Fluorescent Probes for Disease Diagnosis.

The identification and detection of disease-related biomarkers is essential for early clinical diagnosis, evaluating disease progression, and for the development of therapeutics. Possessing the advantages of high sensitivity and selectivity, fluorescent probes have become effective tools for monitoring disease-related active molecules at the cellular level and in vivo. In this review, we describe current fluorescent probes designed for the detection and quantification of key bioactive molecules associated with common diseases, such as organ damage, inflammation, cancers, cardiovascular diseases, and brain disorders. We emphasize the strategies behind the design of fluorescent probes capable of disease biomarker detection and diagnosis and cover some aspects of combined diagnostic/therapeutic strategies based on regulating disease-related molecules. This review concludes with a discussion of the challenges and outlook for fluorescent probes, highlighting future avenues of research that should enable these probes to achieve accurate detection and identification of disease-related biomarkers for biomedical research and clinical applications.

Fluorescent small molecule donors.

Small molecule donors (SMDs) play subtle roles in the signaling mechanism and disease treatments. While many excellent SMDs have been developed, dosage control, targeted delivery, spatiotemporal feedback, as well as the efficiency evaluation of small molecules are still key challenges. Accordingly, fluorescent small molecule donors (FSMDs) have emerged to meet these challenges. FSMDs enable controllable release and non-invasive real-time monitoring, providing significant advantages for drug development and clinical diagnosis. Integration of FSMDs with chemotherapeutic, photodynamic or photothermal properties can take full advantage of each mode to enhance therapeutic efficacy. Given the remarkable properties and the thriving development of FSMDs, we believe a review is needed to summarize the design, triggering strategies and tracking mechanisms of FSMDs. With this review, we compiled FSMDs for most small molecules (nitric oxide, carbon monoxide, hydrogen sulfide, sulfur dioxide, reactive oxygen species and formaldehyde), and discuss recent progress concerning their molecular design, structural classification, mechanisms of generation, triggered release, structure-activity relationships, and the fluorescence response mechanism. Firstly, from the large number of fluorescent small molecular donors available, we have organized the common structures for producing different types of small molecules, providing a general strategy for the development of FSMDs. Secondly, we have classified FSMDs in terms of the respective donor types and fluorophore structures. Thirdly, we discuss the mechanisms and factors associated with the controlled release of small molecules and the regulation of the fluorescence responses, from which universal guidelines for optical properties and structure rearrangement were established, mainly involving light-controlled, enzyme-activated, reactive oxygen species-triggered, biothiol-triggered, single-electron reduction, click chemistry, and other triggering mechanisms. Fourthly, representative applications of FSMDs for trackable release, and evaluation monitoring, as well as for visible in vivo treatment are outlined, to illustrate the potential of FSMDs in drug screening and precision medicine. Finally, we discuss the opportunities and remaining challenges for the development of FSMDs for practical and clinical applications, which we anticipate will stimulate the attention of researchers in the diverse fields of chemistry, pharmacology, chemical biology and clinical chemistry. With this review, we hope to impart new understanding thereby enabling the rapid development of the next generation of FSMDs.

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.

Comprehensive risk factor predictions for 3-year survival among HIV-associated and disseminated cryptococcosis involving lungs and central nervous system.

BACKGROUND: The global mortality rate resulting from HIV-associated cryptococcal disease is remarkably elevated, particularly in severe cases with dissemination to the lungs and central nervous system (CNS). Regrettably, there is a dearth of predictive analysis regarding long-term survival, and few studies have conducted longitudinal follow-up assessments for comparing anti-HIV and antifungal treatments. METHODS: A cohort of 83 patients with HIV-related disseminated cryptococcosis involving the lung and CNS was studied for 3 years to examine survival. Comparative analysis of clinical and immunological parameters was performed between deceased and surviving individuals. Subsequently, multivariate Cox regression models were utilized to validate mortality predictions at 12, 24, and 36 months. RESULTS: Observed plasma cytokine levels before treatment were significantly lower for IL-1RA (p < 0.001) and MCP-1 (p < 0.05) when in the survivor group. Incorporating plasma levels of IL-1RA, IL-6, and high-risk CURB-65 score demonstrated the highest area under curve (AUC) value (0.96) for predicting 1-year mortality. For 1-, 2- and 3-year predictions, the single-factor model with IL-1RA demonstrated superior performance compared to all multiple-variate models (AUC = 0.95/0.78/0.78). CONCLUSIONS: IL-1RA is a biomarker for predicting 3-year survival. Further investigations to explore the pathogenetic role of IL-1RA in HIV-associated disseminated cryptococcosis and as a potential therapeutic target are warranted.

Excited-state dynamics of 4-hydroxyisoindoline-1,3-dione and its derivative as fluorescent probes.

Fluorescent probes have become promising tools for monitoring the concentration of peroxynitrite, which is linked to many diseases. However, despite focusing on developing numerous peroxynitrite based fluorescent probes, limited emphasis is placed on their sensing mechanism. Here, we investigated the sensing mechanism of a peroxynitrite fluorescent probe, named BHID-Bpin, with a focus on the relevant excited state dynamics. The photoexcited BHID-Bpin relaxes to its ground state via an efficient nonradiative process (∼300 ps) due to the presence of a minimum energy conical intersection between its first excited state and ground state. However, upon reacting with peroxynitrite, the Bpin moiety is cleaved from BHID-Bpin and BHID is formed. The formed BHID exhibits strong dual band fluorescence which is caused by an ultrafast excited-state intramolecular proton transfer process (∼1 ps).

The design of small-molecule prodrugs and activatable phototherapeutics for cancer therapy.

Cancer remains as one of the most significant health problems, with approximately 19 million people diagnosed worldwide each year. Chemotherapy is a routinely used method to treat cancer patients. However, current treatment options lack the appropriate selectivity for cancer cells, are prone to resistance mechanisms, and are plagued with dose-limiting toxicities. As such, researchers have devoted their attention to developing prodrug-based strategies that have the potential to overcome these limitations. This tutorial review highlights recently developed prodrug strategies for cancer therapy. Prodrug examples that provide an integrated diagnostic (fluorescent, photoacoustic, and magnetic resonance imaging) response, which are referred to as theranostics, are also discussed. Owing to the non-invasive nature of light (and X-rays), we have discussed external excitation prodrug strategies as well as examples of activatable photosensitizers that enhance the precision of photodynamic therapy/photothermal therapy. Activatable photosensitizers/photothermal agents can be seen as analogous to prodrugs, with their phototherapeutic properties at a specific wavelength activated in the presence of disease-related biomarkers. We discuss each design strategy and illustrate the importance of targeting biomarkers specific to the tumour microenvironment and biomarkers that are known to be overexpressed within cancer cells. Moreover, we discuss the advantages of each approach and highlight their inherent limitations. We hope in doing so, the reader will appreciate the current challenges and available opportunities in the field and inspire subsequent generations to pursue this crucial area of cancer research.

Unveiling the Crucial Roles of O2•- and ATP in Hepatic Ischemia-Reperfusion Injury Using Dual-Color/Reversible Fluorescence Imaging.

Hepatic ischemia-reperfusion injury (HIRI) is mainly responsible for morbidity or death due to graft rejection after liver transplantation. During HIRI, superoxide anion (O2•-) and adenosine-5'-triphosphate (ATP) have been identified as pivotal biomarkers associated with oxidative stress and energy metabolism, respectively. However, how the temporal and spatial fluctuations of O2•- and ATP coordinate changes in HIRI and particularly how they synergistically regulate each other in the pathological mechanism of HIRI remains unclear. Herein, we rationally designed and successfully synthesized a dual-color and dual-reversible molecular fluorescent probe (UDP) for dynamic and simultaneous visualization of O2•- and ATP in real-time, and uncovered their interrelationship and synergy in HIRI. UDP featured excellent sensitivity, selectivity, and reversibility in response to O2•- and ATP, which rendered UDP suitable for detecting O2•- and ATP and generating independent responses in the blue and red fluorescence channels without spectral crosstalk. Notably, in situ imaging with UDP revealed for the first time synchronous O2•- bursts and ATP depletion in hepatocytes and mouse livers during the process of HIRI. Surprisingly, a slight increase in ATP was observed during reperfusion. More importantly, intracellular O2•-─succinate dehydrogenase (SDH)─mitochondrial (Mito) reduced nicotinamide adenine dinucleotide (NADH)─Mito ATP─intracellular ATP cascade signaling pathway in the HIRI process was unveiled which illustrated the correlation between O2•- and ATP for the first time. This research confirms the potential of UDP for the dynamic monitoring of HIRI and provides a clear illustration of HIRI pathogenesis.

Purification and Structure Characterization of the Crude Polysaccharide from the Fruiting Bodies of Butyriboletus pseudospeciosus and Its Modulation Effects on Gut Microbiota.

Polysaccharides from the species of Boletaceae (Boletales, Agaricomycetes, Basidiomycota) are economically significant to both functional foods and medicinal industries. The crude polysaccharide from Butyriboletus pseudospeciosus (BPP) was prepared, and its physicochemical properties were characterized through the use of consecutive experimental apparatus, and its impact on the gut microbiota of Kunming mice was evaluated. Analyses of the structure characteristics revealed that BPP was mainly composed of Man, Glc, and Gal, possessing the pyranose ring and ß/α-glycosidic linkages. TG analysis exhibited that BPP had great heat stability. The SEM observation performed demonstrated that BPP appeared with a rough, dense, and porous shape. Through the BPP intervention, the serum and fecal biochemical index in mice can be improved obviously (p < 0.05). The abundance of beneficial microbiota in the BPP-treated group was significantly increased, while the abundance of harmful microbiota was significantly decreased (p < 0.05). Based on the Tax4Fun, we also revealed the relationship between the species of gut microbiota and showed that the high dose of BPP has significantly changed the functional diversities compared with those in other groups (p < 0.05). The results suggest that B. pseudospeciosus could serve as potential functional food or medicine.

Precision Navigation of Hepatic Ischemia-Reperfusion Injury Guided by Lysosomal Viscosity-Activatable NIR-II Fluorescence.

Hepatic ischemia-reperfusion injury (HIRI) is responsible for postoperative liver dysfunction and liver failure. Precise and rapid navigation of HIRI lesions is critical for early warning and timely development of pretreatment plans. Available methods for assaying liver injury fail to provide the exact location of lesions in real time intraoperatively. HIRI is intimately associated with oxidative stress which impairs lysosomal degradative function, leading to significant changes in lysosomal viscosity. Therefore, lysosomal viscosity is a potential biomarker for the precise targeting of HIRI. Hence, we developed a viscosity-activatable second near-infrared window fluorescent probe (NP-V) for the detection of lysosomal viscosity in hepatocytes and mice during HIRI. A reactive oxygen species-malondialdehyde-cathepsin B signaling pathway during HIRI was established. We further conducted high signal-to-background ratio NIR-II fluorescence imaging of HIRI mice. The contour and boundary of liver lesions were delineated, and as such the precise intraoperative resection of the lesion area was implemented. This research demonstrates the potential of NP-V as a dual-functional probe for the elucidation of HIRI pathogenesis and the direct navigation of HIRI lesions in clinical applications.

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).

Two-photon small-molecule fluorescence-based agents for sensing, imaging, and therapy within biological systems.

In this tutorial review, we will explore recent advances for the design, construction and application of two-photon excited fluorescence (TPEF)-based small-molecule probes. The advantages of TPEF-based probes include deep tissue penetration and minimal photo-damage. We discuss the underlying two-photon (TP) fluorophores including hemicyanine and design strategies such as Förster resonance energy transfer (FRET). Moreover, we emphasize applications for the detection or imaging of cations, anions, small neutral molecules, biomacromolecules, cellular microenvironments, subcellular localization and dual-responsive systems. Examples of photodynamic therapy (PDT) using TP irradiation are also illustrated.

Small molecule based fluorescent chemosensors for imaging the microenvironment within specific cellular regions.

The microenvironment (local environment), including viscosity, temperature, polarity, hypoxia, and acidic-basic status (pH), plays indispensable roles in cellular processes. Significantly, organelles require an appropriate microenvironment to perform their specific physiological functions, and disruption of the microenvironmental homeostasis could lead to malfunctions of organelles, resulting in disorder and disease development. Consequently, monitoring the microenvironment within specific organelles is vital to understand organelle-related physiopathology. Over the past few years, many fluorescent probes have been developed to help reveal variations in the microenvironment within specific cellular regions. Given that a comprehensive understanding of the microenvironment in a particular cellular region is of great significance for further exploration of life events, a thorough summary of this topic is urgently required. However, there has not been a comprehensive and critical review published recently on small-molecule fluorescent chemosensors for the cellular microenvironment. With this review, we summarize the recent progress since 2015 towards small-molecule based fluorescent probes for imaging the microenvironment within specific cellular regions, including the mitochondria, lysosomes, lipid drops, endoplasmic reticulum, golgi, nucleus, cytoplasmic matrix and cell membrane. Further classifications at the suborganelle level, according to detection of microenvironmental factors by probes, including polarity, viscosity, temperature, pH and hypoxia, are presented. Notably, in each category, design principles, chemical synthesis, recognition mechanism, fluorescent signals, and bio-imaging applications are summarized and compared. In addition, the limitations of the current microenvironment-sensitive probes are analyzed and the prospects for future developments are outlined. In a nutshell, this review comprehensively summarizes and highlights recent progress towards small molecule based fluorescent probes for sensing and imaging the microenvironment within specific cellular regions since 2015. We anticipate that this summary will facilitate a deeper understanding of the topic and encourage research directed towards the development of probes for the detection of cellular microenvironments.

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.

Reaction-Based Fluorescent Probes for the Detection and Imaging of Reactive Oxygen, Nitrogen, and Sulfur Species.

This Account describes a range of strategies for the development of fluorescent probes for detecting reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive (redox-active) sulfur species (RSS). Many ROS/RNS have been implicated in pathological processes such as Alzheimer's disease, cancer, diabetes mellitus, cardiovascular disease, and aging, while many RSS play important roles in maintaining redox homeostasis, serving as antioxidants and acting as free radical scavengers. Fluorescence-based systems have emerged as one of the best ways to monitor the concentrations and locations of these often very short lived species. Because of the high levels of sensitivity and in particular their ability to be used for temporal and spatial sampling for in vivo imaging applications. As a direct result, there has been a huge surge in the development of fluorescent probes for sensitive and selective detection of ROS, RNS, and RSS within cellular environments. However, cellular environments are extremely complex, often with more than one species involved in a given biochemical process. As a result, there has been a rise in the development of dual-responsive fluorescent probes (AND-logic probes) that can monitor the presence of more than one species in a biological environment. Our aim with this Account is to introduce the fluorescent probes that we have developed for in vitro and in vivo measurement of ROS, RNS, and RSS. Fluorescence-based sensing mechanisms used in the construction of the probes include photoinduced electron transfer, intramolecular charge transfer, excited-state intramolecular proton transfer (ESIPT), and fluorescence resonance energy transfer. In particular, probes for hydrogen peroxide, hypochlorous acid, superoxide, peroxynitrite, glutathione, cysteine, homocysteine, and hydrogen sulfide are discussed. In addition, we describe the development of AND-logic-based systems capable of detecting two species, such as peroxynitrite and glutathione. One of the most interesting advances contained in this Account is our extension of indicator displacement assays (IDAs) to reaction-based indicator displacement assays (RIAs). In an IDA system, an indicator is allowed to bind reversibly to a receptor. Then a competitive analyte is introduced into the system, resulting in displacement of the indicator from the host, which in turn modulates the optical signal. With an RIA-based system, the indicator is cleaved from a preformed receptor-indicator complex rather than being displaced by the analyte. Nevertheless, without a doubt the most significant result contained in this Account is the use of an ESIPT-based probe for the simultaneous sensing of fibrous proteins/peptides AND environmental ROS/RNS.

Excited-state intramolecular proton-transfer (ESIPT) based fluorescence sensors and imaging agents.

In this review we will explore recent advances in the design and application of excited-state intramolecular proton-transfer (ESIPT) based fluorescent probes. Fluorescence based sensors and imaging agents (probes) are important in biology, physiology, pharmacology, and environmental science for the selective detection of biologically and/or environmentally important species. The development of ESIPT-based fluorescence probes is particularly attractive due to their unique properties, which include a large Stokes shift, environmental sensitivity and potential for ratiometric sensing.

An ESIPT Probe for the Ratiometric Imaging of Peroxynitrite Facilitated by Binding to Aß-Aggregates.

A series of 3-hydroxyflavone (3-HF) ESIPT (excited-state intramolecular proton transfer) boronate-based fluorescent probes have been developed for the detection of peroxynitrite (ONOO-). The dyes are environmentally sensitive, and each probe exhibited a ratiometric response toward ONOO- in a micellar environment. The probes were used to image different aggregation states of amyloid-ß (Aß) in the presence of ONOO-. The 3-HF-OMe probe was found to produce a ratiometric response toward ONOO- when bound to Aß aggregates, resulting in a novel host-guest ensemble, which adds insight into the development of other ESIPT-based probes for the simultaneous sensing of fibrous proteins/peptides and environmental ROS/RNS.

Dual-Modal Colorimetric/Fluorescence Molecular Probe for Ratiometric Sensing of pH and Its Application.

As traditional pH meters cannot work well for minute regions (such as subcellular organelles) and in harsh media, molecular pH-sensitive devices for monitoring pH changes in diverse local heterogeneous environments are urgently needed. Here, we report a new dual-modal colorimetric/fluorescence merocyanine-based molecular probe (CPH) for ratiometric sensing of pH. Compared with previously reported pH probes, CPH bearing the benzyl group at the nitrogen position of the indolium group and the phenol, which is used as the acceptor for proton, could respond to pH changes immediately through both the ratiometric fluorescence signal readout and naked-eye colorimetric observation. The sensing process was highly stable and reversible. Most importantly, the suitable pKa value (6.44) allows CPH to presumably accumulate in lysosomes and become a lysosome-target fluorescent probe. By using CPH, the intralysosomal pH fluctuation stimulated by antimalaria drug chloroquine was successfully tracked in live cells through the ratiometric fluorescence images. Additionally, CPH could be immobilized on test papers, which exhibited a rapid and reversible colorimetric response to acid/base vapor through the naked-eye colorimetric analysis. This proof-of-concept study presents the potential application of CPH as a molecular tool for monitoring intralysosomal pH fluctuation in live cells, as well as paves the way for developing the economic, reusable, and fast-response optical pH meters for colorimetric sensing acid/base vapor with direct naked-eye observation.

A ligand-controlled switch of regioselectivity in ring-opening coupling of diarylmethylenecyclopropa[b]naphthalenes with Grignard reagents.

A ligand-controlled regioselectivity switch of ring-opening coupling reaction of diarylmethylenecyclopropa[b]naphthalenes with Grignard reagents providing differently substituted ß-vinylic naphthalenes in moderate to excellent yields was reported: when Pd(OAc)2 was used, the aromatic group from the Grignard reagent regioselectively coupled to the naphthyl ring after the ring-opening of three-membered cycle, which is different from the Pd(PPh3)2Cl2-catalyzed reaction. Based on a careful NMR study, we concluded that it may be explained by the ligand effect.

Clinical Characteristics of Chlamydia psittaci Infection Diagnosed by Metagenomic Next-Generation Sequencing: A Retrospective Multi-Center Study in Fujian, China.

Objective: This study aimed to describe and compare the epidemiological, demographic, clinical, laboratory and radiological characteristics as well as the complications, treatments, and outcomes of these patients. Methods: We retrospectively investigated clinical data of patients with C. psittaci infection (psittacosis) in eight Grade IIIA hospitals of Fujian. Metagenomic next-generation sequencing (mNGS) was used identify C. psittaci in clinical samples of all included patients. Results: A total of 74 patients (39 severe/35 non-severe) was diagnosed with psittacosis, 25 (33.8%) of whom had history of poultry exposure. Common symptoms included high fever (98% [37/74]), fatigue (52.7% [39/74]), and dyspnea (51.4% [38/74]). Common manifestations in imaging included consolidation (89.2%), pleural effusion (77.0%), and air bronchogram (66.2%). Common complications included acute respiratory distress syndrome (55.4% [41/74]), type I respiratory failure (52.7% [39/74]), acute liver injury (41.9% [31/74]), and secondary infection (27.0% [20/74]). The in-hospital mortality rate was 8.11% (6/74). Conclusion: C. psittaci infection is represents an underestimated cause of CAP. For SCAP patients with poultry and bird contact history, specimens were encouraged to be sended for mNGS test in time. C. psittaci infection can lead to severe, multiple system involvement, and several complications. mNGS facilitate timely diagnosis of C. psittaci infection.