Nontoxic Probe Research Articles

The Enigma of Norbormide, a Rattus-Selective Toxicant.

Norbormide (NRB) is a Rattus-selective toxicant, which was serendipitously discovered in 1964 and formerly marketed as an eco-friendly rodenticide that was deemed harmless to non-Rattus species. However, due to inconsistent efficacy and the emergence of second-generation anticoagulants, its usage declined, with registration lapsing in 2003. NRBs' lethal action in rats entails irreversible vasoconstriction of peripheral arteries, likely inducing cardiac damage: however, the precise chain of events leading to fatality and the target organs involved remain elusive. This unique contractile effect is exclusive to rat arteries and is induced solely by the endo isomers of NRB, hinting at a specific receptor involvement. Understanding NRB's mechanism of action is crucial for developing species-selective toxicants as alternatives to the broad-spectrum ones currently in use. Recent research efforts have focused on elucidating its cellular mechanisms and sites of action using novel NRB derivatives. The key findings are as follows: NRB selectively opens the rat mitochondrial permeability transition pore, which may be a factor that contributes to its lethal effect; it inhibits rat vascular KATP channels, which potentially controls its Rattus-selective vasoconstricting activity; and it possesses intracellular binding sites in both sensitive and insensitive cells, as revealed by fluorescent derivatives. These studies have led to the development of a prodrug with enhanced pharmacokinetic and toxicological profiles, which is currently undergoing registration as a novel efficacious eco-sustainable Rattus-selective toxicant. The NRB-fluorescent derivatives also show promise as non-toxic probes for intracellular organelle labelling. This review documents in more detail these developments and their implications.

Thymol-based Dual Chemosensor for Optical Detection of Cu2+ and Pb2+ ions and Evaluation of its Toxicity Against Plant Growth Promoting Rhizobacteria

Abstract: Thymol is a naturally occurring monoterpenoid phenol, and its derivatives may emerge as eco-friendly materials for the development of chemosensing probes. To prepare a non-toxic chemosensing probe based on thymol moiety for the detection of metal ions. : A chemosensor (TPC) based on thymol was afforded by the reaction of thymol and piperidine in methanol. The structure elucidation of TPC was carried out with UV-Vis, 1H-NMR, 13C-NMR, and FT-IR analysis. The chemosensing properties of the synthesized probe were determined with UVvisible spectroscopy, and further, it was exploited for the determination of the concentration of Cu2+ and Pb2+ ions in spiked tap water samples. The toxicity studies of the chemosensing probe were conducted against the plant growth-promoting rhizobacteria (PGPR), i.e., Rhizobium sp., Pseudomonas sp., and Azospirillum sp. The absorption spectrum of TPC showed a band at 277 nm. The presence of Cu2+ and Pb2+ ions induced an increase in the absorption intensity at 277 nm by 2-fold and 1.5-fold, respectively. The binding constant for both TPC-Cu2+ and TPC-Pb2+ was calculated as 1.55 × 105 M-1 and 1.47 × 105 M-1, respectively. The respective limit of detection (LOD) and limit of quantification (LOQ) for Cu2+ were 623.64 × 10-6 mol L-1 and 1889.85 × 10-6 mol L-1. For Pb2+ ions, LOD and LOQ were calculated as 676.70 × 10-6 mol L-1 and 2050.60 × 10-6 mol L-1, respectively. In spiked tap water samples, percent recovery was observed in the range of 80.1 to 81.0 % and 80.1 to 81.9 % for Cu2+ and Pb2+ ions, respectively. Toxicity studies of the synthesized probe inferred that TPC was non-toxic against the tested PGPR at all the tested concentrations. In this work, we have synthesized a thymolbased chemosensor, which has been evaluated as a non-toxic sensor for Pb2+ and Cu2+ ions.

Application of a Specific and Sensitive NQO1 Turn-On Near-Infrared Fluorescence Probe for Live Cancer Cell and Xenografted Tumor Imaging in Nude Mice.

Sensitive activity stains for enzymes selectively expressed in human cancers offer valuable tools for imaging with wide applications in experimental, diagnostic, and therapeutic settings. The scant expression of the antioxidant enzyme NQO1 in normal tissues and its great abundance in malignant counterparts due to the increased redox stress and hypoxia is one such example. Previously, we described a potent nontoxic probe that remains nonfluorescent but releases an intense fluorogenic compound after intracellular cleavage by NQO1 catalysis. This infrared probe with a 644nm emission has excellent tissue penetrating ability and low background absorption. Described here are methods (fluorescence microscopy, flow cytometry, and in vivo animal imaging) to rapidly image NQO1 activity in hypoxic and non-hypoxic cancer cells and tumors developed in live mouse xenograft models. The specificity of the dye for NQO1 in all three procedures was verified, and the methods should be useful for both in vitro and in vivo studies.

A self-assembled nanoprobe for detecting HSA based on hydrazide Schiff base: Its applications in diseases diagnosis and lysosome targeting imaging

Human serum albumin (HSA) is considered to be a diagnostic index of numerous diseases, and detecting HSA in subcellular environments is of great importance. In this work, we successfully synthesized a fluorescent probe NDIC based on hydrazide Schiff base, which can self-assemble into nanospheres in aqueous solution and produce distinct fluorescence signal in the presence of HSA. NDIC showed advantages including good sensitivity (LOD = 8.44 nM), high selectivity and anti-interference (over different coexisting proteins, amino acids, cations and anions). Job's plot, CD spectra, HSA destruction, site binding replacement and molecular docking provided reliable evidence for the fact that NDIC could combine at drug site I of HSA. Non-toxic probe NDIC could be applied to quantitatively detect HSA contents in urine, and achieve cancer identification by HSA imaging in cancer cells. More importantly, successful confocal experiment proved that NDIC possessed the lysosome selective imaging ability with the aid of HSA. These results showed the probe NDIC met the requirements of biological applications and was expected to explore HSA at the subcellular levels.

Real-Time Detection and Visualization of Amyloid-β Aggregates Induced by Hydrogen Peroxide in Cell and Mouse Models of Alzheimer's Disease.

Oxidative stress, caused by an imbalance between the production and the accumulation of reactive oxygen species (ROS), is a prominent cause of the neurotoxicity induced by aggregated amyloid-β (Aβ) in Alzheimer's disease (AD). Tools that can directly detect and monitor the presence and amount of Aβ-induced ROS are still lacking. We report herein the first Aβ-targeted ratiometric H2O2-responsive fluorescent probe for real-time detection and monitoring of the Aβ-induced H2O2 level in cell and AD mouse models. The H2O2-responsive probe is constructed based on a methylamino-substituted quinolinium-based cyanine as the fluorescence moiety and a phenylboronate ester as the sensing reaction site. This sensing probe exhibits a large emission wavelength shift of ∼87 nm upon reacting with H2O2, a high binding selectivity for Aβ, and a faster response toward H2O2 in the presence of Aβ, concomitant with an enhanced fluorescence intensity, hence greatly boosting the sensitivity of in-situ H2O2 detection. This biocompatible and nontoxic probe is capable of ratiometrically detecting and imaging endogenous H2O2 induced by Aβ in a neuronal cell model. Remarkably, this Aβ-targeted H2O2-responsive probe is also able to detect, monitor, and differentiate different Aβ-induced H2O2 levels in real time in different age groups of transgenic AD mice in which the cerebral H2O2 level increases age dependently concomitant with the plaque contents. Therefore, this smart probe can act as a powerful tool to diagnose high-risk subjects and diseased brains of AD and to further study the role of ROS in AD pathology.

Aqueous synthesis of core/shell/shell ZnSeS/Cu:ZnS/ZnS quantum dots and their use as a probe for the selective photoluminescent detection of Pb2+ in water

Water-dispersible core/shell/shell ZnSeS/Cu:ZnS/ZnS quantum dots (QDs) capped with 3-mercaptopropionic acid (3-MPA) were successfully prepared via a one-pot method. The dots are photostable, exhibit a Cu-related photoluminescence (PL) emission located at 500 nm and a PL quantum yield of 21%. The PL of ZnSeS/Cu:ZnS/ZnS QDs was demonstrated to be selectively quenched by Pb2+ ions and a method for the rapid and sensitive detection of Pb2+ in real water samples was developed. Under the optimized conditions, the PL intensity of ZnSeS/Cu:ZnS/ZnS QDs is almost linearly dependent on the Pb2+ concentration in the range of 0.04 to 6 µM and the detection limit is 21 nM in water. Based on PL lifetime and UV–visible absorption measurements, a dynamic quenching mechanism was demonstrated. ZnSeS/Cu:ZnS/ZnS QDs were also demonstrated to be efficient probes for the detection of Pb2+ in real water samples. The developed method is rapid, simple and sensitive and demonstrates that ZnSeS/Cu:ZnS/ZnS QDs can be used as non-toxic probes to sense Pb2+ ions in aqueous solution.

A π-Stacking Based Fluorescent Probe for Labeling of Flavin Analogues in Live Cells through Unusual FRET Process.

The flavin adenine dinucleotide (FAD) is an indispensable coenzyme in live cells. It acts as a catalyst in many redox responsive metabolic reactions, including oxidative phosphorylation in mitochondria. The real-time monitoring of flavin is important to understand the disorder in the metabolic process, redox system, etc. Thus, we have developed a fluorescent probe CPy-1 that noncovalently binds with flavin to exhibit the FRET process. 1H- NMR and docking study indicated that there is a strong hydrophobic interaction between flavins and CPy-1. Also, a π-π stacking between isoalloxazine ring in flavin and quinoline and coumarin moieties of CPy-1 favors self-assembly. The nontoxic probe CPy-1 could distinguish cancer cells from normal cells based on expressions of endogenous FAD.

Intracellular oxygen metabolism during bovine oocyte and preimplantation embryo development

We report a novel method to profile intrcellular oxygen concentration (icO2) during in vitro mammalian oocyte and preimplantation embryo development using a commercially available multimodal phosphorescent nanosensor (MM2). Abattoir-derived bovine oocytes and embryos were incubated with MM2 in vitro. A series of inhibitors were applied during live-cell multiphoton imaging to record changes in icO2 associated with mitochondrial processes. The uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) uncouples mitochondrial oxygen consumption to its maximum, while antimycin inhibits complex III to ablate mitochondrial oxygen consumption. Increasing oxygen consumption was expected to reduce icO2 and decreasing oxygen consumption to increase icO2. Use of these inhibitors quantifies how much oxygen is consumed at basal in comparison to the upper and lower limits of mitochondrial function. icO2 measurements were compared to mitochondrial DNA copy number analysed by qPCR. Antimycin treatment increased icO2 for all stages tested, suggesting significant mitochondrial oxygen consumption at basal. icO2 of oocytes and preimplantation embryos were unaffected by FCCP treatment. Inner cell mass icO2 was lower than trophectoderm, perhaps reflecting limitations of diffusion. Mitochondrial DNA copy numbers were similar between stages in the range 0.9–4 × 106 copies and did not correlate with icO2. These results validate the MM2 probe as a sensitive, non-toxic probe of intracellular oxygen concentration in mammalian oocytes and preimplantation embryos.

A chromone hydrazide Schiff base fluorescence probe with high selectivity and sensitivity for the detection and discrimination of human serum albumin (HSA) and bovine serum albumin (BSA)

The discrimination and identification of human serum albumin (HSA) and bovine serum albumin (BSA) is very important, which is due to the vital roles of two SAs in biological and pharmaceutical research. Based on structural screening and docking calculation from a series of homologues, a coumarin Schiff base fluorescent probe 3-hydroxy-N′-((4-oxo-4H-chromen-3-yl)methylene)-2-naphthohydrazide (HCNH) has been designed and synthesized, which could effectively discriminate HSA and BSA. The probe HCNH exhibited superior sensitivity toward HSA and BSA with the detection limits of 10.62 nM and 16.03 nM in PBS solution, respectively. The binding mechanism of HCNH with SAs was studied by Job’s plot analysis, SA destruction and displacement assay. Molecular docking and DFT methods were utilized to provide deep insight into the spatial conformation change of HCNH and binding sites in HSA/BSA. The conformation of HCNH was significantly influenced by the microenvironment provided by HSA and BSA, therefore its fluorescence emission was affected correspondingly. Non-toxic probe HCNH could be successfully used for fluorescence bio-imaging of HSA in cancer cells, which is significantly different from normal cells and favors the application in medical diagnosis.

Design, synthesis and evaluation of HepDirect fluorescence probes mediated by asialoglycoprotein receptor

A series of HepDirect pH responsive fluorescence probes of glycosyl-rhodamine are synthesized. The recognition trend of glucoside < lactoside < galactoside illuminates that the galactose is a targeting group toward HepG2 cells and its recognition is affected by the bulk of targeting group. The HepG2 cells binding trend of RDGal1< RDGal2< RDGal3 supports the cluster effect of galactose. RDGal3 with the optimum targeting potential is verified to selectively recognize HepG2 cells via a galactose dependent ASGPR binding mechanism. These nontoxic probes could be controllably activated by the acidic microenvironment of cancer cells, which is very significant to achieve diagnosis of hepatocellular carcinoma and provide a novel HepDirect carrier for precision treatment of hepatocarcinoma.

A novel turn on and reversible sensor for Al3+ and its applications in bioimaging

A new and non-toxic probe 1 is synthesized and acted as fluorogenic sensor for Al3+ detection in CH3CH2OH / H2O ( 1: 1, v / v). Besides the specific Al3+ induced a yellow solution of probe 1 to turn to colorless and with a 33 - fold fluorescence enhancement at 440 nm. Probe 1 is highly selective to Al3+ among other tested metal ions and anions with the detection limit is 0.37 μM. What's more, the binding constant between probe 1 and Al3+ is 7.62 × 106 M−1. The mechanism is confirmed by ESI - MS and a stoichiometric ratio of 1: 1 between probe 1 and AI3+. The reversible ‘‘off-on’’ Al3+ response, fine cell permeability and cell imaging ability displays that its potential application in both environmental and Al3+ polluted biological samples.

Highly selective and sensitive recognition of Zn(ii) by a novel coumarinyl scaffold following spectrofluorometric technique and its application in living cells

A biocompatible coumarinyl Schiff base scaffold (H2L), a nontoxic probe and a fluorescent sensor to Zn2+, with an LOD of 11 nM, was used for living cell imaging.

A novel assay for evaluation of the total antioxidant capacity (TAC) using a nontoxic probe

A novel assay for evaluation of the total antioxidant capacity (TAC) using a nontoxic probe

SERS-active multi-channel fluorescent probe for NO: Guide to discriminate intracellular biothiols

We explored a rational design and synthesis of a Raman-active small molecular fluorescent probe (S1/Int-1) to acquire multicolor fluorescence-images based on intracellular nitric oxide (NO) in cancer cells. A new UV–vis absorption band centered at λab 510 nm for S1 was appeared in the presence of NO. Similarly, with increasing concentration of NO, the emission signal centered at 527 nm increased gradually. The probe S1 reacted with NO to form benzotriazole derivative Int-1 which showed characteristic intense Raman band centered at 1440 cm-1 for NN-. Individually, GSH and Cys reacted with Int-1 to offer a pair of new fluorophore 2 and 3 with different emission band centered at λem = 482 nm and λem = 453 nm respectively. Furthermore, the non-toxic probe S1 facilitated the monitoring of intracellular NO based dual colored imaging in the green and blue channel which intern allowed differentiating the intracellular GSH and Cys levels. Moreover, its cellular “off-on-off” response in the SERS spectral mode indicates that S1 can be a noninvasive tool to detect endogenous NO, GSH/Cys. Therefore, probe (S1/Int-1) could allow us monitoring of intracellular NO formation through dual channel fluorescence imaging with synchronous discrimination of thiols.

A BODIPY-based two-photon fluorescent probe validates tyrosinase activity in live cells.

Herein, we report rational design, synthesis, and application of a two-photon fluorescent probe (Tyro-1) for tracking intracellular tyrosinase activity. The chemoselective detection of tyrosinase is precluded from interference of other competitive omnipresent oxidizing entities in cellular milieu. The probe showed 12.5-fold fluorescence enhancement at λem = 450 nm in the presence of tyrosinase. The nontoxic probe Tyro-1 provides information about H2O2-mediated upregulation of tyrosinase through cellular imaging. Its two-photon imaging ability makes it a noninvasive tool for validating the expression of tyrosinase in the live cells.

Direct Measurements of Oxygen Gradients in Spheroid Culture System Using Electron Parametric Resonance Oximetry.

Advanced in vitro culture from tissues of different origin includes three-dimensional (3D) organoid micro structures that may mimic conditions in vivo. One example of simple 3D culture is spheroids; ball shaped structures typically used as liver and tumour models. Oxygen is critically important in physiological processes, but is difficult to quantify in 3D culture: and the question arises, how small does a spheroid have to be to have minimal micro-environment formation? This question is of particular importance in the growing field of 3D based models for toxicological assessment. Here, we describe a simple non-invasive approach modified for the quantitative measurement and subsequent evaluation of oxygen gradients in spheroids developed from a non-malignant fish cell line (i.e. RTG-2 cells) using Electron Paramagnetic Resonance (EPR) oximetry. Sonication of the paramagnetic probe Lithium phthalocyanine (LiPc) allows for incorporation of probe particulates into spheroid during its formation. Spectra signal strength after incorporation of probe into spheroid indicated that a volume of 20 μl of probe (stock solution: 0.10 mg/mL) is sufficient to provide a strong spectra across a range of spheroid sizes. The addition of non-toxic probes (that do not produce or consume oxygen) report on oxygen diffusion throughout the spheroid as a function of size. We provide evidence supporting the use of this model over a range of initial cell seeding densities and spheroid sizes with the production of oxygen distribution as a function of these parameters. In our spheroid model, lower cell seeding densities (∼2,500 cells/spheroid) and absolute size (118±32 μm) allow control of factors such as pre-existing stresses (e.g. ∼ 2% normoxic/hypoxic interface) for more accurate measurement of treatment response. The applied methodology provides an elegant, widely applicable approach to directly characterize spheroid (and other organoid) cultures in biomedical and toxicological research.

Xenobiotic Receptor-Mediated Regulation of Intestinal Barrier Function and Innate Immunity

The molecular basis for the regulation of the intestinal barrier is a very fertile research area. A growing body of knowledge supports the targeting of various components of intestinal barrier function as means to treat a variety of diseases, including the inflammatory bowel diseases. Herein, we will summarize the current state of knowledge of key xenobiotic receptor regulators of barrier function, highlighting recent advances, such that the field and its future are succinctly reviewed. We posit that these receptors confer an additional dimension of host-microbe interaction in the gut, by sensing and responding to metabolites released from the symbiotic microbiota, in innate immunity and also in host drug metabolism. The scientific evidence for involvement of the receptors and its molecular basis for the control of barrier function and innate immunity regulation would serve as a rationale towards development of non-toxic probes and ligands as drugs.

Generation of Internal-Image Functional Aptamers of Okadaic Acid via Magnetic-Bead SELEX.

Okadaic acid (OA) is produced by Dinophysis and Prorocentrum dinoflagellates and primarily accumulates in bivalves, and this toxin has harmful effects on consumers and operators. In this work, we first report the use of aptamers as novel non-toxic probes capable of binding to a monoclonal antibody against OA (OA-mAb). Aptamers that mimic the OA toxin with high affinity and selectivity were generated by the magnetic bead-assisted systematic evolution of ligands by exponential enrichment (SELEX) strategy. After 12 selection rounds, cloning, sequencing and enzyme-linked immunosorbent assay (ELISA) analysis, four candidate aptamers (O24, O31, O39, O40) were selected that showed high affinity and specificity for OA-mAb. The affinity constants of O24, O31, O39 and O40 were 8.3 × 108 M−1, 1.47 × 109 M−1, 1.23 × 109 M−1 and 1.05 × 109 M−1, respectively. Indirect competitive ELISA was employed to determine the internal-image function of the aptamers. The results reveal that O31 has a similar competitive function as free OA toxin, whereas the other three aptamers did not bear the necessary internal-image function. Based on the derivation of the curvilinear equation for OA/O31, the equation that defined the relationship between the OA toxin content and O31 was Y = 2.185X − 1.78. The IC50 of O31 was 3.39 ng·mL−1, which was close to the value predicted by the OA ELISA (IC50 = 4.4 ng·mL−1); the IC10 was 0.33 ng·mL−1. The above data provides strong evidence that internal-image functional aptamers could be applicable as novel probes in a non-toxic assay.

Fluorescent kapakahines serve as non-toxic probes for live cell Golgi imaging

There is an ongoing need for fluorescent probes that specifically-target select organelles within mammalian cells. This study describes the development of probes for the selective labeling of the Golgi apparatus and offers applications for live cell and fixed cell imaging. The kapakahines, characterized by a common C(3)-N(1') dimeric tryptophan linkage, comprise a unique family of bioactive marine depsipeptide natural products. We describe the uptake and subcellular localization of fluorescently-labeled analogs of kapakahine E. Using confocal microscopy, we identify a rapid and selective localization within the Golgi apparatus. Comparison with commercial Golgi stains indicates a unique localization pattern, which differs from currently available materials, therein offering a new tool to monitor the Golgi in live cells without toxic side effects. This study identifies a fluorescent analog of kapakahine E that is rapidly uptaken in cells and localizes within the Golgi apparatus. The advance of microscopic methods is reliant on the parallel discovery of next generation molecular probes. This study describes the advance of stable and viable probe for staining the Golgi apparatus.

Gated Luminescence Imaging of Silicon Nanoparticles.

The luminescence lifetime of nanocrystalline silicon is typically on the order of microseconds, significantly longer than the nanosecond lifetimes exhibited by fluorescent molecules naturally present in cells and tissues. Time-gated imaging, where the image is acquired at a time after termination of an excitation pulse, allows discrimination of a silicon nanoparticle probe from these endogenous signals. Because of the microsecond time scale for silicon emission, time-gated imaging is relatively simple to implement for this biocompatible and nontoxic probe. Here a time-gated system with ∼10 ns resolution is described, using an intensified CCD camera and pulsed LED or laser excitation sources. The method is demonstrated by tracking the fate of mesoporous silicon nanoparticles containing the tumor-targeting peptide iRGD, administered by retro-orbital injection into live mice. Imaging of such systemically administered nanoparticles in vivo is particularly challenging because of the low concentration of probe in the targeted tissues and relatively high background signals from tissue autofluorescence. Contrast improvements of >100-fold (relative to steady-state imaging) is demonstrated in the targeted tissues.