Research Progress of Cyanine-based Near-Infrared Fluorescent Probes for Biological Application.

Cyanine-based near-infrared (NIR) fluorescent probes have played vital roles in biological application due to their low interference from background fluorescence, deep tissue penetration, high sensitivity, and minimal photodamage to biological samples. They are widely utilized in molecular recognition, medical diagnosis, biomolecular detection, and biological imaging. Herein, we provide a review of recent advancements in cyanine-based NIR fluorescent probes for the detection of pH, cells, tumor as well as their application in photothermal therapy (PTT) and photodynamic therapy (PDT).

Cellular Uptake of Cell-Penetrating Peptides Activated by Amphiphilic p-Sulfonatocalix[4]arenes.

We report the synthesis of a series of amphiphilic p-sulfonatocalix[4]arenes with varying alkyl chain lengths (CX4-Cn) and their application as efficient counterion activators for membrane transport of cell-penetrating peptides (CPPs). The enhanced membrane activity is confirmed with the carboxyfluorescein (CF) assay in vesicles and by the direct cytosolic delivery of CPPs into CHO-K1, HCT 116, and KTC-1 cells enabling excellent cellular uptake of the CPPs into two cancer cell lines. Intracellular delivery was confirmed by fluorescence microscopy after CPP entry into live cells mediated by CX4-Cn, which was also quantified after cell lysis by fluorescence spectroscopy. The results present the first systematic exploration of structure-activity relationships for calixarene-based counterion activators and show that CX4-Cn are exceptionally effective in cellular delivery of CPPs. The dodecyl derivative, CX4-C12, serves as best activator. A first mechanistic insight is provided by efficient CPP uptake at 4 °C and in the presence of the endocytosis inhibitor dynasore, which indicates a direct translocation of the CPP-counterion complexes into the cytosol and highlights the potential benefits of CX4-Cn for efficient and direct translocation of CPPs and CPP-conjugated cargo molecules into the cytosol of live cells.

De Novo Green Fluorescent Protein Chromophore-Based Probes for Capturing Latent Fingerprints Using a Portable System.

Rapid visualization of latent fingerprints, preferably at their point of origin, is essential for effective crime scene evaluation. Here, we present a new class of green fluorescent protein chromophore-based fluorescent dyes (LFP-Yellow and LFP-Red) that can be used for real-time visualization of LFPs within 10 s. Compared with traditional chemical reagents for LFPs, these fluorescent dyes are completely water-soluble, exhibit low cytotoxicity, and are harmless to users. Level 1-3 details of the LFPs could be clearly revealed through "off-on" fluorescence signal readout. Additionally, the fluorescent dyes were constructed based on an imidazolinone core and so do not contain pyridine groups or metal ions, which ensures that the DNA is not contaminated during extraction and identification after the LFPs are treated with the dyes. Combined with our as-developed portable system for capturing LFPs, LFP-Yellow and LFP-Red enabled the rapid capture of LFPs. Therefore, these green fluorescent protein chromophore-based probes provide an approach for the rapid identification of individuals who were present at a crime scene.

In2O3/Bi2S3 S-scheme Heterojunction-Driven Molecularly Imprinted Photoelectrochemical Sensor for Ultrasensitive Detection of Florfenicol.

Florfenicol (FF) raises significant human health and environmental concerns due to its toxicity to the hematology system and the potential spread of antibiotic-resistant genes. Here, a highly sensitive molecularly imprinted photoelectrochemical (PEC) sensor, featuring an In2O3/Bi2S3 S-scheme heterojunction, is proposed to detect FF without an external voltage supply. Compared with conventional II-type heterojunctions, S-scheme heterojunctions efficiently promote carrier separation and enhance the redox capability of the photocatalytic system. This allows more dissolved O2 and H2O molecules to participate in the redox reaction, resulting in an amplified and stabilized photocurrent response. The electron transfer in the S-scheme heterojunction is confirmed via electron spin resonance (ESR). With the molecular imprinting technique, this PEC platform exhibits exceptional selectivity, wide linear range (1.0 × 10-4-1.0 × 104 ng mL-1), low detection limit (6.4 × 10-5 ng mL-1), and applicability in real milk and chicken samples. This work not only showcases a PEC platform for accurately and portably detecting drugs but also proposes a viable approach for designing S-scheme heterojunctions in sensing analysis.

Chemoselective Fluorogenic Bioconjugation of Vicinal Dithiol-Containing Proteins for Live Cellular Imaging via Small Molecular Conjugate Acceptors.

To develop small molecular fluorogenic tools for the chemoselective labeling of vicinal dithiol-containing proteins (VDPs) in live cells is important for studying intracellular redox homeostasis. With this research, we developed small molecule-based fluorescent probes, achieving selective labeling of VDPs through thiol-thiol substitutions on bisvinylogous thioester conjugated acceptors (IDAs). Initially, IDAs demonstrated its ability to bridge vicinal cysteine-sulfhydryls on a peptide as a mimic. Then, the peptide complex could be decoupled to recover the original peptide-SH in the presence of dithiothreitol. Furthermore, fluorometric signal amplification of the fluorescent probes occurred with high sensitivity, low limit of detection, and selectivity toward vicinal dithiols on reduced bovine serum albumin, as an example of real world VDPs. More importantly, the probes were utilized successfully for labeling of endogenous VDPs at different redox states in live cells. Thus, the bisvinylogous thioester-based receptor as a functional probe represents a new platform for uncovering the function of VDPs in live cells.

An ultrasensitive cathodic electrochemiluminescence immunoassay for thrombomodulin based on Ru(bpy)32+ encapsulated by MIL-NH2-101(Al) nanocomposites.

The rapid and reliable determination of thrombomodulin (TM) is of great significance for the diagnosis of disseminated intravascular coagulation, thrombosis and others. This work exhibits an electrochemiluminescent (ECL) sensor, which was prepared using Ru(bpy)32+ encapsulated by MIL-NH2-101(Al) nanocomposites for the sensitive detection of the new-thrombus marker thrombomodulin (TM) for the first time. Specifically, on one hand, with the advantages of high specific surface area, large hollow porous structure and favorable biocompatibility, MIL-NH2-101(Al) could load a large amount of luminescent Ru(bpy)32+ and thereby greatly enhance the ECL signal of the immunosensor. On the other hand, K2S2O8 is used as co-reactant to form a reduction-oxidation ECL system for cathodic ECL detection with strong anti-interference capacity. The experimental results show that the ECL signal intensity of the Ru(bpy)32+@MIL-NH2-101(Al)-based immunosensor decreased with the immunocapturing of TM, exhibiting a linear detection concentration ranging from 1 × 10-5 to 10 µg mL-1 and the limit of detection (LOD) of 8.2 × 10-6 µg mL-1 (S/N = 3). With its ideal stability, selectivity and reproducibility, the proposed ECL immunosensor can provide excellent aid and shows great promise for the detection of TM.

An endoplasmic reticulum targeting green fluorescent protein chromophore-based probe for the detection of viscosity.

The occurrence of endoplasmic reticulum (ER) stress is the main cause of a variety of biological processes that are closely related to numerous diseases. The homeostasis of the ER microenvironment can be disrupted under ER stress. In this research, by linking a pentafluorophenyl to the green fluorescent protein chromophore, we have developed a new ER-targeting fluorescent probe (GE-Y) for measuring changes of intracellular ER viscosity caused by ER stress. Importantly, an increase in ER viscosity was observed using GE-Y in cells undergoing autophagy. As such, our research provides an ideal tool for studying ER stress and autophagy.

A HBDI-Based Fluorescent Probe for Labeling Endoplasmic Reticulum in Living Cells.

The endoplasmic reticulum (ER) is an important organelle in eukaryotic cells and is closely involved in the synthesis and processing of proteins, as well as the storage, regulation, and release of calcium. A series of signaling pathways within the ER play a crucial part in the pathogenesis of various diseases, including cancer. Thus, it is necessary to design ER-targeting probes to monitor these signaling pathways. Additionally, precision medicine also requires new ER-targeting group to facilitate the delivery of drug cargoes to the ER. However, only a limited number of ER-targeting groups have been used for the design of fluorescent probes for ER imaging in living cells, as well as the development of ER-targeted drug delivery systems. Herein, a new ER-targeting fluorescent probe (BDI-ER) was designed and prepared. BDI-ER contains the hydrophilic fluorophore, derived from the core structure of GFP, and two hydrophobic octadecane chains. The amphipathic nature of BDI-ER facilitates localization in the ER. Live cell imaging demonstrated selective localization of BDI-ER towards ER compared to other organelles. Additionally, co-localization imaging in various cell lines indicate that BDI-ER is effective at targeting the ER.

Integrating potential-resolved electrochemiluminescence with molecularly imprinting immunoassay for simultaneous detection of dual acute myocardial infarction markers.

A novel potential-resolved molecularly imprinted electrochemical luminescence (ECL) immunosensor has been developed for the first time for the dual sensitive detection of markers of acute myocardial infarction (AMI): cardiac troponin I (cTnI) and myoglobin (Mb). In this work, cost-effective and robust molecularly imprinted polymer (MIP) as biomimetic antibody was used to construct the immunosensors through electropolymerization and elution to form polydopamine (PDA)-MIP modified electrode. In the presence of AMI biomarkers, two ECL probes including Ru(bpy)32+@ MOCs and MoS2 QDs functionalized by cTnI antibody and Mb aptamer could be specifically captured respectively. And two potential distinct ECL signals will be generated in one potential scan. The intensity of ECL reflects the concentrations of cTnI and Mb. The two ECL probes were characterized with field emission scanning electron microscopy, X-ray diffraction, FT-IR spectrum and UV-Vis diffuse reflectance spectroscopy. The prepared sensor exhibited a wide linear range (0.05-104 ng/mL) and a low detection limit (0.0184 ng/mL for cTnI and 0.0492 ng/mL for Mb). Additionally, the MIP-ECL sensor displayed excellent anti-interference, sensitivity and stability to detect cTnI and Mb. Therefore, it will be conducive to accelerate more precise and credible early diagnosis for AMI.

Dual-Channel Fluorescent Probe for the Simultaneous Monitoring of Peroxynitrite and Adenosine-5'-triphosphate in Cellular Applications.

Changes in adenosine triphosphate (ATP) and peroxynitrite (ONOO-) concentrations have been correlated in a number of diseases including ischemia-reperfusion injury and drug-induced liver injury. Herein, we report the development of a fluorescent probe ATP-LW, which enables the simultaneous detection of ONOO- and ATP. ONOO- selectively oxidizes the boronate pinacol ester of ATP-LW to afford the fluorescent 4-hydroxy-1,8-naphthalimide product NA-OH (λex = 450 nm, λem = 562 nm or λex = 488 nm, λem = 568 nm). In contrast, the binding of ATP to ATP-LW induces the spirolactam ring opening of rhodamine to afford a highly emissive product (λex = 520 nm, λem = 587 nm). Due to the differences in emission between the ONOO- and ATP products, ATP-LW allows ONOO- levels to be monitored in the green channel (λex = 488 nm, λem = 500-575 nm) and ATP concentrations in the red channel (λex = 514 nm, λem = 575-650 nm). The use of ATP-LW as a combined ONOO- and ATP probe was demonstrated using hepatocytes (HL-7702 cells) in cellular imaging experiments. Treatment of HL-7702 cells with oligomycin A (an inhibitor of ATP synthase) resulted in a reduction of signal intensity in the red channel and an increase in that of the green channel as expected for a reduction in ATP concentrations. Similar fluorescence changes were seen in the presence of SIN-1 (an exogenous ONOO- donor).

Green Fluorescent Protein GFP-Chromophore-Based Probe for the Detection of Mitochondrial Viscosity in Living Cells.

Viscosity is a pivotal factor for indicating the dysfunction of the mitochondria. To date, most of the fluorescent probes developed for mitochondrial viscosity have been designed using BODIPY, hemicyanine, or pyridine-based molecular rotors as part of the core structure. Our aim with this research was to extend the range of suitable fluorophores available for the construction of such fluorescent molecular rotors for evaluating the viscosity of mitocondria. Herein, we have developed a green fluorescent protein (GFP)-chromophore-based fluorescent probe (MIT-V) for the detection of mitochondrial viscosity in live cells. MIT-V exhibited a high sensitivity toward viscosity (from 7.9 cP to 438.4 cP). The "off-on" sensing mechanism of MIT-V was ascribed to the restricted rotation of single bonds and excited-state C═C double bonds of MIT-V. Cell studies indicated that MIT-V targets the mitochondria and that it was able to monitor real-time changes in the viscosity of live HeLa cell mitochondria. Therefore, we propose that MIT-V can be used as an effective chemosensor for the real-time imaging of mitochondrial viscosity in live cells. Our results clearly demonstrate the utility of such GFP-chromophore-based derivatives for the development of viscosity-sensitive systems.

Supramolecular ensembles modified by near-infrared dyes and their biological applications.

Near-infrared dyes possess the qualities of lower interference with biological autofluorescence, low photon scattering, and deep tissue penetration, and are being increasingly involved in the development of biomaterials for sensing and precision medicine. However, dyes usually suffer from the disadvantages of poor water solubility and photobleaching, factors that limit their application in vivo. The introduction of supramolecular ensembles can provide an ideal solution. This review presents recently developed supramolecular ensembles modified by near-infrared dyes. Compared with small-molecule fluorophores, the specific size of a supramolecular-based fluorophore endows it with longer circulation time in the bloodstream, increasing its chances of reaching a specific target. In addition, the construction of supramolecule-based fluorophores with versatile functions can be achieved by simple encapsulation or doping, instead of by complicated chemical synthesis. Thus, supramolecular-complex-based fluorophores offer high potential in diagnosis and therapy. This review outlines four different species of near-infrared dye based ensembles in terms of their method of formation, including simple encapsulation or doping and copolymerisation. Recently, a new technology has employed modified fluorophores for in situ self-assembly that form supramolecular ensembles at a specific position, thus solving the problem of poor uptake of nanoparticles by cells, and is included in this review. Finally, the future of this field is considered.

Molecularly imprinted photoelectrochemical sensing based on ZnO/polypyrrole nanocomposites for acrylamide detection.

A highly sensitive quenching molecular imprinting (MIP) photoelectrochemical (PEC) sensor was proposed to detect acrylamide (AM) by using the photoactive composite of ZnO and polypyrrole (PPy) as the PEC signal probe. ZnO, with high electron mobility, excellent chemical and thermal stability as well as good biocompatibility, was selected as the photoelectrically active material. A polypyrrole film was formed on the nanodisk ZnO by electrochemical polymerization, and the recognition site of AM was left on the surface of the PPy film by elution, enabling the specific detection of AM. The transfer of electrons will be hindered when AM is adsorbed on the ZnO/PPy nanocomposites surface, which results in the decrease of photocurrent signal. The proposed molecularly imprinted PEC sensor exhibits significant detection performance of AM in the range of 10-1 M-2.5 × 10-9 M with a LOD of 2.147 × 10-9 M (S/N = 3). The use of photoelectrochemical technology combined with molecular imprinting technology enables the PEC sensor to have excellent selectivity, superior repeatability, preferable stability, low cost, and easy construction, providing a new method for the detection of AM. The high recovery rate in the detection of real samples of potato chips and biscuits indicates that the proposed PEC sensor has potential in monitoring the emerging food safety risks.

A ratiometric electrochemiluminescence resonance energy transfer platform based on novel dye BODIPY derivatives for sensitive detection of lactoferrin.

A ratiometric electrochemiluminescence resonance energy transfer (ECL-RET) platform depended on novel dye BODIPY derivatives was proposed for rapid detection of lactoferrin. This ECL-RET platform is composed of aptamer decorated BODIPY composites and C60@BSA, in which BODIPY derivative is the ECL probe and can generate significant resonance energy transfer with K2S2O8. BODIPY derivative and K2S2O8 are used as built-in reference signal and calibration signal respectively to eliminate background signal and abnormal change signal by double signal self-calibration process. At the same time, C60, as the accelerator of K2S2O8, can effectively increase the ECL signal and further transfer as much energy as possible to BODIPY derivative. Under optimal conditions, the constructed ECL-RET platform exhibited sensitive detection of lactoferrin in the wide linear range of 10-4- 850 ng/mL with a LOD of 42 fg/mL. Meanwhile, the proposed ECL-RET aptasensor demonstrated superior stability, specificity and reproducibility, displaying favorable application value in practical diagnosis of this method.

Förster resonance energy transfer (FRET)-based small-molecule sensors and imaging agents.

In this tutorial review, we will explore recent advances in the construction and application of Förster resonance energy transfer (FRET)-based small-molecule fluorescent probes. The advantages of FRET-based fluorescent probes include: a large Stokes shift, ratiometric sensing and dual/multi-analyte responsive systems. We discuss the underlying energy donor-acceptor dye combinations and emphasise their applications for the detection or imaging of cations, anions, small neutral molecules, biomacromolecules, cellular microenvionments and dual/multi-analyte responsive systems.

A new mitochondria-targeting fluorescent probe for ratiometric detection of H2O2 in live cells.

With this research we presented a ratiometric and mitochondria-target fluorescent probe (Mito-HT) for detection of H2O2 both in vitro and in live cells. Mito-HT was constructed by direct conjugation of aryl boronate to fluorophore with three synthetic steps. The borate group is cleaved from Mito-HT in the presence of H2O2, resulting in the exposure of the hydroxyl group of the electron donating group. Then the ICT mechanism was turned on, and the fluorescence emission of Mito-HT at 493 nm was red-shifted to 562 nm, thereby achieving radiometric detection of H2O2. Mito-HT exhibited a highly selectivity towards H2O2, and this interaction can be completed within 40 min. Mito-HT could be used for quantitative detection of H2O2 (0-200 µM) through ratiometric fluorescence signal readout. And limit of detection (LOD) is approximately 0.33 µM. The relatively high stability and medium fluorescence quantum yield of Mito-HT (0.39) and Mito-HT-OH (0.43) enable clear mitochondria localization and dual-channel fluorescence imaging of H2O2 in live cells with confocal microscopy.

A new long-wavelength fluorescent probe for tracking peroxynitrite in live cells and inflammatory sites of zebrafish.

Peroxynitrite (OONO-), as a reactive oxygen species (ROS), would be mostly profoundly implicated in diseases such as inflammation in organisms. However, bioimaging of ONOO- still faces difficulties owing to the shortage of bioimaging and real-time dynamic tracking distribution of ROS in inflammation. To address this challenge, we designed and synthesized a long-wavelength fluorescent probe based on tricyanofuran (ACDM-BE), which exhibits a fast response (response time is 40 s), high selectivity and great sensitivity (LOD is approximately 21 nM) towards ONOO-. ACDM-BE was shown to be capable of detecting ONOO- in living cells and monitor the changes in ONOO- levels under the stimulus of various concentrations of SIN-1 (from 100 to 700 µM), which was successfully tracked by the fluorescence changes in live cells. It is worth noting that ACDM-BE further demonstrated its ability to track the dynamic changes of the level of ONOO- in the inflammatory sites of larval zebrafish. Thus, ACDM-BE could be employed as an efficient tool for exploiting the role of ONOO- in inflammation in living biosystems.

Two-isophorone fluorophore-based design of a ratiometric fluorescent probe and its application in the sensing of biothiols.

2,4-Dinitrobenzenesulfonyl (DNBS) has been widely used for the design of small fluorescent probes for biothiols due to its high reactivity. However, most DNBS-based fluorescent probes exhibit "off-on" fluorescence response towards biothiols due to the strong quenching effects of DBNS on the fluorophores. Herein, we present an alternative design of a ratiometric fluorescent probe based on DNBS for biothiols. A new fluorophore bearing two isophorone malononitrile structures was conjugated with DNBS to provide a target probe (CHT), which exhibited a ratiometric sensing behavior towards biothiols. The sensing process is rapid and highly selective. Most importantly, CHT has high stability in the quantitative detection of Cys compared to the control probe CHM, which performed an "off-on" sensing for biothiols. Endogenous biothiols were successfully monitored with CHT in live cells through the ratiometric fluorescence signal. This new fluorophore bearing two isophorone malononitrile moieties will pave a new avenue to design ratiometric fluorescent probes for imaging and quantitative detection.

Correction: ESIPT-based ratiometric fluorescence probe for the intracellular imaging of peroxynitrite.

Correction for 'ESIPT-based ratiometric fluorescence probe for the intracellular imaging of peroxynitrite' by Luling Wu et al., Chem. Commun., 2018, 54, 9953-9956.

A versatile probe for serum albumin and its application for monitoring wounds in live zebrafish.

Serum albumins perform various biological functions in bioorganisms, and abnormal levels of serum albumin are predictors of many diseases; contrary to the various approaches developed for the detection of serum albumins in blood and urine samples, limited tools are available for tracing and exploring endogenous serum albumins in living bioorganisms. Specifically, fluorescent probes have not been used for the exploration of endogenous serum albumins in zebrafish. Herein, we presented a versatile fluorescent probe (C7H) that highly specifically interacted with the site I of serum albumins. We succeeded in developing C7H as a visualized tool for tracing endogenous serum albumins in living larval zebrafish. Furthermore, C7H could be used as a fluorescent probe for the real-time monitoring of the wound area in larval zebrafish. Our results suggest that the larval zebrafish has a strong self-repair capacity for wound areas. Thus, C7H could be an efficient probe for studying wound healing in live zebrafish. Moreover, C7H can significantly expand the further understanding of wound healing in zebrafish, which may also promote the research on wound healing in human beings.