Theranostic Fluorescent Probes.

The ability to gain spatiotemporal information, and in some cases achieve spatiotemporal control, in the context of drug delivery makes theranostic fluorescent probes an attractive and intensely investigated research topic. This interest is reflected in the steep rise in publications on the topic that have appeared over the past decade. Theranostic fluorescent probes, in their various incarnations, generally comprise a fluorophore linked to a masked drug, in which the drug is released as the result of certain stimuli, with both intrinsic and extrinsic stimuli being reported. This release is then signaled by the emergence of a fluorescent signal. Importantly, the use of appropriate fluorophores has enabled not only this emerging fluorescence as a spatiotemporal marker for drug delivery but also has provided modalities useful in photodynamic, photothermal, and sonodynamic therapeutic applications. In this review we highlight recent work on theranostic fluorescent probes with a particular focus on probes that are activated in tumor microenvironments. We also summarize efforts to develop probes for other applications, such as neurodegenerative diseases and antibacterials. This review celebrates the diversity of designs reported to date, from discrete small-molecule systems to nanomaterials. Our aim is to provide insights into the potential clinical impact of this still-emerging research direction.

Selective FRET nano probe based on carbon dots and naphthalimide-isatin for the ratiometric detection of peroxynitrite in drug-induced liver injury.

Drug-induced liver injury (DILI) is the most common cause for acute liver failure in the USA and Europe. However, most of DILI cases can recover or be prevented if treatment by the offending drug is discontinued. Recent research indicates that peroxynitrite (ONOO-) can be a potential indicator to diagnose DILI at an early stage. Therefore, the establishment of an assay to detect and track ONOO- in DILI cases is urgently needed. Here, a FRET-based ratiometric nano fluorescent probe CD-N-I was developed to detect ONOO- with high selectivity and excellent sensitivity. This probe consists of carbon dots and a naphthalimide-isatin peroxynitrite sensing system assembled based on electrostatic interactions. Using CD-N-I we were able to detect exogenous ONOO- in live cells and endogenous ONOO- in APAP-induced liver injury of HepG2 cells.

The development of logic gate-based fluorescent probes that respond to intracellular hydrogen peroxide and pH in tandem.

Logic gate-based fluorescent probes are powerful tools for the discriminative sensing of multiple signaling molecules that are expressed in concert during the progression of many diseases such as inflammation, cancer, aging, and other disorders. To achieve logical sensing, multiple functional groups are introduced to the different substitution sites of a single fluorescent dye, which increases the complexity of chemical synthesis. Herein, we report a simple strategy that incorporates just one responsive unit into a hemicyanine dye achieving the logic gate-based sensing of two independent analytes. We introduce boronic acid to hemicyanine to quench the fluorescence, and in the presence of hydrogen peroxide (H2O2), the fluorescence is recovered due to removal of the boronate. Interestingly, the subsequent decrease in pH turned the red fluorescence of hemicyanine to green emissive because of protonation of the phenolic alcohol. This unique feature of the probe enables us to construct "INHIBIT" and "AND" logical gates for the accurate measuring of intracellular H2O2 and acidic pH in tandem. This study offers insight into the simple construction of logic-gate based fluorescent probes for the tandem sensing of multiple analytes that are correlatively produced during disease progression.

Metal-organic framework (MOF) hybridized gold nanoparticles as a bifunctional nanozyme for glucose sensing.

Inspired by natural enzymes that possess multiple catalytic activities, here we develop a bifunctional metal-organic frame-work (MOF) for biosensing applications. Ultrasmall gold nano-particles (AuNPs) are grown in the internal cavities of an iron (Fe) porphyrin-based MOF to produce a hybridized nanozyme, AuNPs@PCN-224(Fe), in which AuNPs and PCN-224(Fe) exhibit the catalytic activity of glucose oxidase (GOx) and horseradish peroxidase (HRP), respectively. We established that the bifunctional nanozyme was capable of a cascade reaction to generate hydrogen peroxide in the presence of d-glucose and oxygen in situ, and subsequently activate a colorimetric or chemiluminescent substrate through HRP-mimicking catalytic activity. The nanozyme was selective over a range of other saccharides, and 93% of the catalytic activity was retained after being recycled five times.

Orthogonally Engineered Albumin with Attenuated Macrophage Phagocytosis for the Targeted Visualization and Phototherapy of Liver Cancer.

The five-year survival rate of hepatocellular carcinoma (HCC) remains unsatisfactory. This reflects, in part, the paucity of effective methods that allow the target-specific diagnosis and therapy of HCC. Here, we report a strategy based on engineered human serum albumin (HSA) that permits the HCC-targeted delivery of diagnostic and therapeutic agents. Covalent cysteine conjugation combined with the exploitation of host-guest chemistry was used to effect the orthogonal functionalization of HSA with two functionally independent peptides. One of these peptides targets glypican-3 (GPC-3), an HCC-specific biomarker, while the second reduces macrophage phagocytosis through immune-checkpoint stimulation. This orthogonally engineered HSA proved effective for the GPC-3-targeted delivery of near-infrared fluorescent and phototherapeutic agents, thus permitting target-specific optical visualization and photodynamic ablation of HCC in vivo. This study thus offers new insights into specificity-enhanced fluorescence-guided surgery and phototherapy of HCC through the orthogonal engineering of biocompatible proteins.

A TCF-based fluorescent probe to determine nitroreductase (NTR) activity for a broad-spectrum of bacterial species.

A nitroreductase (NTR) responsive fluorescent probe with long wavelength fluorescence emission was used to determine the NTR activity of a selection of bacterial species under a range of different bacterial growth conditions ensuring applicability under multiple complex clinical environments, where sensitivity, reaction time, and the detection accuracy were suitable for planktonic cultures and biofilms.

Human serum albumin-based supramolecular host-guest boronate probe for enhanced peroxynitrite sensing.

Peroxynitrite (ONOO-) is an important oxygen/nitrogen reactive species implicated in a number of physiological and pathological processes. However, due to the complexity of the cellular micro-environment, the sensitive and accurate detection of ONOO- remains a challenging task. Here, we developed a long-wavelength fluorescent probe based on the conjugation between a TCF scaffold and phenylboronate; the resulting conjugate is capable of supramolecular host-guest assembly with human serum albumin (HSA) for the fluorogenic sensing of ONOO-. The probe exhibited an enhanced fluorescence over a low concentration range of ONOO- (0-9.6 µM), whist the fluorescence was quenched when the concentration of ONOO- exceeded 9.6 µM. In addition, when human serum albumin (HSA) was added, the initial fluorescence of the probe was significantly enhanced, which enabled the more sensitive detection of low-concentrations of ONOO- in aqueous buffer solution and in cells. The molecular structure of the supramolecular host-guest ensemble was determined using small-angle X-ray scattering.

Phenotyping of Methicillin-Resistant Staphylococcus aureus Using a Ratiometric Sensor Array.

Chemical tools capable of classifying multidrug-resistant bacteria (superbugs) can facilitate early-stage disease diagnosis and help guide precision therapy. Here, we report a sensor array that permits the facile phenotyping of methicillin-resistant Staphylococcus aureus (MRSA), a clinically common superbug. The array consists of a panel of eight separate ratiometric fluorescent probes that provide characteristic vibration-induced emission (VIE) profiles. These probes bear a pair of quaternary ammonium salts in different substitution positions around a known VIEgen core. The differences in the substituents result in varying interactions with the negatively charged cell walls of bacteria. This, in turn, dictates the molecular conformation of the probes and affects their blue-to-red fluorescence intensity ratios (ratiometric changes). Within the sensor array, the differences in the ratiometric changes for the probes result in "fingerprints" for MRSA of different genotypes. This allows them to be identified using principal component analysis (PCA) without the need for cell lysis and nucleic acid isolation. The results obtained with the present sensor array agree well with those obtained using polymerase chain reaction (PCR) analysis.

Selective detection of peroxynitrite using an isatin receptor and a naphthalimide fluorophore.

Peroxynitrite is a reactive oxygen and nitrogen species that participates in various biological reactions. Therefore, it is important to readily detect and track peroxynitrite in biological systems. Here, a novel turn-on probe encapsulated in PEG DSPE-PEG/HN-I was used to fluorescently detect ONOO- rapidly. The encapsulation of HN-I using DSPE-PEG2000 optimizes the sensing performances of the naphthalimide probe and avoids ACQ. Using DSPE-PEG/HN-I to detect changes in the levels of exogenous ONOO- in HepG2 cells and endogenous ONOO- induced by LPS in RAW 267.4 cells was demonstrated.

A Highly Sensitive and Selective Near-Infrared Fluorescent Probe for Imaging Peroxynitrite in Living Cells and Drug-Induced Liver Injury Mice.

Drug-induced liver injury (DILI) is a major clinical issue associated with the majority of commercial drugs. During DILI, the peroxynitrite (ONOO-) level is upregulated in the liver. However, traditional methods are unable to timely monitor the dynamic changes of the ONOO- level during DILI in vivo. Therefore, ONOO--activated near-infrared (NIR) fluorescent probes with high sensitivity and selectivity are key to the early diagnosis of DILI in situ. Herein, we report a novel ONOO--responsive NIR fluorescent probe, QCy7-DP, which incorporates a donor-dual-acceptor π-electron cyanine skeleton with diphenyl phosphinate. The ONOO--mediated highly selective hydrolytic cleavage via an addition-elimination pathway of diphenyl phosphinate produced the deprotonated form of QCy7 in physiological conditions with a distinctive extended conjugated π-electron system and ∼200-fold enhancement in NIR fluorescence emission at 710 nm. Moreover, the probe QCy7-DP was successfully used for the imaging of the endogenous and exogenous ONOO- concentration changes in living cells. Importantly, in vivo fluorescence imaging tests demonstrated that the probe can effectively detect the endogenous generation of ONOO- in an acetaminophen (APAP)-induced liver injury mouse model. This study provides insight into the design of highly selective NIR fluorescent probes suitable for spatiotemporal monitoring of ONOO- under different pathological conditions.

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.

Targeted photothermal release of antibiotics by a graphene nanoribbon-based supramolecular glycomaterial.

Here, we report the simple construction of a supramolecular glycomaterial for the targeted delivery of antibiotics to P. aeruginosa in a photothermally-controlled manner. A galactose-pyrene conjugate (Gal-pyr) was developed to self-assemble with graphene nanoribbon-based nanowires via π-π stacking to produce a supramolecular glycomaterial, which exhibits a 1250-fold enhanced binding avidity toward a galactose-selective lectin when compared to Gal-pyr. The as-prepared glycomaterial when loaded with an antibiotic that acts as an inhibitor of the bacterial folic acid biosynthetic pathway eradicated P. aeruginosa-derived biofilms under near-infrared light irradiation due to the strong photothermal effect of the nanowires accelerating antibiotic release.

Fluorescent probes for the detection of chemical warfare agents.

Chemical warfare agents (CWAs) are toxic chemicals that have been intentionally developed for targeted and deadly use on humans. Although intended for military targets, the use of CWAs more often than not results in mass civilian casualties. To prevent further atrocities from occurring during conflicts, a global ban was implemented through the chemical weapons convention, with the aim of eliminating the development, stockpiling, and use of CWAs. Unfortunately, because of their relatively low cost, ease of manufacture and effectiveness on mass populations, CWAs still exist in today's world. CWAs have been used in several recent terrorist-related incidents and conflicts (e.g., Syria). Therefore, they continue to remain serious threats to public health and safety and to global peace and stability. Analytical methods that can accurately detect CWAs are essential to global security measures and for forensic analysis. Small molecule fluorescent probes have emerged as attractive chemical tools for CWA detection, due to their simplicity, ease of use, excellent selectivity and high sensitivity, as well as their ability to be translated into handheld devices. This includes the ability to non-invasively image CWA distribution within living systems (in vitro and in vivo) to permit in-depth evaluation of their biological interactions and allow potential identification of therapeutic countermeasures. In this review, we provide an overview of the various reported fluorescent probes that have been designed for the detection of CWAs. The mechanism for CWA detection, change in optical output and application for each fluorescent probe are described in detail. The limitations and challenges of currently developed fluorescent probes are discussed providing insight into the future development of this research area. We hope the information provided in this review will give readers a clear understanding of how to design a fluorescent probe for the detection of a specific CWA. We anticipate that this will advance our security systems and provide new tools for environmental and toxicology monitoring.

Fluorescent probes for the detection of disease-associated biomarkers.

Fluorescent probes have emerged as indispensable chemical tools to the field of chemical biology and medicine. The ability to detect intracellular species and monitor physiological processes has not only advanced our knowledge in biology but has provided new approaches towards disease diagnosis. In this review, we detail the design criteria and strategies for some recently reported fluorescent probes that can detect a wide range of biologically important species in cells and in vivo. In doing so, we highlight the importance of each biological species and their role in biological systems and for disease progression. We then discuss the current problems and challenges of existing technologies and provide our perspective on the future directions of the research area. Overall, we hope this review will provide inspiration for researchers and prove as useful guide for the development of the next generation of fluorescent probes.

TCF-based fluorescent probe for monitoring superoxide anion produced in bacteria under chloramphenicol- and heat-induced stress.

We report on a superoxide anion (O2˙-) responsive fluorescent probe called TCF-OTf. TCF-OTf is able to monitor O2˙- production when the bacterial species Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Enterococcus faecalis are exposed to chloramphenicol and heat shock at 50 and 58 °C.

Molecularly engineered AIEgens with enhanced quantum and singlet-oxygen yield for mitochondria-targeted imaging and photodynamic therapy.

Luminogens characteristic of aggregation-induced emission (AIEgens) have been extensively exploited for the development of imaging-guided photodynamic therapeutic (PDT) agents. However, intramolecular rotation of donor-acceptor (D-A) type AIEgens favors non-radiative decay of photonic energy which results in unsatisfactory fluorescence quantum and singlet oxygen yields. To address this issue, we developed several molecularly engineered AIEgens with partially "locked" molecular structures enhancing both fluorescence emission and the production of triplet excitons. A triphenylphosphine group was introduced to form a D-A conjugate, improving water solubility and the capacity for mitochondrial localization of the resulting probes. Experimental and theoretical analyses suggest that the much higher quantum and singlet oxygen yield of a structurally "significantly-locked" probe (LOCK-2) than its "partially locked" (LOCK-1) and "unlocked" equivalent (LOCK-0) is a result of suppressed AIE and twisted intramolecular charge transfer. LOCK-2 was also used for the mitochondrial-targeting, fluorescence image-guided PDT of liver cancer cells.

Metabolically Specific In Situ Fluorescent Visualization of Bacterial Infection on Wound Tissues.

The ability to effectively detect bacterial infection in human tissues is important for the timely treatment of the infection. However, traditional techniques fail to visualize bacterial species adhered to host cells in situ in a target-specific manner. Dihydropteroate synthase (DHPS) exclusively exists in bacterial species and metabolically converts p-aminobenzoic acid (PABA) to folic acid (FA). By targeting this bacterium-specific metabolism, we have developed a fluorescent imaging probe, PABA-DCM, based on the conjugation of PABA with a long-wavelength fluorophore, dicyanomethylene 4H-pyran (DCM). We confirmed that the probe can be used in the synthetic pathway of a broad spectrum of Gram-positive and negative bacteria, resulting in a significantly extended retention time in bacterial over mammalian cells. We validated that DHPS catalytically introduces a dihydropteridine group to the amino end of the PABA motif of PABA-DCM, and the resulting adduct leads to an increase in the FA levels of bacteria. We also constructed a hydrogel dressing containing PABA-DCM and graphene oxide (GO), termed PABA-DCM@GO, that achieves target-specific fluorescence visualization of bacterial infection on the wounded tissues of mice. Our research paves the way for the development of fluorescent imaging agents that target species-conserved metabolic pathways of microorganisms for the in situ monitoring of infections in human tissues.

The Key Role of Magnetic Resonance Imaging in the Detection of Neurodegenerative Diseases-Associated Biomarkers: A Review.

Neurodegenerative diseases (NDs), including chronic disease such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis, and acute diseases like traumatic brain injury and ischemic stroke are characterized by progressive degeneration, brain tissue damage and loss of neurons, accompanied by behavioral and cognitive dysfunctions. So far, there are no complete cures for NDs; thus, early and timely diagnoses are essential and beneficial to patients' treatment. Magnetic resonance imaging (MRI) has become one of the advanced medical imaging techniques widely used in the clinical examination of NDs due to its non-invasive diagnostic value. In this review, research published in English in current decade from PubMed electronic database on the use of MRI to detect specific biomarkers of NDs was collected, summarized, and discussed, which provides valuable suggestions for the early diagnosis, prevention, and treatment of NDs in the clinic.

Fluorescence Analysis of Circulating Exosomes for Breast Cancer Diagnosis Using a Sensor Array and Deep Learning.

Emerging liquid biopsy methods for investigating biomarkers in bodily fluids such as blood, saliva, or urine can be used to perform noninvasive cancer detection. However, the complexity and heterogeneity of exosomes require improved methods to achieve the desired sensitivity and accuracy. Herein, we report our study on developing a breast cancer liquid biopsy system, including a fluorescence sensor array and deep learning (DL) tool AggMapNet. In particular, we used a 12-unit sensor array composed of conjugated polyelectrolytes, fluorophore-labeled peptides, and monosaccharides or glycans to collect fluorescence signals from cells and exosomes. Linear discriminant analysis (LDA) processed the fluorescence spectral data of cells and cell-derived exosomes, demonstrating successful discrimination between normal and different cancerous cells and 100% accurate classification of different BC cells. For heterogeneous plasma-derived exosome analysis, CNN-based DL tool AggMapNet was applied to transform the unordered fluorescence spectra into feature maps (Fmaps), which gave a straightforward visual demonstration of the difference between healthy donors and BC patients with 100% prediction accuracy. Our work indicates that our fluorescent sensor array and DL model can be used as a promising noninvasive method for BC diagnosis.