are still lacking. This review provides a general introduction to fluorescence imaging technology, the sensing mechanisms of fluorescent probes, lysosomes, and design strategies for lysosome-targetable fluorescent ROS probes. In addition, the latest advancements in organic small-molecule fluorescent probes for ROS detection within lysosomes are discussed. Finally, the main challenges and future perspectives for developing effective lysosome-targetable fluorescent ROS probes for biomedical applications are presented.

Development of a fluorescent chemical probe with the ability to visualize nascent phase-separated stress granules

Stress granules (SGs) are a fascinating type of membrane-less organelle that form through liquid-liquid phase separation in eukaryotic cells. Our previous investigations have been instrumental in the development of the first small-molecule fluorescent probe, named TASG, which can selectively identify SGs. However, the practical utility of TASG for monitoring SG dynamics in live cells has been hindered by the difficulty in visualizing small nascent SGs. To overcome this obstacle, the present study systematically modified the structure of TASG, with the aim of discovering fluorescent chemical probes that exhibit superior performance. Among the evaluated candidates, TASG-8 exhibited the most promising ability to image SGs. Importantly, TASG-8 possessed a critical advantage over TASG, in that it could effectively label small nascent SGs. The structure-activity relationship of the candidates was analyzed. Additionally, the underlying mechanism responsible for this enhancement was extensively explored. Collectively, our research presents a rational strategy for the development of innovative fluorescent probes that selectively target SGs, and introduces a powerful new tool, TASG-8, which has the potential to facilitate more comprehensive and efficient investigations into the functions of SGs in cells.

A Functionalizable Analog of the Yariv Reagent for AGP Imaging using Fluorescence Microscopy.

Small molecule fluorescent probes that bind selectively to plant cell wall polysaccharides have been instrumental in elucidating the localization and function of these glycans. Arabinogalactan proteins (AGPs) are cell wall proteoglycans implicated in essential functions such as cell signaling, plant growth, and programmed cell death. There is currently no small molecule probe capable of fluorescently labeling AGPs. The Yariv reagents are the only small molecules that bind AGPs, and have been used to study AGP function and isolate AGPs via precipitation of an AGP-Yariv complex. However, the Yariv reagents are not fluorescent, rendering them ineffective for localization studies using fluorescence microscopy. A fluorescent version of a Yariv reagent that is capable of both binding as well as imaging AGPs would provide a powerful tool for studying AGPs in planta. Herein, we describe the synthesis of an azido analog of the Yariv reagent that can be further functionalized with a fluorophore to provide a glycoconjugate that binds AGPs and is fluorescent. We show that the modified reagent binds gum arabic in in vitro binding assays when used in conjunction with the βGlcYariv reagent. Fluorescent imaging of AGPs in fixed maize leaf tissue enables localization of AGPs to cell walls in the leaf. Significantly, imaging can also be carried out using fresh tissue. This represents the first small molecule probe that can be used to visualize AGPs using fluorescence microscopy.

Advances in Small-Molecule Fluorescent pH Probes for Monitoring Mitophagy.

Mitochondria play a crucial role in regulating cellular energy homeostasis and cell death, making them essential organelles. Maintaining proper cellular functions relies on the removal of damaged mitochondria through a process called mitophagy. Mitophagy is associated with changes in the pH value and has implications for numerous diseases. To effectively monitor mitophagy, fluorescent probes that exhibit high selectivity and sensitivity based on pH detection have emerged as powerful tools. In this review, we present recent advancements in the monitoring of mitophagy using small-molecule fluorescence pH probes. We focus on various sensing mechanisms employed by these probes, including intramolecular charge transfer (ICT), fluorescence resonance energy transfer (FRET), through bond energy transfer (TBET), and photoelectron transfer (PET). Additionally, we discuss disease models used for studying mitophagy and summarize the design requirements for small-molecule fluorescent pH probes suitable for monitoring the mitophagy process. Lastly, we highlight the remaining challenges in this field and propose potential directions for the future development of mitophagy probes.

Harnessing single fluorescent probe to image deoxyribonucleic acid and ribonucleic acid in cells

The roles of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in cells are closely related. However, the absence of molecular tools for simultaneous imaging of the two nucleic acids has prevented scientists from elucidating the regulatory mechanisms of nucleic acid interaction. The nucleic acid probes developed in recent years have ignored the regulatory relationship between DNA and RNA. Simultaneously imaging RNA and DNA in cells through a single small-molecule fluorescent probe is important. In this study, we propose a strategy for developing fluorescent probes localized to DNA and RNA to investigate their detection and imaging characteristics. The novel probe Bptp-RD has been successfully used for DNA and RNA imaging in cells. We investigated the detection and imaging characteristics of this nucleic acid probe and discovered the following: 1) the differences in the detection results of this nucleic acid probe for DNA and RNA come from the structural differences of the nucleic acids rather than chemical composition differences; 2) through using small-molecule probes to image a nucleic acid in cells, another nucleic acid can be visualized by reducing the fluorescence signal caused by DNA or RNA; 3) the order of response of the small-molecule fluorescent probe with intercalation and binding mechanisms to the type of nucleic acid structure is single chain, double chain, and ring. This work will help improve the understanding of RNA and DNA probes, and the novel probe has high potential to explore the interaction between RNA and DNA in cells.

A Cys-regulated fluorescent probe targeting cancer cells and their application in inflammation detection

Cysteine (Cys) plays an important role in physiological and pathological processes as an essential amino acid in the human body. Cys is often used as a biomarker for cancer cells. Therefore, it is very important to detect and analyze Cys in the cancer cell environment. However, small molecule fluorescent probes that can be used to target Cys in cancer cells have rarely been reported. In this paper, a novel fluorescent probe BN-C with the function of targeted detection of Cys in cancer cells was designed and synthesized based on the ICT (intramolecular charge transfer) mechanism. BN-C exhibits low cytotoxicity, high sensitivity and high selectivity. The probe has been shown to be useful for fluorescence imaging of Cys in cancer cells and zebrafish. In addition, the probe BN-C can detect the process of inflammation production in cells, which can be used as a potential tool for diagnosis and prevention of related diseases.

Fluorescent probe for highly selective detection of cysteine in living cells.

Cys play an important physiological role in the human body. Abnormal Cys concentration can cause many diseases. Therefore, it is of great significance to detect Cys with high selectivity and sensitivity in vivo. Because homocysteine (Hcy) and glutathione (GSH) have similar reactivity and structure to cysteine, few fluorescent probes have been reported to be specific and efficient for cysteine. In this study, we designed and synthesized an organic small molecule fluorescent probe ZHJ-X based on cyanobiphenyl, which can be used to specifically recognize cysteine. The probe ZHJ-X exhibits specific selectivity for cysteine, high sensitivity, short reaction response time, good anti-interference ability, and has a low detection limit of 3.8×10-6 M. The probe ZHJ-X was successfully applied to the visualization of Cys in living cells and had great application prospects in cell imaging and detection.

A simple benzothiazole-based turn-on fluorescent probe for detecting H2S in HeLa cells

H2S is an endogenously produced gaseous signaling molecule that plays a vital role in many biological processes. Small molecule fluorescent probes as a powerful tool can be used to in vivo and in vitro visualize the H2S with many advantages. At present, many fluorescence probes were reported based on the nucleophilicity of H2S, which were susceptible to interference from other nucleophilic species and possessed poor selectivity. In this work, we designed a simple benzothiazole-based turn-on fluorescence probe HBT-PBA that was modified with pyridyl disulfide as a recognition group and had good selectivity towards H2S over biothiols. Additionally, HBT-PBA was utilized for fluorescence images of the H2S in the lysosomes and endoplasmic reticulum of HeLa cells.

Acidic tumor microenvironment-activatable fluorescent diagnostic probe for the rapid identification and resection of human tumors via spraying

A fluorescent diagnostic probe for real-time intraoperative image-guided tumor resection can significantly improve the efficiency and quality of oncological therapy, but their development is challenging. Herein, a novel fluorescent diagnostic probe called HLTC based on β-carboline was designed and synthesized. HLTC was found to show a ∼10-fold enhancement of fluorescence quantum field with pH from 7.4 to 4.0, indicating its imaging potential in acid environment which is a typical hallmark of the tumor microenvironment (TME). Following fluorescence microscopy imaging showed HLTC could emit specific signals in cancer cells and sections, by both one-photon excitation and two-photon excitation. Importantly, HLTC enabled the precise and rapid delineation of both transplanted tumor and clinical tumor tissues within several minutes of simple topical spray. The tumor-to-background ratio (TBR) was up to 10.2 ± 1.0 at clinical liver cancer tissues and 9.9 ± 0.3 at clinical colon cancer tissues, allowing precise tumor margin identification and the effective guidance of surgical tumor resection. Furthermore, CCK8 assay, pharmacokinetic evaluation, blood analysis and H&E staining were performed, which verified high biocompatibility and biosafety of HLTC at working concentration. These results reveal the exciting potential of this small-molecule fluorescent diagnostic probe for real-time fluorescence-based navigation during surgical tumor resection.

Debut of a novel AIE-based fluorescent probe as tyrosinase tracer to image skin pigmentary disorders

Tyrosinase (TYR) functions as the critical enzyme in biosynthesis of melanin, and its abnormality is an important hallmark for skin pigmentary disorders. This renders the visualization of tyrosinase dynamics a strategically vital way to facilitate clinical diagnostic and monitoring. However, the use of small molecule fluorescent probes to assess tyrosinase levels and evaluate related therapeutics and drugs has been limited. This is mainly due to the aggregation-induced quenching (ACQ) effects of existing fluorescent probes. In this study, we introduce the first aggregation-induced emission (AIE) small molecule fluorescent probe, BTFTYR, for simple, highly sensitive, and effective detection of tyrosinase activity. The specific fluorescence signal generated by tyrosinase catalytic reaction enables the imaging of pigment cells. We demonstrate its application in visualizing tyrosinase with high contrast in living vertebrates, benefiting from the inherent photobleaching resistance of AIE materials, large Stokes shift, and considerable tissue permeability. We also show the outstanding skin penetration capability of BTFTYR in a skin model. Moreover, we present the first comprehensive evaluation of relevant clinical therapeutic approaches or drugs, including ultraviolet B (UVB) irradiation, kojic acid, and α-MSH, by interpreting the tyrosinase variation with BTFTYR. Finally, our studies confirm that BTFTYR neither causes skin inflammation nor produces visceral toxicity, making it a promising chemical tool for medical diagnosis and drug screening. This tool may further help elucidate the pathomechanisms associated with tyrosinase.

Rapid detection of serum calcium based on supramolecular aggregates of a cationic perylene in vitro

The steady-state level of serum calcium is of great significance to human body. Accurate and rapid detection of serum calcium is a crucial component of clinical diagnosis and blood calcium census. Current detection of Ca2+ in living environment mainly focus on intracellular imaging, and there are few probes appropriate for serum calcium detection. In this work, we designed a fluorescence sensing system based on the indicator displacement assays (IDAs) strategy using supramolecular complex formed by 3,4,9,10-tetra-(4-trimethylaminobutoxy-carbonyl)-perylene (PBTMA) and casein phosphopeptides (CPPs). Due to the synergistic effect of non-covalent interactions, the complex can be induced by Ca2+ to disintegrate, thus realizing the visual and rapid detection of serum calcium. The quantitative range is 0.01–3.0 mM, while the limit of detection (LOD) and the visual detection limit are 8.4 μM and 0.1 mM, respectively. This method features good stability, selectivity and anti-interference. It can be used for serum samples (recovery rate: 103.4%–106.3%; relative standard deviation (RSD): 2.21%–3.46%). Furthermore, a portable device for rapid serum calcium measurement was designed, which could substantially improve the applicability of this method. To our knowledge, this is the first organic small molecule fluorescent probe capable of application in the range of serum calcium concentrations.

Assessing the pollution level of a subtropical lake by using a novel hydrogen sulfide fluorescence technology

Hydrogen sulfide (H2S) is an important environmental toxin with bi-directional biological effects on organisms. In natural waters, H2S complexes with heavy metal ions in an anaerobic environment influence heavy metals' bioavailability and induce phosphorus release and eutrophication in water columns. Traditional detection techniques, such as colorimetric, electrochemical, and chromatographic, cannot simultaneously detect H2S and pollution assessment of subtropical lakes. To address these technical defects, we developed small-molecule fluorescent probes to evaluate the pollution level in natural water bodies. This method relies on the combination of the probes’ response signals to raw water and the water quality index, thereby enhancing the accuracy and reliability of water quality assessments. Furthermore, this novel material has a large Stokes shift. It can detect complex levels of H2S concentrations in natural water bodies by correlating the degree of contamination and fluorescence signals. The development of this visual research tool for detecting environmental H2S levels in natural water bodies is expected to have meaningful, practical applications.

Probing mitochondrial damage using a fluorescent probe with mitochondria-to-nucleolus translocation

Mitochondrial damage is closely related to the occurrence of many diseases. However, accurate monitoring and reporting of mitochondrial damage are not easy. Here, we developed a small molecule fluorescent probe named CB-Cl, which has splendid spectral properties (large Stokes shift, strong affinity for RNA, etc.) and excellent targeting ability to intracellular mitochondria. After mitochondria were damaged by external stimuli, CB-Cl would light up the nucleolus as a signal reporter. The cascade imaging of mitochondria and nucleolus using CB-Cl can monitor and visualize the mitochondrial status in living cells in real-time. Based on the above advantages, the probe CB-Cl has reference significance for the related research of mitochondrial damage and the prevention and treatment of related diseases.

An ICT-PET dual-controlled strategy for improving molecular probe sensitivity: Application to photoactivatable fluorescence imaging and H2S detection

The organic small molecule fluorescent probes are very effective tool for accurately analyzing target molecules and super-resolution microscopy biological imaging. Generally, reducing the interference of background fluorescence is one of the effective strategies to improve the resolution and signal-to-noise ratio of biological imaging. Herein, in this work, using the synergistic effect of the ICT-PET dual quenching mechanism to reduce background fluorescence, the fluorescent probe Np-N3 with naphthalimide and small size azide as photocage and the recognition site of H2S was designed and synthesized. The synergistic mechanism of ICT-PET was computationally demonstrated by TD-DFT. The Np-N3 showed low background fluorescence, high sensitivity and high selectivity for visible light and H2S, respectively. Meanwhile, under visible light irradiation (465 nm), the azide was easily reduced to amino in situ without complicated separation steps, restoring strong emission in a short time (<3 min). Importantly, the spatiotemporal-controlled imaging of live HeLa cells was successfully achieved. In addition, the probe was also applied to detect and image H2S in solution and living HeLa cells.

Pyridine-Based Small-Molecule Fluorescent Probes as Optical Sensors for Benzene and Gasoline Adulteration

Here we present simple fluorophores based on the pyridine core, obtained with straightforward synthetic methodologies. These compounds present in solution absorption maxima in the UV region and fluorescence emission of between 300 and 450 nm, depending on the solvent and chemical structure of the fluorophore. The nature of the solvent was shown to play a fundamental role in their excite-state deactivation, which allowed successful exploration of these compounds as optical sensors for benzene and fuel adulteration in gasoline. In ethanolic solution, upon the addition of benzene, in general the fluorophores presented fluorescence quenching, where a linear correlation between the emission intensity and the amount of benzene (quencher) was observed. In addition, the application of an optical sensor for the detection of fuel adulteration in commercial standard and premium gasoline was successfully presented and discussed. Theoretical calculations were also applied to better understand the solvent–fluorophore interactions.

A near-infrared HBT-based fluorescent probe for tracking carbon monoxide-releasing CORM-3

Tricarbonylchloro(glycinato)ruthenium (CORM-3) has important application in the treatment of clinical diseases because of anti-inflammatory and heart-protecting properties, and is also used to study the metabolic mechanism of endogenous carbon monoxide as a safe and convenient carbon monoxide-releasing agent in vivo due to good water solubility and releasing controlled amounts of CO. Thus the development of effective detection methods to achieve the analysis of CORM-3 is of great significance in monitoring CORM-3 and studying the specific mechanism of endogenous CO in biological samples. Herein a small molecule fluorescent probe was developed by selecting 2-(2-hydroxyphenyl)benzothiazole (HBT) derivative as the optical signal unit and 4-nitrobenzyl moiety as the reactive unit. It could quickly detect CORM-3 within 15 min by the enhanced near-infrared fluorescence at 720 nm without the participation of heavy metal ions, accompanied by the large Stokes movement of 195 nm. The detection limit and linear range of the probe toward CORM-3 were achieved 0.11 µM and 0 – 175 µM, respectively. Besides, this probe displayed a red-edge effect, and the excitation wavelength and emission wavelength may be adjusted reasonably according to the test requirement to avoid interference signal. More importantly, it has been successfully used for the detection of CORM-3 with high resolution of fluorescence imaging in living cells and Zebrafish.

Fluorescent Probes for Biological Species and Microenvironments: from Rational Design to Bioimaging Applications.

ConspectusSome important biological species and microenvironments maintain a complex and delicate dynamic balance in life systems, participating in the regulation of various physiological processes and playing indispensable roles in maintaining the healthy development of living bodies. Disruption of their homeostasis in living organisms can cause various diseases and even death. Therefore, real time monitoring of these biological species and microenvironments during different physiological and pathological processes is of great significance. Fluorescent-probe-based techniques have been recognized as one of the most powerful tools for real time imaging in biological samples. In this Account, we introduce the representative works from our group in the field of fluorescent probes for biological imaging capable of detecting metal ions, small bioactive molecules, and the microenvironment. The design strategies of small molecule fluorescent probes and their applications in biological imaging will be discussed. By regulating the design strategy and mechanism (e.g., ICT, PeT, and FRET) of the electronic and spectral characteristics of the fluorescent platforms, these chemical probes show high selectivity and diverse functions, which can be used for imaging of various physiological and pathological processes. Through the exploration of the rational response mechanism and design strategy, combined with a variety of imaging techniques, such as super-resolution imaging, photoacoustic (PA) imaging, etc., we have realized multimode imaging of the important biological analytes from the subcellular level to the in vivo level, which provides powerful means to study the physiological and pathological functions of these species and microenvironments. This Account aims to offer insights and inspiration for the development of novel fluorescent probes for biological imaging, which could provide powerful tools for the study of chemical biology. Overall, we represent a series of turn-on/turn-off/ratiometric fluorescent/PA probes to visually and dynamically trace biological species and microenvironments in cells and even in vivo that seek higher resolution and depth molecular imaging to improve diagnostic methods and clarify new discoveries related to chemical biology. Our future efforts will be devoted to developing multiorganelle targeted fluorescent probes to study the mechanism of subcellular organelle interaction and employing various dual-mode probes of NIR II and PA imaging to investigate the development of related diseases and treat the related diseases at subcellular and in vivo levels.

AIE-based fluorescence probes with different torsional configurations for monitoring human serum albumin

Human serum albumin (HSA) levels can reflect a variety of health issues in the human body, and quantitative examination of HSA is of great significance for disease diagnosis. Herein, we rationally designed and developed two novel AIE small molecule fluorescent probes, AE-BP and AE-AQ. Two probes have various torsion configurations and can be applied to the detection of exogenous HSA. They have high signal-to-noise ratio and selectivity and can enter the HSA cavity smoothly. Both probes can combine with the IB site, successfully avoiding most drug interference and having different detection effects on HSA. Meanwhile, the rational mechanism of AE-BP and AE-AQ to perform target detection was investigated using density functional theory. Furthermore, the scientific problems associated with the high signal-to-noise ratio and low aggregation of fluorescent probes were explained by investigating the effects of different torsional configurations on the detection effect. The main factors to be concerned in designing HSA fluorescent probes were proposed. This work provides methods and concepts for designing fluorescent probes for HSA based on the AIE strategy, as well as an effective manner for fluorescent probes to detect HSA. We anticipate that AE-BP and AE-AQ will be employed as new tools for detecting HSA at the preclinical stage.