Emerging applications of near-infrared fluorescent metal nanoclusters for biological imaging

Recent advances in the development of near-infrared fluorescent metal nanoclusters for bioimaging applications have been thoroughly overviewed.     

双光子荧光探针研究及其应用

双光子荧光显微成像兼具诸如近红外激发、暗场成像、避免荧光漂白和光致毒、定靶激发、高横向分辨率与纵向分辨率、降低生物组织吸光系数及降低组织自发荧光干扰等特点而显著地优于单光子荧光显微成像,为生命科学研究提供了更为锐利的工具。而用于像离子的含量及其对生理的影响、离子参与的生理活动机制、离子与分子的作用、特定分子的分布及其相互作用等方面研究的双光子荧光探针,是实现成像的关键。双光子荧光探针的研究旨在促进双光子荧光显微镜应用的发展,促进生命科学、医学科学的快速发展,同时也带动双光子荧光探针所隶属的化学这一学科的发展。因此对双光子荧光探针的研究具有重要的理论和实践意义。该文综述了双光子荧光显微成像的优点、双光子荧光探针设计的原理及双光子荧光探针在离子分析方面的应用,并展望了这类荧光探针的发展趋势与应用前景。     

Sensitive Near-Infrared Fluorescent Probes for Thiols Based on Se?N Bond Cleavage: Imaging in Living Cells and Tissues

Cy-NiSe and Cy-TfSe were designed and synthesized as sensitive near-infrared (NIR) fluorescent probes for detecting thiols on the basis of Se N bond cleavage both in cells and in tissues. Since a donor-excited photoinduced electron transfer (d-PET) process occurs between the modulator and the fluorophore, Cy-NiSe and Cy-TfSe have weak fluorescence. On titration with glutathione, the free dye exhibits significant fluorescence enhancement. The two probes are sensitive and selective for thiols over other relevant biological species. They can function rapidly at pH 7.4, and their emission lies in the NIR region. Confocal imaging confirms that Cy-NiSe and Cy-TfSe can be used for detecting thiols in living cells and tissues.     

A Fluorescent Probe for Rapid,High‐Contrast Visualization of Folate‐Receptor‐Expressing Tumors In Vivo

Folate receptors (FRs) are membrane proteins involved in folic acid uptake, and the alpha isoform (FR‐α) is overexpressed in ovarian and endometrial cancer cells. For fluorescence imaging of FRs in vivo, the near‐infrared (NIR) region (650–900 nm), in which tissue penetration is high and autofluorescence is low, is optimal, but existing NIR fluorescent probes targeting FR‐α show high non‐specific tissue adsorption, and require prolonged washout to visualize tumors. We have designed and synthesized a new NIR fluorescent probe, FolateSiR‐1 , utilizing a Si‐rhodamine fluorophore having a carboxy group at the benzene moiety, coupled to a folate ligand moiety through a negatively charged tripeptide linker. This probe exhibits very low background fluorescence and afforded a tumor‐to‐background ratio (TBR) of up to 83 in FR‐expressing tumor‐bearing mice within 30 min. Thus, FolateSiR‐1 has the potential to contribute to the research in the field of biology and the clinical medicine.     

Near‐Infrared Fluorescent Molecular Probe for Sensitive Imaging of Keloid

Early detection of skin diseases is imperative for their effective treatment. However, fluorescence molecular probes that allow this are rare. The first activatable near‐infrared (NIR) fluorescent molecular probe is reported for sensitive imaging of keloid cells, skin cells from abnormal scar fibrous lesions. As keloid cells have high expression levels of fibroblast activation protein‐alpha (FAPα), the probe (FNP1) is designed to have a caged NIR dye and a FAPα‐cleavable peptide substrate linked by a self‐immolative segment. FNP1 can quickly and specifically turn on its fluorescence at 710 nm by 45‐fold in the presence of FAPα, allowing it to effectively recognize keloid cells from normal skin cells. Integration of FNP1 with a simple microneedle‐assisted topical application enables sensitive detection of keloid cells in metabolically‐active human skin tissue with a theoretical limit of detection down to 20 000 cells.     

Rational Design of a Near‐infrared Fluorescence Probe for Ca2+ Based on Phosphorus‐substituted Rhodamines Utilizing Photoinduced Electron Transfer

Fluorescence imaging in the near‐infrared (NIR) region (650–900 nm) is useful for bioimaging because background autofluorescence is low and tissue penetration is high in this range. In addition, NIR fluorescence is useful as a complementary color window to green and red for multicolor imaging. Here, we compared the photoinduced electron transfer (PeT)‐mediated fluorescence quenching of silicon‐ and phosphorus‐substituted rhodamines (SiRs and PRs) in order to guide the development of improved far‐red to NIR fluorescent dyes. The results of density functional theory calculations and photophysical evaluation of a series of newly synthesized PRs confirmed that the fluorescence of PRs was more susceptible than that of SiRs to quenching via PeT. Based on this, we designed and synthesized a NIR fluorescence probe for Ca²⁺, CaPR‐1 , and its membrane‐permeable acetoxymethyl derivative, CaPR‐1 AM , which is distributed to the cytosol, in marked contrast to our previously reported Ca²⁺ far‐red to NIR fluorescence probe based on the SiR scaffold, CaSiR‐1 AM , which is mainly localized in lysosomes as well as cytosol in living cells. CaPR‐1 showed longer‐wavelength absorption and emission (up to 712 nm) than CaSiR‐1 . The new probe was able to image Ca²⁺ at dendrites and spines in brain slices, and should be a useful tool in neuroscience research.     

Design strategy for a near-infrared fluorescence probe for matrix metalloproteinase utilizing highly cell permeable boron dipyrromethene

Near-infrared (NIR) fluorescence probes are especially useful for simple and noninvasive in vivo imaging inside the body because of low autofluorescence and high tissue transparency in the NIR region compared with other wavelength regions. However, existing NIR fluorescence probes for matrix metalloproteinases (MMPs), which are tumor, atherosclerosis, and inflammation markers, have various disadvantages, especially as regards sensitivity. Here, we report a novel design strategy to obtain a NIR fluorescence probe that is rapidly internalized by free diffusion and well retained intracellularly after activation by extracellular MMPs. We designed and synthesized four candidate probes, each consisting of a cell permeable or nonpermeable NIR fluorescent dye as a F?rster resonance energy transfer (FRET) donor linked to the NIR dark quencher BHQ-3 as a FRET acceptor via a MMP substrate peptide. We applied these probes for detection of the MMP activity of cultured HT-1080 cells, which express MMP2 and MT1-MMP, by fluorescence microscopy. Among them, the probe incorporating BODIPY650/665, BODIPY-MMP, clearly visualized the MMP activity as an increment of fluorescence inside the cells. We then applied this probe to a mouse xenograft tumor model prepared with HT-1080 cells. Following intratumoral injection of the probe, MMP activity could be visualized for much longer with BODIPY-MMP than with the probe containing SulfoCy5, which is cell impermeable and consequently readily washed out of the tissue. This simple design strategy should be applicable to develop a range of sensitive, rapidly responsive NIR fluorescence probes not only for MMP activity, but also for other proteases.     

An Activatable Polymeric Reporter for Near-Infrared Fluorescent and Photoacoustic Imaging of Invasive Cancer

Discriminative detection of invasive and noninvasive breast cancers is crucial for their effective treatment and prognosis. However, activatable probes able to do so in vivo are rare. Herein, we report an activatable polymeric reporter (P-Dex) that specifically turns on near-infrared (NIR) fluorescent and photoacoustic (PA) signals in response to the urokinase-type plasminogen activator (uPA) overexpressed in invasive breast cancer. P-Dex has a renal-clearable dextran backbone that is linked with a NIR dye caged with an uPA-cleavable peptide substrate. Such a molecular design allows P-Dex to passively target tumors, activate NIR fluorescence and PA signals to effectively distinguish invasive MDA-MB-231 breast cancer from noninvasive MCF-7 breast cancer, and ultimately undergo renal clearance to minimize the toxicity potential. Thus, this polymeric reporter holds great promise for the early detection of malignant breast cancer.     

Synthesis and characterization of glucosamine-bound near-infrared probes for optical imaging

[structure: see text] Two novel near-infrared (NIR) fluorescent probes have been synthesized by linking a carbocyanine fluorophore and glucosamine through different linkers. These probes demonstrated a high quantum yield, low cytotoxicity, reversible pH-dependent fluorescence in the physiological pH range, and a decreased aggregation tendency in aqueous solutions. In vitro NIR optical imaging studies revealed cellular uptake and strong intracellular NIR fluorescence of these two probes in four breast epithelial cell lines.     

An Activatable Polymeric Reporter for Near‐Infrared Fluorescent and Photoacoustic Imaging of Invasive Cancer

Discriminative detection of invasive and noninvasive breast cancers is crucial for their effective treatment and prognosis. However, activatable probes able to do so in vivo are rare. Herein, we report an activatable polymeric reporter (P‐Dex) that specifically turns on near‐infrared (NIR) fluorescent and photoacoustic (PA) signals in response to the urokinase‐type plasminogen activator (uPA) overexpressed in invasive breast cancer. P‐Dex has a renal‐clearable dextran backbone that is linked with a NIR dye caged with an uPA‐cleavable peptide substrate. Such a molecular design allows P‐Dex to passively target tumors, activate NIR fluorescence and PA signals to effectively distinguish invasive MDA‐MB‐231 breast cancer from noninvasive MCF‐7 breast cancer, and ultimately undergo renal clearance to minimize the toxicity potential. Thus, this polymeric reporter holds great promise for the early detection of malignant breast cancer.     

Design and synthesis of squaraine based near infrared fluorescent probes

A novel class of dialkylanthracene containing squaraine dyes (Sq1-3) possessing intense absorption and emission in the NIR region has been synthesized. Structural and electronic features investigated using DFT methods suggest that the significant bathochromic shifts observed on replacing dialkylaniline by dialkylanthracene in this class of molecules can be attributed to a reduction in the HOMO-LUMO gap mainly due to enhanced hydrogen bonding between the carbonyl group of the cyclobutane ring and the neighboring aromatic hydrogen in the dyes containing the anthracene moiety. The absence of fluorescence in aqueous media and high fluorescence when encapsulated into hydrophobic domains make this class of dyes especially useful as probes for mapping such domains in biological systems.     

A near-infrared fluorescent probe for monitoring fluvastatin-stimulated endogenous H2S production

Most reported fluorescent probes have limitations in practical applications in living systems due to the strong autofluorescence background,construction of probes with near-infrared(NIR) fluorescence emission is an accessible approach for addressing this challenge.We here designed a NIR fluorescent probe for monitoring the endogenous production of H_2S in living cells.The designed probe showed significant NIR fluorescence turn-on response to H_2S with high selectivity,enabling the sensitive detection H_2S.Importantly,the probe could be applied in monitoring the endogenous production of H_2S in raw 264.7 macrophages.This study showed that fluvastatin can promote the activity of cystathionineγ-lyase(CSE) for generation H_2S.     

Highly sensitive near-infrared fluorescent probes for nitric oxide and their application to isolated organs

Novel near-infrared (NIR) fluorescent probes for nitric oxide (NO) have been designed, synthesized, and evaluated. Their NIR fluorescence was increased in an NO concentration-dependent manner under physiological conditions, and their reaction efficiency with NO was at least 53 times higher than that of a widely used NO probe, DAF-2. They were confirmed to function in isolated intact rat kidneys. Because NIR light can penetrate deeply into tissues, these probes may have potential for in vivo NO imaging.     

手术导航用荧光探针

荧光成像技术因具有操作简便、分辨率高、安全性好且可实时成像等优势,在术中导航中具有广阔的应用前景.虽然目前还没有靶向荧光探针在临床上得到批准,但已经有相当一部分荧光探针进入了临床试验阶段.最早进入临床试验的是一些偶联肿瘤靶向配体的荧光染料,例如近红外菁染料(IRDye800CW)标记的肿瘤特异抗体,叶酸标记的异硫氰酸荧...     

Thieno–Pyrrole‐Fused BODIPY Intermediate as a Platform to Multifunctional NIR Agents

We report the synthesis, photophysical and electrochemical properties, and in vivo fluorescence imaging of a series of new thieno–pyrrole‐fused near‐infrared (NIR) BODIPY agents by using a versatile intermediate as a building block. The versatile thieno–pyrrole‐fused BODIPY intermediate was rationally designed to bear bromo‐substituents and absorb in the mid‐red region (635 nm) to act as an organic electrophile for the development of NIR multifunctional agents. The use of subsequent palladium‐catalyzed and nucleophilic substitution reactions afforded highly conjugated NIR BODIPYs. The novel BODIPYs exhibit long‐wavelength absorptions in the NIR region (650–840 nm). The agents produce sharp fluorescence bands, and most of them display respectable quantum yields of fluorescence (0.05–0.87) useful for biomedical imaging, as demonstrated by in vivo imaging with SBDPiR740 . Interestingly, a number of agents in the series that are non‐halogenated were reactive to O₂ at the triplet photo‐excited state coupled with a favorable redox potential and decent fluorescence, and hence could be potential candidates for use as photosensitizers in fluorescence‐guided photodynamic therapy. Furthermore, the synthetic approach allows further functionalization of the highly conjugated NIR BODIPYs to tune the excited states (PET, ICT) and to conjugate targeting moieties for enhanced biological applications.     

Rhodamine-inspired far-red to near-infrared dyes and their application as fluorescence probes

Probes to dye for: Rhodamine-inspired Si-pyronine, Si-rhodamine, Te-rhodamine, and Changsha NIR dyes have been developed recently. These dyes show fluorescence in the far-red to near-infrared region, while retaining the advantages of the original rhodamines, such as high fluorescence quantum yield, tolerance to photobleaching, good water solubility, and exhibit great potential for biological application.     

A unique class of near-infrared functional fluorescent dyes with carboxylic-acid-modulated fluorescence ON/OFF switching: rational design, synthesis, optical properties, theoretical calculations, and applications for fluorescence imaging in living animals

Fluorescence imaging is one of the most powerful techniques for monitoring biomolecules in living systems. Fluorescent sensors with absorption and emission in the near-infrared (NIR) region are favorable for biological imaging applications in living animals, as NIR light leads to minimum photodamage, deep tissue penetration, and minimum background autofluorescence interference. Herein, we have introduced a new strategy to design NIR functional dyes with the carboxylic-acid-controlled fluorescence on-off switching mechanism by the spirocyclization. Based on the design strategy, we have developed a series of Changsha (CS1-6) NIR fluorophores, a unique new class of NIR functional fluorescent dyes, bearing excellent photophysical properties including large absorption extinction coefficients, high fluorescence quantum yields, high brightness, good photostability, and sufficient chemical stability. Significantly, the new CS1-6 NIR dyes are superior to the traditional rhodamine dyes with both absorption and emission in the NIR region while retaining the rhodamine-like fluorescence ON-OFF switching mechanism. In addition, we have performed quantum chemical calculations with the B3LYP exchange functional employing 6-31G* basis sets to shed light on the structure-optical properties of the new CS1-6 NIR dyes. Furthermore, using CS2 as a platform, we further constructed the novel NIR fluorescent TURN-ON sensor 7, which is capable of imaging endogenously produced HClO in the living animals, demonstrating the value of our new CS NIR functional fluorescent dyes. We expect that the design strategy may be extended for development of a wide variety of NIR functional dyes with a suitable fluorescence-controlled mechanism for many useful applications in biological studies.     

Phenoxazine‐based Near‐infrared Fluorescent Probes for the Specific Detection of Copper (II) Ions in Living Cells

Abstract : It is well known that copper ions play a critical role in various physiological processes. However, a variety of human diseases are tightly correlated with copper overload. Although there are numerous fluorescent probes capable of detecting copper ions, most of them are “turn‐off” probes owing to copper (II) ions fluorescence quenching effect, resulting in poor sensitivity. Herein, a novel “turn‐on” near‐infrared (NIR) fluorescent probe PZ‐N based on phenoxazine was designed and synthesized for the selective detection of copper (II) ions (Cu²⁺). Upon the addition of Cu²⁺, the probe could quickly react with Cu²⁺ and emit strong fluorescence, along with colour change from colourless to obvious blue. Moreover, the probe PZ‐N showed good water solubility, high selectivity, and excellent sensitivity with low limit of detection (1.93 nM) towards copper (II) ions. More importantly, PZ‐N was capable of effectively detecting Cu²⁺ in living cells.     

Sensitive near-infrared fluorescent probes for thiols based on sen bond cleavage: imaging in living cells and tissues

Cy-NiSe and Cy-TfSe were designed and synthesized as sensitive near-infrared (NIR) fluorescent probes for detecting thiols on the basis of Se?N bond cleavage both in cells and in tissues. Since a donor-excited photoinduced electron transfer (d-PET) process occurs between the modulator and the fluorophore, Cy-NiSe and Cy-TfSe have weak fluorescence. On titration with glutathione, the free dye exhibits significant fluorescence enhancement. The two probes are sensitive and selective for thiols over other relevant biological species. They can function rapidly at pH?7.4, and their emission lies in the NIR region. Confocal imaging confirms that Cy-NiSe and Cy-TfSe can be used for detecting thiols in living cells and tissues.     

荧光素类荧光分子探针

荧光素衍生物是重要的荧光探针,在检测和生物成像等领域中显示出巨大的前景。因此,急需对功能性荧光素结构探针的设计策略进行深入研究。通常通过引入醛基或酯化到荧光素呫吨环和苯部分来构建探针,由于其高活性,这些衍生物可以与分析物复合以发生颜色和荧光强度的变化。本文总结了荧光素的修饰位点及方法,介绍了荧光素探针的合成、性质及应用,并对近五年荧光素探针对不同分析物(包括金属阳离子、阴离子、小分子和生物大分子)的检测进行分类说明,旨在为高灵敏度荧光素探针的筛选和生物检测提供参考,并推动其在分析物传感和检测中的进一步应用。