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Abnormal physiological processes and diseases can lead to content or activity fluctuations of biocomponents in organelles and whole blood. However, precise monitoring of these abnormalities remains extremely challenging due to the insufficient sensitivity and accuracy of available fluorescence probes, which can be attributed to the background fluorescence arising from two sources, 1) biocomponent autofluorescence (BCAF) and 2) probe intrinsic fluorescence (PIF). To overcome these obstacles, we have re‐engineered far‐red to NIR II rhodol derivatives that possess weak BCAF interference. And a series of "zero" PIF sensing‐platforms were created by systematically regulating the open‐loop/spirocyclic forms. Leveraging these advancements, we devised various ultra‐sensitive NIR indicators, achieving substantial fluorescence boosts (190 to 1300‐fold). Among these indicators, 8‐LAP demonstrated accurate tracking and quantifying of leucine aminopeptidase (LAP) in whole blood at various stages of tumor metastasis. Furthermore, coupling 8‐LAP with an endoplasmic reticulum‐targeting element enabled the detection of ERAP1 activity in HCT116 cells with p53 abnormalities. This delicate design of eliminating PIF provides insights into enhancing the sensitivity and accuracy of existing fluorescence probes toward the detection and imaging of biocomponents in abnormal physiological processes and diseases. [ABSTRACT FROM AUTHOR]

Abnormal physiological processes and diseases can lead to content or activity fluctuations of biocomponents in organelles and whole blood. However, precise monitoring of these abnormalities remains extremely challenging due to the insufficient sensitivity and accuracy of available fluorescence probes, which can be attributed to the background fluorescence arising from two sources, 1) biocomponent autofluorescence (BCAF) and 2) probe intrinsic fluorescence (PIF). To overcome these obstacles, we have re‐engineered far‐red to NIR II rhodol derivatives that possess weak BCAF interference. And a series of "zero" PIF sensing‐platforms were created by systematically regulating the open‐loop/spirocyclic forms. Leveraging these advancements, we devised various ultra‐sensitive NIR indicators, achieving substantial fluorescence boosts (190 to 1300‐fold). Among these indicators, 8‐LAP demonstrated accurate tracking and quantifying of leucine aminopeptidase (LAP) in whole blood at various stages of tumor metastasis. Furthermore, coupling 8‐LAP with an endoplasmic reticulum‐targeting element enabled the detection of ERAP1 activity in HCT116 cells with p53 abnormalities. This delicate design of eliminating PIF provides insights into enhancing the sensitivity and accuracy of existing fluorescence probes toward the detection and imaging of biocomponents in abnormal physiological processes and diseases. [ABSTRACT FROM AUTHOR]

Fluorescence imaging utilizing traditional organic fluorophores is extensively applied in both cellular and in vivo studies. However, it faces significant obstacles, such as low signal‐to‐background ratio (SBR) and spurious positive/negative signals, primarily due to the facile diffusion of these fluorophores. To cope with this challenge, orderly self‐assembled functionalized organic fluorophores have gained significant attention in the past decades. These fluorophores can create nanoaggregates via a well‐ordered self‐assembly process, thus prolonging their residency time within cells and in vivo settings. The development of self‐assembled‐based fluorophores is an emerging field, and as such, in this review, we present a summary of the progress and challenges of self‐assembly fluorophores, focusing on their development history, self‐assembly mechanisms, and biomedical applications. We hope that the insights provided herein will assist scientists in further developing functionalized organic fluorophores for in situ imaging, sensing, and therapy. [ABSTRACT FROM AUTHOR]

Fluorescence imaging utilizing traditional organic fluorophores is extensively applied in both cellular and in vivo studies. However, it faces significant obstacles, such as low signal‐to‐background ratio (SBR) and spurious positive/negative signals, primarily due to the facile diffusion of these fluorophores. To cope with this challenge, orderly self‐assembled functionalized organic fluorophores have gained significant attention in the past decades. These fluorophores can create nanoaggregates via a well‐ordered self‐assembly process, thus prolonging their residency time within cells and in vivo settings. The development of self‐assembled‐based fluorophores is an emerging field, and as such, in this review, we present a summary of the progress and challenges of self‐assembly fluorophores, focusing on their development history, self‐assembly mechanisms, and biomedical applications. We hope that the insights provided herein will assist scientists in further developing functionalized organic fluorophores for in situ imaging, sensing, and therapy. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: Fluorophores play a significant role in achieving high‐contrast imaging in complex physiological environment. Herein, we present the results of our studies on the development of various fluorophores, such as anti‐solvatochromic fluorophores, long wavelength emission two‐photon fluorophores, large Stokes shift fluorophores and solid‐state fluorophores, to achieve high‐contrast bioimaging in physiological environments. In this study, we have primarily focused on the design, optical performance, imaging mechanisms, and the applications of these fluorophores with respect to complex physiological conditions. What is the most favorite and original chemistry developed in your research group? Orderly assembly of small‐molecule dyes and their applications for long‐term in situ bioimaging. How do you get into this specific field? Could you please share some experiences with our readers? We have long been engaged in the field of fluorescence imaging for biomedical applications, and realized that obtaining the target's in situ information is urgent and challenging. Our exploration revealed that the orderly assembly of specific molecular dyes is an effective strategy to overcome this problem. Keeping curiosity and thinking is the key to solving problems in scientific research. What is the most important personality for scientific research? Clear mind and perseverance. What's your hobby? Communicating with Youngs. How do you keep balance between research and family? To make a proper allotment of time. How do you supervise your students? Let them know what they want and fight for it. [ABSTRACT FROM AUTHOR]

Abstract: The design and synthesis of a porphyrin-appended terpyridine, 5-(4-([2,2′:6′,2″]-terpyridin-4-yl-carboxyamidyl)phenyl)-10,15,20-triphenylporphyrin (H2TPPTPy) and its application as potential fluoroionophore for recognition of metal ions are reported. For preparation of the fluoroionophore, a novel simple strategy with improved total yield has been applied for the synthesis of 2,2′:6′,2″-terpyridine-4′-carboxylic acid as a ligand. H2TPPTPy shows chelation-enhanced fluorescence effect with cadmium ion via the interruption of photoinduced electron transfer (PET) process, which has been utilized as the basis of the fabrication of the Cd(II)-sensitive fluorescent chemosensor. The analytical performance characteristics of the proposed Cd(II)-sensitive chemosensor were investigated. It shows a linear response toward Cd(II) in the concentration range of 3.2×10−6 to 3.2×10−4 M with a limit of detection of 1.2×10−6 M. The chemosensor shows good selectivity for Cd(II) over a large number of cations, such as alkali, alkali earth and transitional metal ions except Cu(II) and Zn(II). The sensor has been used for determination of Cd(II) in water samples with satisfactory recoveries. [Copyright &y& Elsevier]

The in situ and real-time supervision of reactive oxygen species (ROS) generated during photodynamic therapy (PDT) is of great significance for lessening nonspecific damage and guiding personalized therapy. However, photosensitizers frequently fail to deliver successful treatment accompanying the ROS-related imaging signals produced, impeding simple treatment outcome predictions and therapeutic schedule adjustments. Here, we report a two-photon fluorescence self-reporting strategy for the in situ and real-time monitoring of treatment response via a novel black phosphorus-based two-photon nanoprobe (TPBP). TPBP effectively generated singlet oxygen (1O2) under near-infrared laser irradiation for PDT, and 1O2 stimulated a two-photon molecule to emit fluorescence signals for feedback of 1O2 generation, which facilitated the regulation of treatment parameters to achieve precise and personalized medicine in deep tissue. [ABSTRACT FROM AUTHOR]

A FRET-based probe for mapping the fluctuation of ONOO− in cisplatin-induced acute kidney injury was constructed. It exhibits ratiometric near infrared fluorescence and a dramatic decrease of its peak absorbance at 719 nm upon addition of ONOO− that is converted into remarkable signal changes in fluorescence and photoacoustic images respectively. [ABSTRACT FROM AUTHOR]

Abstract A novel ratiometric and reversible chemosensor 4-((2-(Benzo[d]thiazol-2-yl)phenyl)ethynyl)- N,N -diethylaniline (BT-1) based on ortho -arylethynyl benzothiazole with large Stokes shift (Δλ≈190 nm) was designed and synthesized to recognize Cu2+. Copper ion induces a remarkable fluorescence enhancement and causes formation of a BT-1-Cu complex. The clamp-on coordination mode of BT-1 to Cu2+ was demonstrated using Job's plot, mass spectrum (MS) and DFT calculations. The calculations also indicate that Cu2+ was chelated to BT-1 through N and alkyne instead of S and alkyne. The probe could quantify Cu2+ with the detection limit of 3.2 × 10−9 M. The in vitro imaging results indicated that the probe BT-1 was membrane-permeable and could be applied into the recognition of Cu2+ ions in living cells. Graphical abstract Image 1 Highlights 1. A novel ratiometric and reversible fluorescence probe with a large stokes shift based on a new clamp-on unit. 2. It is the first example that utilizes aryl alkyne coordination in the determination of copper ions. 3. The probe BT-1 was membrane-permeable and could be applied into the recognition of Cu2+ ions in living cells. [ABSTRACT FROM AUTHOR]

Fluorescent probes have become an indispensable tool in the detection and imaging of biological and disease‐related analytes due to their sensitivity and technical simplicity. In particular, fluorescent probes with far‐red to near‐infrared (FR‐NIR) emissions are very attractive for biomedical applications. However, many available FR‐NIR fluorophores suffer from small Stokes shifts and sometimes low quantum yields, resulting in self‐quenching and low contrast. In this work, we describe the rational design and engineering of FR‐NIR 2,4,6‐triphenylpyrylium‐based fluorophores (TPP‐Fluors) with the help of theoretical calculations. Our strategy is based on the appending of electron‐donating substituents and fusing groups onto 2,4,6‐triphenylpyrylium. In contrast to the parent TPP with short emission wavelength, weak quantum yields, and low chemical stability, the obtained novel TPP‐Fluors display some favorable properties, such as long‐wavelength emission, large Stokes shifts, moderate to high quantum yields, and chemical stability. TPP‐Fluors demonstrate their biological value as mitochondria‐specific labeling reagents due to their inherently positive nature. In addition, TPP‐Fluors can also be applied to develop ratiometric fluorescent probes, as the electron‐donating ability of the 2,6‐phenyl substituents is closely correlated with their emission wavelength. A proof‐of‐concept ratiometric probe has been developed by derivatizing the amino groups of TPP‐Fluor for the detection and imaging of nitroreductase in vitro and in hypoxic cells. [ABSTRACT FROM AUTHOR]

Abstract: Two‐photon imaging is an emerging tool for biomedical research and clinical diagnostics. Electron donor–acceptor (D–A) type molecules are the most widely employed two‐photon scaffolds. However, current D–A type fluorophores suffer from solvatochromic quenching in aqueous biological samples. To address this issue, we devised a novel class of D–A type green fluorescent protein (GFP) chromophore analogues that form a hydrogen‐bond network in water to improve the two‐photon efficiency. Our design results in two‐photon chalcone (TPC) dyes with 0.80 quantum yield and large two‐photon action cross section (210 GM) in water. This strategy to form hydrogen bonds can be generalized to design two‐photon materials with anti‐solvatochromic fluorescence. To demonstrate the improved in vivo imaging, we designed a sulfide probe based on TPC dyes and monitored endogenous H2S generation and scavenging in the cirrhotic rat liver for the first time. [ABSTRACT FROM AUTHOR]

Abstract: Two‐photon imaging is an emerging tool for biomedical research and clinical diagnostics. Electron donor–acceptor (D–A) type molecules are the most widely employed two‐photon scaffolds. However, current D–A type fluorophores suffer from solvatochromic quenching in aqueous biological samples. To address this issue, we devised a novel class of D–A type green fluorescent protein (GFP) chromophore analogues that form a hydrogen‐bond network in water to improve the two‐photon efficiency. Our design results in two‐photon chalcone (TPC) dyes with 0.80 quantum yield and large two‐photon action cross section (210 GM) in water. This strategy to form hydrogen bonds can be generalized to design two‐photon materials with anti‐solvatochromic fluorescence. To demonstrate the improved in vivo imaging, we designed a sulfide probe based on TPC dyes and monitored endogenous H2S generation and scavenging in the cirrhotic rat liver for the first time. [ABSTRACT FROM AUTHOR]

Superoxide anion radical (O 2 − ), the “primary” reactive oxygen species (ROS) in living systems, is linked to a variety of physiological and pathological processes. Therefore, developing an effective strategy to monitor the fluctuation of O 2 − in biological systems is of great importance. This paper describes a new turn-on two-photon fluorescent probe for endogenous O 2 − detection and imaging, which was rationally designed and synthesized via a non-redox strategy. In the presence of O 2 − , the probe exhibited notable fluorescence enhancement (∼235-fold) with a low detection limit down to 1 nM, indicating a high signal-to-background ratio and excellent sensitivity. In addition, short response time, good biocompatibility, low cytotoxicity, long-term stability against light illumination, specificity to O 2 − over general reductants, and pH stability were demonstrated, indicating that the requirements for cellular O 2 − determination are met. Furthermore, the probe was successfully applied in two-photon fluorescence imaging of endogenous O 2 − in living cells and tissues and showed high imaging resolution and a deep-tissue imaging depth of ∼150 μm, illustrating the promising potential for practical applications in complex biosystems and providing a valuable theoretical basis and technical support for the study of physiological and pathological functions of O 2 − . [ABSTRACT FROM AUTHOR]

Two-dimensional graphitic carbon nitride nanosheets (CNNSs) with planar graphene-like structure have stimulated increasingly research interest in recent years due to their unique physicochemical properties. CNNSs possess superior stability, high fluorescence quantum yield, low-toxicity, excellent biocompatibility, unique electroluminescent and photoelectrochemical properties, which make them appropriate candidates for biosensing. In this review, we first introduce the preparation and unique properties of CNNSs, with emphasis on their superior properties for biosensing. Then, recent advances of CNNSs in photoelectrochemical biosensing, electrochemiluminescence biosensing and fluorescence biosensing are highlighted. An additional attention is paid to the marriage of CNNSs and nucleic acids, which exhibits great potentials in both biosensing and intracellular imaging. Finally, current challenges and opportunities of this 2D material are outlined. Inspired by the unique properties of CNNSs and their advantages in biological applications, we expect that more attention will be drawn to this promising 2D material and extensive applications can be found in bioanalysis and diseases diagnosis. [ABSTRACT FROM AUTHOR]

SO 2 and its derivatives (bisulfite/sulfite) play crucial roles in several physiological processes. Therefore, development of reliable analytical methods for monitoring SO 2 and its derivatives in biological systems is very significant. In this paper, a FRET-based two-photon fluorescent turn-on probe, A-HCy , was proposed for specific detection of SO 2 derivatives through the bisulfite/sulfite-promoted Michael addition reaction. In this FRET system, an acedan (2-acetyl-6-dialkylaminonaphthalene) moiety was selected as a two-photon donor and a hemicyanine derivative served as both the quencher and the recognition unit for bisulfite/sulfite. A-HCy exhibited excellent selectivity and rapid response to HSO 3 − with a detection limit of 0.24 μM. More importantly, probe A-HCy was first successfully applied in two-photon fluorescence imaging of biological SO 2 derivatives in living cells and tissues, suggesting its great potential for practical application in biological systems. [ABSTRACT FROM AUTHOR]

In this work we use a water-soluble cationic perylene derivative (compound 1) as the G-quadruplex (G4) structure fluorescence indicator to construct a fluorescent biosensor for simple, rapid and label-free detection of Pb 2+ . In the absence of Pb 2+ , strong electrostatic interactions between compound 1 and the G-rich DNA probe (PW17) induced the aggregation of compound 1 and resulted in the fluorescence quenching. In the presence of Pb 2+ , the PW17 formed Pb 2+ -stabilized G4 structure, which reduced the aggregation of compound 1 and gave rise to high fluorescence. This allowed us to use convenient “mix-and-detect” protocol for quantitative analysis of Pb 2+ . Since Pb 2+ can specially induce PW17 to form compact DNA fold, our proposed biosensor displayed high selectivity for Pb 2+ . It also exhibited a high sensitivity to Pb 2+ , with a limit of detection of 5.0 nM observed. Furthermore, the biosensor was applied for the detection of Pb 2+ in urine and paint samples, and both showed satisfactory results. [ABSTRACT FROM AUTHOR]

DNAzyme-based catalytic beacons have been widely studied for both in vivo and in vitro molecular detection. However, only a few label-free catalytic beacons with excellent analytical performance have been reported so far. In this work, by combining a catalytic DNAzyme for amplified sensing through enzymatic turnover with cleavage-induced G-quadruplex formation, a label-free DNAzyme biosensor was developed for amplified “turn-on” fluorescence detection of Pb 2+ with a detection limit of 3 nM. The method is very competitive compared to many other labeled or label-free methods with or without signal amplification. Due to the inherent specificity of the GR-5 DNAzyme, the method also exhibits excellent selectivity. This biosensor successfully detected Pb 2+ in river water samples with high sensitivity and selectivity. Such a method might provide a universal DNAzyme-based sensing platform for sensitive detection of various targets both in environmental and biomedical fields. [ABSTRACT FROM AUTHOR]

Abstract: Fluorescent sensing systems based on the quenching of fluorophores have found wide applications in bioassays. An efficient quencher will endow the sensing system a high sensitivity. The frequently used quenchers are based on organic molecules or nanomaterials, which usually need tedious synthesizing and modifying steps, and exhibit different quenching efficiencies to different fluorophores. In this work, we for the first time report that aggregated perylene derivative can serve as a broad-spectrum and label-free quencher that is able to efficiently quench a variety of fluorophores, such as green, red and far red dyes labeled on DNA. By choosing nucleases as model biomolecules, such a broad-spectrum quencher was then employed to construct a multiplexed bioassay platform through a label-free manner. Due to the high quenching efficiency of the aggregated perylene, the proposed platform could detect nuclease with high sensitivity, with a detection limit of 0.03U/mL for EcoRV, and 0.05U/mL for EcoRI. The perylene quencher does not affect the activity of nuclease, which makes it possible to design post-addition type bioassay platform. Moreover, the proposed platform allows simultaneous and multicolor analysis of nucleases in homogeneous solution, demonstrating its value of potential application in rapid screening of multiple bio-targets. [Copyright &y& Elsevier]

Abstract: In this work, based on the fact that pyrophosphate (PPi) could regulate the activity of Zn2+-dependent DNAzyme, we for the first time report a fluorescence turn-on sensing system for alkaline phosphatase (ALP) with improved sensitivity via nonprotein-enzymatic signal amplification. A catalytic and molecular beacon (CAMB) design was employed to further improve its sensitivity. Taking advantage of the strong interactions between PPi and the Zn2+, the cofactor Zn2+ was caged, and the DNAzyme activity was effectively inhibited. The introduction of ALP, however, could catalyze the hydrolysis of PPi and release free Zn2+, resulting in the activation of DNAzyme to catalyze the cleavage of the molecular beacon substrate with a remarkable increase of fluorescent signal. These optimized designs together allow a high sensitivity for ALP, with a detection limit of 20pM observed, much lower than previously reported methods. It has also been used for detection of ALP in human serum with satisfactory results, demonstrating its potential applications in clinical diagnosis. [Copyright &y& Elsevier]