Organ-Targeted Ionizable Lipid Nanoparticles Facilitate Sequence-Activated Fluorogenic Probe for Precise Imaging of Inflammatory Liver Disease.

Activatable near-infrared (NIR) fluorogenic probes offer a potent tool for real-time, in situ detection of hepatic biomarkers, significantly advancing the precision in diagnosing inflammatory liver disease (ILD). However, the limited distribution of small molecule fluorogenic probes in the liver and their rapid clearance impair the accuracy of fluorescence imaging and in ILD diagnosis. In this study, an effective utilization of ionizable lipid nanoparticles (iLNPs) is presented as liver-targeted carriers for efficient delivery of fluorogenic probes, aiming to overcome biodistribution barriers and achieve accurate detection of hepatic biomarkers. Based on this strategy, a liver-targeted NIR fluorogenic nanoprobe hCy-H2O2@iLNP is prepared using hCy-H2O2 as a small molecule reporter for visualizing the over-produced hydrogen peroxide (H2O2) in situ of liver. Notably, iLNPs not only significantly enhance probe accumulation in the liver, but also enable sequence activation of fluorescent nanoprobes. This response is achieved through primary liposome-dissociation release and secondary hCy-H2O2 response with pathological H2O2, enabling high-precision detection of oxidative stress in hepatocytes. These distinctive features facilitate accurate early diagnosis of acetaminophen (APAP)-induced inflammatory liver injury as well as lipopolysaccharide (LPS)-induced hepatitis. Therefore, the organ-targeted nanoprobe design strategy showcasts great potential for early and accurate diagnosis of lesions in situ in different organs.

A fluorogenic nitric oxide donor induced by yellow LED light for cells proliferation inhibition and imaging.

Nitric oxide (NO), as a vital cellular signalling molecule in physiological processes, has been found to play an important role in various biological functions. In this study, we rationally designed three NO donors by tethering nitrobenzene derivatives to three fluorescent chromophores. NX-NO was found to release NO and exhibit a high fluorescence turn-on signal ratio upon exposure to LED yellow light. Additionally, it had excellent photo-stability and good inhibitory activity against cancer cell proliferation, and was successfully applied to cell imaging. Moreover, we detected the release of NO and fluorescence response in the blood of a mouse, suggesting its potential therapeutic application in living organisms.

Real-Time Live Imaging of Osteoclast Activation via Cathepsin K Activity in Bone Diseases.

Intravital fluorescence imaging of functional osteoclasts within their intact disease context provides valuable insights into the intricate biology at the microscopic level, facilitating the development of therapeutic approaches for osteoclast-associated bone diseases. However, there is a lack of studies investigating osteoclast activity within deep-seated bone lesions using appropriate fluorescent probes, despite the advantages offered by the multi-photon excitation system in enhancing deep tissue imaging resolution. In this study, we report on the intravital tracking of osteoclast activity in three distinct murine bone disease models. We utilized a cathepsin K (CatK)-responsive two-photon fluorogenic probe (CatKP1), which exhibited a notable fluorescence turn-on response in the presence of active CatK. By utilizing CatKP1, we successfully monitored a significant increase in osteoclast activity in hindlimb long bones and its attenuation through pharmacological intervention without sacrificing mice. Thus, our findings highlight the efficacy of CatKP1 as a valuable tool for unraveling pathological osteoclast behavior and exploring novel therapeutic strategies.

Rapid genotyping of 32 insertion/deletion panel for human identification using fluorogenic probes-based multiplex real-time PCR.

BACKGROUND: Insertion and deletion (InDel) polymorphisms have considerable potential in the field of forensic genetics because of their low mutation rate and small amplicons. At present, InDel polymorphisms detection based on the technique of capillary electrophoresis is the main technique used in forensic DNA laboratory. However, this method is complicated and time-consuming, and is not suitable for rapid on-site paternity and personal identification. Next-generation sequencing analysis of InDels polymorphisms requires expensive instruments, large upfront reagent and supply costs, computational requirements and complex bioinformatics, increased the time to obtain results. Thus, there is an urgent need to establish a method to provide reliable, rapid, sensitive and economical genotyping for InDels. METHOD: A rapid InDels (32 InDels) panel was established using fluorogenic probes-based multiplex real-time PCR with microfluidic test cartridge and portable real-time PCR instrument. Then, we performed several validation studies including concordance, accuracy, sensitivity, stability, species specificity. RESULTS: It showed that the complete genotypes could be obtained from ≥100 pg of input DNA and from a series of challenging samples with high accuracy and specificity within 90 min. CONCLUSION: This method provides a rapid and cost-effective solution for InDels genotyping and personal identification in portable format.

Ultrafast Detection of Monoamine Oxidase A in Live Cells and Clinical Glioma Tissues Using an Affinity Binding-Based Two-Photon Fluorogenic Probe.

Abnormal expression of monoamine oxidase A (MAO-A) has been implicated in the development of human glioma, making MAO-A a promising target for therapy. Therefore, a rapid determination of MAO-A is critical for diagnosis. Through in silico screening of two-photon fluorophores, we discovered that a derivative of N,N-dimethyl-naphthalenamine (pre-mito) can effectively fit into the entrance of the MAO-A cavity. Substitutions on the N-pyridine not only further explore the MAO-A cavity, but also enable mitochondrial targeting ability. The aminopropyl substituted molecule, CD1, showed the fastest MAO-A detection (within 20 s), high MAO-A affinity and selectivity. It was also used for in situ imaging of MAO-A in living cells, enabling a comparison of the MAO-A content in human glioma and paracancerous tissues. Our results demonstrate that optimizing the affinity binding-based fluorogenic probes significantly improves their detection rate, providing a general approach for rapid detection probe design and optimization.

Multifunctional fluorescent probes for high-throughput characterization of hexosaminidase enzyme activity.

Microbial polysaccharides composed of N-acetylglucosamine (GlcNAc), such as chitin, peptidoglycan and poly-ß-(1 â†’ 6)-GlcNAc (dPNAG), play a critical role in maintaining cell integrity or in facilitating biofilm formation in numerous fungal and bacterial pathogens. Glycosyl hydrolase enzymes that catalyze the degradation of these ß-GlcNAc containing polysaccharides play important roles in normal microbial cell physiology and can also be exploited as biocatalysts with applications as anti-fungal, anti-bacterial, or biofilm dispersal agents. Assays to rapidly detect and characterize the activity of such glycosyl hydrolase enzymes can facilitate their development as biocatalyst, however, currently available probes such as 4-methylumbelliferyl-ß-GlcNAc (4MU-GlcNAc) are not universally accepted as substrates, and their fluorescent signal is sensitive to changes in pH. Here, we present the development of a new multifunctional fluorescent substrate analog for the detection and characterization of hexosaminidase enzyme activity containing a 7-amino-4-methyl coumarin (AMC) carbamate aglycone. This probe is widely tolerated as a substrate for exo-acting ß-hexosaminidase, family 19 endo-chitinase, and the dPNAG hydrolase enzyme Dispersin B (DspB) and enables detection of hexosaminidase enzyme activity via either single wavelength fluorescent measurements or ratiometric fluorescent detection. We demonstrate the utility of this probe to screen for recombinant DspB activity in Escherichia coli cell lysates, and for the development of a high-throughput assay to screen for DspB inhibitors.

An Oxazine-Based Fluorogenic Probe with Changeable π-Conjugation to Eliminate False-Positive Interference of Albumin and Its Application to Sensing Aminopeptidase†N.

Many fluorophores/probes suffer from the interference of albumin in biosystems. Herein, we propose an effective strategy to overcome this interference by virtue of both an albumin-insensitive fluorophore and its changeable π-conjugation, and demonstrate the strategy by designing an oxazine-based fluorogenic probe for aminopeptidase N (APN). The modification on the N atom in the oxazine fluorophore with alanine through a cleavable linker locks the resulting probe in a non-conjugated, colorless and non-fluorescent state, so the non-specific interaction of albumin produces no spectroscopic response. APN can selectively cleave the alanine moiety, restoring the large π-conjugation and strong fluorescence. The capability of the probe to eliminate the albumin influence has been demonstrated by imaging APN in different cell lines, and by quantitatively determining APN in human serum and mouse urine. The present strategy may be useful for developing more specific fluorogenic probes for other enzymes.

Design, synthesis and mechanistic studies of a TICT based fluorogenic probe for lighting up protein HSA.

Human serum albumin (HSA) in blood serves as an important biomarker for clinical diagnosis, and fluorescence sensing method has attracted extensive attention. In this work, a small organic molecule probe, YS8, involving twisted intramolecular charge transfer (TICT) characteristic, was designed and investigated to detect HSA. YS8 kept silent state in fluorescence under physiological conditions, but the encapsulation of YS8 in the hydrophobic subdomain IB region of HSA inhibited the TICT state and produced a clear light-up fluorescent signal. Especially, YS8 was demonstrated to be an efficient fluorogenic probe to discriminate HSA from other proteins including the bovine serum albumin (BSA). Moreover, YS8/HSA complex could be applied in fluorescence imaging in living cells and is also useful in the study of artificial fluorescent protein (AFP).

4-Azidocinnoline-Cinnoline-4-amine Pair as a New Fluorogenic and Fluorochromic Environment-Sensitive Probe.

A new type of fluorogenic and fluorochromic probe based on the reduction of weakly fluorescent 4-azido-6-(4-cyanophenyl)cinnoline to the corresponding fluorescent cinnoline-4-amine was developed. We found that the fluorescence of 6-(4-cyanophenyl)cinnoline-4-amine is strongly affected by the nature of the solvent. The fluorogenic effect for the amine was detected in polar solvents with the strongest fluorescence increase in water. The environment-sensitive fluorogenic properties of cinnoline-4-amine in water were explained as a combination of two types of fluorescence mechanisms: aggregation-induced emission (AIE) and excited state intermolecular proton transfer (ESPT). The suitability of an azide-amine pair as a fluorogenic probe was tested using a HepG2 hepatic cancer cell line with detection by fluorescent microscopy, flow cytometry, and HPLC analysis of cells lysates. The results obtained confirm the possibility of the transformation of the azide to amine in cells and the potential applicability of the discovered fluorogenic and fluorochromic probe for different analytical and biological applications in aqueous medium.

Fluorogenic Probes/Inhibitors of ß-Lactamase and their Applications in Drug-Resistant Bacteria.

ß-Lactam antibiotics are generally perceived as one of the greatest inventions of the 20th century, and these small molecular compounds have saved millions of lives. However, upon clinical application of antibiotics, the ß-lactamase secreted by pathogenic bacteria can lead to the gradual development of drug resistance. ß-Lactamase is a hydrolase that can efficiently hydrolyze and destroy ß-lactam antibiotics. It develops and spreads rapidly in pathogens, and the drug-resistant bacteria pose a severe threat to human health and development. As a result, detecting and inhibiting the activities of ß-lactamase are of great value for the rational use of antibiotics and the treatment of infectious diseases. At present, many specific detection methods and inhibitors of ß-lactamase have been developed and applied in clinical practice. In this Minireview, we describe the resistance mechanism of bacteria producing ß-lactamase and further summarize the fluorogenic probes, inhibitors of ß-lactamase, and their applications in the treatment of infectious diseases. It may be valuable to design fluorogenic probes with improved selectivity, sensitivity, and effectiveness to further identify the inhibitors for ß-lactamases and eventually overcome bacterial resistance.

Performance of a Novel Fluorogenic Assay for Detection of Carbapenemase-Producing Enterobacteriaceae from Bacterial Colonies and Directly from Positive Blood Cultures.

Rapid and accurate detection of carbapenemase-producing Enterobacteriaceae (CPE) is critical for appropriate treatment and infection control. We compared a rapid fluorogenic assay using a carbapenem-based fluorogenic probe with other phenotypic assays: modified carbapenem inactivation method (mCIM), Carba NP test (CNP), and carbapenemase inhibition test (CIT). A total of 217 characterized isolates of Enterobacteriaceae were included as follows: 63 CPE; 48 non-carbapenemase-producing carbapenem-resistant Enterobacteriaceae (non-CP-CRE); 53 extended-spectrum ß-lactamase producers; and 53 third-generation-cephalosporin-susceptible isolates. The fluorogenic assay using bacterial colonies (Fluore-C) was conducted by lysing the isolates followed by centrifugation and mixing the supernatant with fluorogenic probe. In addition, for the fluorogenic assay using spiked blood culture bottles (Fluore-Direct), pellets were obtained via the saponin preparation method, which can directly identify the pathogens using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The fluorescence signal was measured over 50 min using a fluorometer. The fluorescent signal of CPE was significantly higher than that of non-CPE in both Fluore-C (median relative fluorescence units [RFU] [range], 5,814 [240 to 32,009] versus 804 [36 to 2,480], respectively; P < 0.0001) and Fluore-Direct (median RFU [range], 10,355 [1,689 to 31,463] versus 1,068 [428 to 2,155], respectively; P < 0.0001) tests. Overall, positive and negative percent agreements of Fluore-C, mCIM, CNP, CIT, and Fluore-Direct were 100% and 98.7%, 98.3% and 97.5%, 88.1% and 100%, 96.4% and 98.7%, and 98.3% and 98.1%, respectively. The relatively lower positive percent agreement (PPA) of CNP was mainly observed in OXA-type CPE. The fluorogenic assay showed excellent performance with bacterial colonies and also directly from positive blood cultures. We included many non-CP-CRE isolates for strict evaluation. The fluorogenic assay will be a useful tool for clinical microbiology laboratories.

Detection of esterase activity by chromogenic and fluorogenic probe based on an O-nitrobenzoxadiazole (O-NBD) unit.

We designed a conjugated molecule bearing an O-nitrobenzoxadiazole (O-NBD) unit and an acetylated trimethyl lock as a chromogenic and fluorogenic probe for the detection of esterase activity. The designed molecule was briefly synthesized from a commercially available compound in two steps. Several experiments revealed that the conjugated molecule serves as a sensitive chromogenic and fluorogenic probe for the detection of porcine liver esterase activity. Mechanistic studies indicated that an intramolecular O- to N-NBD migration is involved in the chromogenic/fluorogenic phenomena. The results here would be helpful for designing other O-NBD-based chromogenic/fluorogenic probes in future.

Fluorogenic approach to evaluating prodrug hydrolysis and stability in live cells.

Fluorescein diester which is conjugated with cell membrane permeable Arg9 peptide was proposed as probe for ester prodrug stability and drug release study in living cells. α-Amino protected d-Val and l-Ala which bear differently hindered side chains were used to afford model diesters of 5-maleimide-fluorescein. Such fluorescein diesters were further conjugated with a Cys containing cell membrane permeable Arg9 peptide via thiol-ene crosslink reaction. The resulted conjugates of fluorescein diester and Arg9 peptide were purified with HPLC and characterized with MALDI-TOF MS. Upon incubation with cultured cells, the fluorescein diesters were delivered into the cells, the following hydrolysis of fluorescein diesters and release of fluorescein inside living cells were observed by monitoring the fluorescence accumulation. Fluorescence microscopic imaging studies of HeLa cells treated with fluorescein l-Ala diester show strong fluorescence accumulation in 30 min indicating fast hydrolysis of fluorescein diester and fluorescein release; in contrast d-Val diester remains stable inside cells evidenced by margin fluorescence formation. Further flowcytometry studies on the fluorescein diester-Arg9 conjugate treated cells show that the hydrolysis t1/2 for l-Ala diester is 15 min. The results also show that Arg9 peptide not only transports the ester probes into cell efficiently but also can retain and concentrate hydrolytic product fluorescein inside cells so that the accumulated fluorescence can be accurately quantified. This fluorogenic probe approach provides feasible applications in dynamic studies on ester prodrug hydrolysis and release, facilitating screening and optimization of prodrug structures in living cell settings.

Design and synthesis of fluorogenic substrate-based probes for detecting Cathepsin B activity.

Cathepsin B plays key roles in tumor progression with its overexpression being associated with invasive and metastatic phenotypes and is a primary target of protease activated antibody-directed prodrug therapy. It therefore represents a potential therapeutic and diagnostic target and effort has been made to develop fluorescent probes to report on Cathepsin B activity in cells and animal models of cancer. We have designed, synthesized, and thoroughly evaluated four novel "turn on" probes that employ a lysosomotropic dansylcadaverine dye to report on Cathepsin B activity. Enzyme activity assays using a recombinant human enzyme and cancer cell lysates coupled with confocal microscopy experiments demonstrated that one of the probes, derivatized with the self-immolative prodrug linker p-aminobenzyl alcohol, can selectively report on Cathepsin B in biological samples including live cells.

Chromo-Fluorogenic Detection of Soman and Its Simulant by Thiourea-Based Rhodamine Probe.

Here, we introduced a novel thiourea-based rhodamine compound as a chromo-fluorogenic indicator of nerve agent Soman and its simulant diethyl chlorophosphate (DCP). The synthesized probe N-(rhodamine B)-lactam-2-(4-cyanophenyl) thiourea (RB-CT), which has a rhodamine core linked by a cyanophenyl thiosemicarbazide group, enabled a rapidly and highly sensitive response to DCP with clear fluorescence and color changes. The detection limit was as low as 2 × 10-6 M. The sensing mechanism showed that opening of the spirolactam ring following the phosphorylation of thiosemicarbazides group formed a seven-membered heterocycle adduct, according to MS analysis and TD-DFT calculations. RB-CT exhibited high detecting selectivity for DCP, among other organophosphorus compounds. Moreover, two test kits were employed and successfully used to detect real nerve agent Soman in liquid and gas phase.

Maytansinoid-BODIPY Conjugates: Application to Microscale Determination of Drug Extinction Coefficients and for Quantification of Maytansinoid Analytes.

Determining drug to antibody ratios (DAR) for antibody-drug conjugates (ADCs) in early research and development can be hampered by difficulties in accurate weighing of the effector payload and subsequent determination of its extinction coefficient. Two maytansinoids, DM1 and DM4, potent antimitotic agents used in clinical ADCs, were derivatized with the compact fluorophore BODIPY FL using two different linker designs. We identified DM1-mal-BODIPY as a conjugate with little through-space interaction between the maytansinoid and BODIPY chromophores. The 1:1 stoichiometry between the maytansinoid and BODIPY makes the molar concentration of both components equal and the extinction coefficient of the maytansinoid in proportion with the known BODIPY chromophore according to Beer's Law. By only derivatizing 50 µg of unpurified DM1 and analyzing about 25 µg of DM1-mal-BODIPY by UV-vis, we determined εDM1 252 nm and εDM1 280 nm as 26 355 ± 360 and 5230 ± 160 cm(-1) M(-1), respectively. These values are nearly identical to those accepted for DM1 based on weighing >100 mg of pure sample. Surprisingly, some of the maytansinoid-BODIPY conjugates that were synthesized were partially or completely fluorescence-quenched. The green fluorescence of quenched DM4-acetamide-BODIPY could be fully restored in the presence of an antibody designed to tightly bind maytansine. We exploited this observation to develop a simple "mix and read" fluorogenic immunoassay for detection of nanogram quantities of maytansinoids.

Development of surface immobilized 3-azidocoumarin-based fluorogenic probe via strain promoted click chemistry.

A new class of imaging probe, a fluorogenic version of 1, 3-dipolar cycloaddition of azides and alkynes has been developed. 3-azidocoumarin scaffolds were selectively immobilized on the DBCO modified bead surface via SPAAC and provide direct and strong fluorescence in fluorescence microscopy. This developed click-on beads could be applied to label various biomolecules, such as nucleic acids, proteins and other molecules. To this end, 5'(7-hydroxy 3-azido coumarin) labelled DNA primer also displayed strong fluorescence upon successful immobilization on the bead surface.

Marine natural products as inhibitors of cystathionine beta-synthase activity.

A library consisting of characterized marine natural products as well as synthetic derivatives was screened for compounds capable of inhibiting the production of hydrogen sulfide (H2S) by cystathionine beta-synthase (CBS). Eight hits were validated and shown to inhibit CBS activity with IC50 values ranging from 83 to 187µM. The majority of hits came from a series of synthetic polyandrocarpamine derivatives. In addition, a modified fluorogenic probe for H2S detection with improved solubility in aqueous solutions is reported.

A Near-Infrared Fluorogenic Probe for Rapid, Specific, and Ultrasensitive Detection of Sphingosine in Living Cells and In Vivo.

Sphingosine (Sph) plays important roles in various complex biological processes. Abnormalities in Sph metabolism can result in various diseases, including neurodegenerative disorders. However, due to the lack of rapid and accurate detection methods, understanding sph metabolic in related diseases is limited. Herein, a series of near-infrared fluorogenic probes DMS-X (X = 2F, F, Cl, Br, and I) are designed and synthesized. The fast oxazolidinone ring formation enables the DMS-2F to detect Sph selectively and ultrasensitively, and the detection limit reaches 9.33 ± 0.41 nm. Moreover, it is demonstrated that DMS-2F exhibited a dose- and time-dependent response to Sph and can detect sph in living cells. Importantly, for the first time, the changes in Sph levels induced by Aß42 oligomers and H2 O2 are assessed through a fluorescent imaging approach, and further validated the physiological processes by which Aß42 oligomers and reactive oxygen species (ROS)-induce changes in intracellular Sph levels. Additionally, the distribution of Sph in living zebrafish is successfully mapped by in vivo imaging of a zebrafish model. This work provides a simple and efficient method for probing Sph in living cells and in vivo, which will facilitate investigation into the metabolic process of Sph and the connection between Sph and disease pathologies.

Antibody-based near-infrared fluorogenic probes for wash-free imaging of cell-surface proteins.

BACKGROUND: Cell-surface proteins, which are closely associated with various physiological and pathological processes, have drawn much attention in drug discovery and disease diagnosis. Thus, wash-free imaging of the target cell-surface protein under its native environment is critical and helpful for early detection and prognostic evaluation of diseases. RESULTS: To minimize the interference from autofluorescence and fit the penetration depth towards tissue samples, we developed a fluorogenic antibody-based probe, Ab-Cy5.5, which will liberate > 5-fold turn-on near-infrared (NIR) emission in the presence of its target antigen within 10 min. SIGNIFICANCE: By taking advantage of the fluorescence-quenched dimeric H-aggregation of Cy5.5, Ab-Cy5.5 with Cy5.5 attached at the N-terminus showed negligible background signal, allowing direct imaging of the target cell-surface protein in both living cells and tissue samples without washing.