Near-infrared (NIR) activatable fluorescent probes have been considered to be the effective edge tools for the investigation of cell biology and disease diagnosis because of their outstanding advantages. Related genes involved in tumor genesis and progression regulate the overexpression of certain enzymes. Owing to the distinctive characteristics of quick reaction time and favorable pharmacokinetics, enzyme-reactive NIR optical probes have shown great potential in the diagnosis of tumorigenesis and in image-guided intraoperative surgeries with high signal-to-noise ratios. In this review, we mainly summarize the latest advancements in enzyme-reactive NIR fluorescent probes from design strategy to biomedical application. Specifically, some challenges and prospects in this field are presented at the end of the review, which will be beneficial to innovatively construct new multifunctional fluorescent probes and actively promote their clinical transformation in the future.

Activity‐Based NIR Enzyme Fluorescent Probes for the Diagnosis of Tumors and Image‐Guided Surgery

Self-immolative chemistry features a cascade of disassembly reactions in response to external stimuli, which provides great opportunities to design new self-immolative chemosensors with advanced performance and/or functions Self-immolative spacers in these chemosensors not only facilitate the linkage of designed triggers to various chromophores or fluorophores, but can also be used to solve inherent problems associated with native chemosensors, such as low reactivities, poor stabilities and slow response times Their capacity for stimuli-responsive release through operation of a self-immolative reaction further enables integration of sophisticated functions into chemosensors, including signal amplification, enzyme activity localization, and drug monitoring Significant advances have been made in the field of self-immolative chemosensors, leading to intriguing applications to sensitive detection of analytes, bioimaging and cancer theranostics This tutorial review summarizes this recent progress with a focus on their design strategies and sensing mechanisms

Self-immolative colorimetric, fluorescent and chemiluminescent chemosensors

Emerging from the origins of supramolecular chemistry and the development of selective chemical receptors that rely on lock-and-key binding, activity-based sensing (ABS)-which utilizes molecular reactivity rather than molecular recognition for analyte detection-has rapidly grown into a distinct field to investigate the production and regulation of chemical species that mediate biological signaling and stress pathways, particularly metal ions and small molecules. Chemical reactions exploit the diverse chemical reactivity of biological species to enable the development of selective and sensitive synthetic methods to decipher their contributions within complex living environments. The broad utility of this reaction-driven approach facilitates application to imaging platforms ranging from fluorescence, luminescence, photoacoustic, magnetic resonance, and positron emission tomography modalities. ABS methods are also being expanded to other fields, such as drug and materials discovery.

Activity‐Based Sensing: A Synthetic Methods Approach for Selective Molecular Imaging and Beyond

Anthracyclines rank among the most efficacious anticancer medications However, their clinical utility and oncologic efficacy are severely compromised by the cardiotoxicity risk facing the early-diagnosis difficulty and their unclear molecular mechanism Herein, a two-photon-excitable and near-infrared-emissive fluorescent probe, TPNIR-FP, was fabricated and endowed with extraordinary specificity and sensitivity and a rapid response toward peroxynitrite (ONOO-), as well as mitochondria-targeting ability With the aid of TPNIR-FP, we demonstrate that mitochondrial ONOO- is upregulated in the early stage and contributes to the onset and progression of anthracycline cardiotoxicity in cardiomyocyte and mouse models; therefore, it represents an early biomarker to predict subclinical cardiotoxicity induced by drug challenge Furthermore, TPNIR-FP is proved to be a robust imaging tool to provide critical insights into drug-induced cardiotoxicity and other ONOO--related pathophysiological processes

Mitochondrial Peroxynitrite Mediation of Anthracycline-Induced Cardiotoxicity as Visualized by a Two-Photon Near-Infrared Fluorescent Probe.

Gasotransmitters and related reactive sulfur, oxygen, and nitrogen species such as nitric oxide (NO), hydrogen sul-fide (H2S), carbon monoxide (CO), hydrogen peroxide (H2O2) and downstream products like peroxynitrite (ONOO–), polysulfides (HSnH), hydroxyl radical (HO•), and azanone (nitroxyl, HNO) are produced under physiological condi-tions and impact cellular signaling and stress. Evolution-ary pressures have driven life to develop sophisticated mechanisms to control the formation and response to this complex reactive species interactome, making it a critical fulcrum that balances health and disease in human physiol-ogy. Driven by a need to understand and measure these types of reactive species, the last decade has witnessed ex-tensive research activity in developing reaction-based probes for optical detection and imaging of reactive sulfur, oxygen, and nitrogen species in living systems. – The cen-tral advantage of this approach is that it is compatible with living cells and animals, thereby enabling t...

Reaction-Based Luminescent Probes for Reactive Sulfur, Oxygen, and Nitrogen Species: Analytical Techniques and Recent Progress.

Detection of nitroxyl (HNO), the transient one-electron reduced form of nitric oxide, is a significant challenge owing to its high reactivity with biological thiols (with rate constants as high as 109 M–1 s–1). To address this, we report a new thiol-based HNO-responsive trigger that can compete against reactive thiols for HNO. This process forms a common N-hydroxysulfenamide intermediate that cyclizes to release a masked fluorophore leading to fluorescence enhancement. To ensure that the cyclization step is rapid, our design capitalizes on two established physical organic phenomena; the alpha-effect and the Thorpe-Ingold effect. Using this new trigger, we developed NitroxylFluor, a selective HNO-responsive fluorescent probe. Treatment of NitroxylFluor with an HNO donor results in a 16-fold turn-on. This probe also exhibits excellent selectivity over various reactive nitrogen, oxygen, and sulfur species and efficacy in the presence of thiols (e.g., glutathione in mM concentrations). Lastly, we successfully...

NitroxylFluor: A Thiol-Based Fluorescent Probe for Live-Cell Imaging of Nitroxyl

Cancer stem cells (CSCs) are progenitor cells that contribute to treatment-resistant phenotypes during relapse. CSCs exist in specific tissue microenvironments that cell cultures and more complex models cannot mimic. Therefore, the development of new approaches that can detect CSCs and report on specific properties (e.g., stem cell plasticity) in their native environment have profound implications for studying CSC biology. Herein, we present AlDeSense, a turn-on fluorescent probe for aldehyde dehydrogenase 1A1 (ALDH1A1) and Ctrl-AlDeSense, a matching nonresponsive reagent. Although ALDH1A1 contributes to the detoxification of reactive aldehydes, it is also associated with stemness and is highly elevated in CSCs. AlDeSense exhibits a 20-fold fluorescent enhancement when treated with ALDH1A1. Moreover, we established that AlDeSense is selective against a panel of common ALDH isoforms and exhibits exquisite chemostability against a collection of biologically relevant species. Through the application of surfa...

Surveillance of Cancer Stem Cell Plasticity Using an Isoform-Selective Fluorescent Probe for Aldehyde Dehydrogenase 1A1

Controlled light-mediated delivery of biological analytes can enable the investigation of highly reactivity molecules within living systems. As many biological effects are concentration dependent, it is critical to determine the location, time, and quantity of analyte donation. In this work, we have developed the first photoactivatable donor for formaldehyde (FA). Our optimized photoactivatable donor, photoFAD-3, is equipped with a fluorescence readout that enables monitoring of FA release with a concomitant 139-fold fluorescence enhancement. Tuning of photostability and cellular retention enabled quantification of intracellular FA release through cell lysate calibration. Application of photoFAD-3 uncovered the concentration range necessary for arresting wound healing in live cells. This marks the first report where a photoactivatable donor for any analyte has been used to quantify intracellular release.

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Nicholas W. Pino

Papers

NitroxylFluor: A Thiol-Based Fluorescent Probe for Live-Cell Imaging of Nitroxyl

Surveillance of Cancer Stem Cell Plasticity Using an Isoform-Selective Fluorescent Probe for Aldehyde Dehydrogenase 1A1

A Photoactivatable Formaldehyde Donor with Fluorescence Monitoring Reveals Threshold To Arrest Cell Migration

Thienylpiperidine Donor NIR Xanthene-Based Dye for Photoacoustic Imaging.