Florescent Probe Research Articles

A rapid fluorescence detecting platform: applicable to sense carnitine and chloramphenicol in food samples

A new long-wavelength latent florescent probe, termed “BCC” for sensitive determination of coenzyme A, carnitine and chloramphenicol.

Abstract 4515: Improvement of tissue distribution by the treatment of novel different double-arms polyethyleneglycol modified liposome encapsulating doxorubicin.

Abstract [Purpose] Polyethyleneglycol (PEG) modification on liposomal membrane increases therapeutic index by improvement of targeting based on prolonged blood stream circulation. In contrast, some accumulation of drug after liposome treatment has also been observed in normal tissues, it is necessary to improve liposomal composition on the decrease of adverse reactions. Particularly, doxorubicin (DOX) encapsulated liposome has some adverse reactions as cardiac toxicity or hand-foot syndrome,etc. To contribute superior liposome formulation, different double arms PEG in a molecule(DDA-PEG) was synthesized and was clarified effectiveness in our previous reports. In this study, we examined physical properties of novel synthesized DDA-PEG, and the tissue distribution of the DOX encapsulated liposome with modification of these DDA-PEGs. [Methods] Two DDA-PEGs with (PEG1000 and PEG500) or (PEG2000 and PEG500) were synthesized. Critical micelle concentration (cmc) in 10 mM Tris-HCl buffer (pH7.4) and 10 mM lactate buffer (pH4.0) was measured by the method of florescent probe using the 8-anilinonaphthalene-1-sulfonate (ANS). Octanol-buffer partitioning value was determined. DOX encapsulated liposomes with the modification of each DDA-PEGs were prepared by DSPC/cholesterol/DSPG-Na/DOX/PEG-lipid =100:100:60:18:7.5 μmol according to the method of Bangham. M5076 ovarian sarcoma cells were transplanted into the backs of BDF1 mice, and each liposome was injected intravenously at a dose of 2.5 mg DOX/kg on 15, 18 and 21 day after tumor inoculation. The mice were sacrificed after 2 day from last administration, and the tumor was removed and weighted. Moreover, DOX concentrations in each tissue were determined. [Results and discussion] The cmc of all PEG-lipids were same level. In PEG(2000, 500)-modified liposome, DOX concentrations in tumors were equal to that in PEG2000-modified liposome. On the other hand, DOX concentration in the heart after PEG(2000, 500) modified liposome and PEG(1000,500)-modified liposome treatments were one second and one sixth compared with DOX solution group, respectively. According to these results, it was expected that novel DDA-PEG-modified liposome was able to reduce the cardiac toxicity which is adverse reaction by DOX. The DOX concentration in the liver was showed a low level by PEG (1000, 500) modified liposome. It was suggested that novel DDA- PEG-modified liposome had the effect of avoiding the uptake into the liver. In conclusion, it was suggested that PEG(1000,500) modified liposome could decrease the DOX level in heart, and had ability of accumulation into tumor and avoidance from reticuloendothelial cells as same as PEG2000-modified liposome as known liposome. Citation Format: Mai Yagi, Ikumi Sugiyama, Yasuyuki Sadzuka. Improvement of tissue distribution by the treatment of novel different double-arms polyethyleneglycol modified liposome encapsulating doxorubicin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4515. doi:10.1158/1538-7445.AM2013-4515

DNase activity in kidney cell pyknosis induced by serum deprivation

Pyknosis (nuclear shrinkage) is an important feature of apoptosis, which is preceded by activation of apoptotic pathways and is followed by karyorrhexis (nuclear fragmentation). The mechanism of pyknosis remains a mystery. The goals of this study were to determine if nuclear membrane ruptures when DNA fragmentation starts during pyknosis and to determine the involvement of DNases, cellular compartments, and chaperone proteins in pyknosis. A novel florescent probe to measure DNase activity was used in live kidney tubular epithelial NRK‐52E cells. Pyknosis was induced by serum‐deprivation; DNA breaks were measured by TUNEL; and immunocytochemistry of nuclear membrane, DNases, cellular compartments and chaperones was applies to characterize potential mechanisms. We demonstrated that: (a) DNase activity asymmetrically concentrates on one side of the nucleus, which changes the shape of the nucleus; (b) DNA fragmentation occurs but; (c) nuclear membrane does not break during pyknosis; (d) no measurable DNases were observed in the nucleus; (e) DNase activity colocalized with Golgi, ER, vesicles and HSP60, all of which seem to contribute to the mechanism of pyknosis. NIH R01 DK078908 and VA Merit Review grants to AGB.

Detection of RNA Viruses: Current Technologies and Future Perspectives

RNA viruses constitute one of the major classes of pathogenic organisms causing human diseases, with varying degrees of severity. This review summarizes the conventional and emerging technologies that are available for the detection of these organisms. Cell culture-based techniques for viral detection have been popular since their inception and continue to be the gold standard against which all other techniques. Over many years, these techniques have undergone some radical changes, reducing the total time needed for detection and improving sensitivity, although even with their reliability and improved features they are being slowly replaced by nucleic acid-based technologies. These molecular detection techniques have revolutionized the area of viral detection by their high sensitivity, selectivity, and short detection time. The majority of nucleic acid-based techniques depend on amplifying viral RNA; however, there are some newer emerging techniques that detect viral RNA in live cells using various configurations of florescent probes. In addition, nucleic acid-based technology has made it possible for multiviral detection with either multiplex polymerase chain reaction assays or microarrays. Every technique described in this review has its own unique abilities, making them indispensable for viral detection. However, we believe that nucleic acid-based technologies will find widespread use after being standardized, limiting other technologies to very specific uses.

Critical issues in detecting viable Listeria monocytogenes cells by real-time reverse transcriptase PCR.

Rapid and specific detection of viable Listeria monocytogenes cells, particularly in processed foods, is a major challenge in the food industry. To assess the suitability of using RNA-based detection methods to detect viable cells, several sets of PCR primers and florescent probes were designed targeting the 16S rRNA, internalin A, and ribosomal protein L4 genes. One-step real-time reverse transcriptase (RT) PCR assays were conducted using RNAs extracted from control and heat-treated L. monocytogenes samples. The cycle threshold values were significantly higher in heat-treated cells than in controls. However, real-time RT-PCR amplification signals were still detected even in samples stored at room temperature for 24 h after lethal treatments, and the intensity of the signals was correlated with the cell population. The 16S rRNA molecules were the most stable of the three targets evaluated, and the impact on detection efficacy of the relative positions of the PCR primers within the target genes was limited under the experimental conditions. These results suggest that real-time RT-PCR assays have advantages over conventional PCR assays for assessing viable L. monocytogenes cells, but the results are affected by the stability of the RNA molecules targeted. These findings could have a major impact on interpretation of RNA-based detection data and gene expression studies.

Bio-capture of S. Typhimurium from surface water by aptamer for culture-free quantification

In this study, a DNA aptamer was used to bio-capture Salmonella enterica serovar Typhimurium from surface water collected from highly endemic zone prior to culture-free detection through Molecular-Beacon based real-time PCR assay targeting invA gene. The assay could detect S. Typhimurium cells (1CFU/PCR or 100CFU/ml) selectively captured by serovar specific DNA aptamer. The observations indicate that all the water samples (n=40) collected from the river Gomti were contaminated by S. Typhimurium (31400–1×107CFU/100ml). The pre-analytical step in the form of serovar specific DNA aptamer based bio-capture of the bacterial cell was found to enhance the sensitivity of the florescent probe based real-time PCR assay during detection of S. Typhimurium in environmental samples exhibiting natural PCR inhibitors and high background bacterial flora. The assay could be used for the regular monitoring of surface waters for forecasting and management of non-typhoidal Salmonellosis in south Asia.

A facile transport assay for H+coupled membrane transport using fluorescence probes

A facile activity assay for an H+-coupled transporter using florescent probes was developed with an H+-coupled manganese transporter (MntH) as a model. Making use of coupled-proton transport, the transport activity (H+/Mn2+ cotransport) can be directly determined via fluorescence intensity changes of the probe, 5-(and-6)-carboxyfluorescein (5(6)-FAM). The approach of using highly sensitive fluorescence probes provides a more simple and convenient assay method for the determination of proton-coupled metal-ion uptake by transporters.

Facile bulk production of highly blue fluorescent graphitic carbon nitride quantum dots and their application as highly selective and sensitive sensors for the detection of mercuric and iodide ions in aqueous media

Highly blue fluorescent graphitic carbon nitride quantum dots (g-CNQDs) were synthesized by a simple microwave mediated method from formamide (HCONH2). The fluorescence emission of g-CNQDs is found to be strongly dependent on solvent, pH as well as on excitation wavelengths; the life time of the emission decreases with increasing polarity of the solvent. These quantum dots are highly sensitive and selective florescent probes for mercuric ions in aqueous media due to the “superquenching” of fluorescence. The complex formation of Hg2+ ions with the CNx sheet involving π delocalized electron moieties of the latter, in fact, is responsible for the quenching of fluorescence. The addition of iodide ions abstracts the bound Hg2+ forming HgI2 and gives back the fluorescence characteristic of g-CNQDs. Thus, g-CNQDs can play a dual role for selective and sensitive detection of mercuric ions as well as iodide ions in aqueous media via “ON–OFF–ON” fluorescence response.

2-(2'- Chloro Phenyl)- 5- (2'-Hydroxyl Phenyl)-1,3,4-Oxadiazole as a Florescent Probe for DNA Determination

In recent years, fluorescence spectrometry was widely used in quantitative determination of DNA. In this paper, a convenient synthesis of a new fluorescent 2-(2'- Chloro phenyl)- 5- (2'- hydroxyl phenyl)-1,3,4-oxadiazole (HOXD) was realized. Experimental data showed that fluorescence of HOXD could be quenched by DNA and the decreased fluorescence intensity of HOXD resulting from fluorescence quenching is proportional to DNA concentrations suggesting that HOXD could be used as a new fluorescent probe for quantitative determination of DNA. Optimal experimental conditions for DNA analysis were also studied in the paper.

Pyrroloquinoxaline hydrazones as fluorescent probes for amyloid fibrils

Here we describe the identification and preliminary characterization of a new class of pyrrolo(imidazo)quinoxaline hydrazones as florescent probes for Aβ(1-42) fibrils. All the newly developed compounds were able to bind amyloid fibrils formed in vitro and some of them displayed an increase of their fluorescence upon binding. When tested on brain tissue preparations presenting Aβ deposits, the described hydrazones selectively stained amyloid structures and did not display aspecific binding. The hydrazones did not show antifibrillogenic activity and electron microscopy analysis revealed that they do not interfere with fibrils structure. The described pyrrolo(imidazo)quinoxalines could be useful for studying amyloid structures in vitro. Moreover, their experimentally proven ability to cross the blood-brain barrier in mouse opens the possibility of developing these compounds as potential amyloid imaging agents for in vivo applications.

Simultaneous Measurement Of Force And Fluorescence In Single Guinea-pig Cardiac Myofibrils

Kinetic studies of force activation and relaxation in the single myofibril preparation have enhanced our understanding of mechanisms that underlie regulation of muscle force. Likewise, stop-flow and steady-state measurement using fluorescently labeled TnC upon Ca2+ activation in reconstituted protein systems have provided data regarding the kinetics of thin filament regulation. However, to develop a detailed understanding and fully characterize muscle regulation, an integration of mechanical, biochemical, and structural information is necessary. We present the development of a new apparatus which allows simultaneous measurement of florescent probe signals from labeled contractile proteins (such as TnC), and force kinetics under active isometric force development to allow direct correlation between these parameters in a single myofibril. Single guinea-pig cardiac myofibrils were prepared in relaxing solution (PCa8.0) by mechanical homogenization; endogenous TnC was exchanged for recombinant mutant labeled human TnC (2.5 mg/ml; overnight 4°C; Alexa-350 label conjugated to cys-84). Myofibrils were attached to glass micro-tools with one serving as a cantilever of known stiffness to assess force (painted black for improved contrast) and the other attached to a high speed motor. Myofibrils were activated with a Ca2+ pulse delivered via a double barreled perfusion pipette attached to a stepper motor (∼5 ms). Excitation UV-light (270-350 nm), chopped at 52 kHz using a photo-elastic modulator, was projected (400nm DC mirror) onto the attached myofibril. Force probe deflection was measured in bright-field using red light (670nm) while fluorescence emission was measured (420nm LP filter) using a photomultiplier. Data was sampled by a high-speed (2MHZ) A/D converter and demodulated using custom designed software. Preliminary results indicate that using this apparatus and techniques, ultra-low intensity TnC fluorescence signals can be obtained at high temporal resolution in parallel with bright-field optical measurement of force development.

Luminescence from Surfactant-Free ZnO Quantum Dots Prepared by Laser Ablation in Liquid

Highly transparent, luminescent and biocompatible ZnO quantum dots were prepared in water, methanol, and ethanol usingliquid-phase pulsed laser ablation technique without using any surfactant. Transmission electron microscopy analysis confirmedthe formation of good crystalline ZnO quantum dots with a uniform size distribution of 7 nm. The emission wavelength could bevaried by varying the native defect chemistry of ZnO quantum dots and the laser fluence. Highly luminescent nontoxic ZnOquantum dots have exciting application potential as florescent probes in biomedical applications.© 2007 The Electrochemical Society. DOI: 10.1149/1.2820767 All rights reserved.Manuscript submitted September 4, 2007; revised manuscript received November 6, 2007.Available electronically December 17, 2007.

Mitochondrial mechanism of microvascular endothelial cells apoptosis in hyperhomocysteinemia

An elevated level of homocysteine (Hcy) limits the growth and induces apoptosis. However, the mechanism of Hcy-induced programmed cell death in endothelial cells is largely unknown. We hypothesize that Hcy induces intracellular reactive oxygen species (ROS) production that leads to the loss of transmembrane mitochondrial potential (Deltapsi(m)) accompanied by the release of cytochrome-c from mitochondria. Cytochrome-c release contributes to caspase activation, such as caspase-9, caspase-6, and caspase-3, which results in the degradation of numerous nuclear proteins including poly (ADP-ribose) polymerase (PARP), which subsequently leads to the internucleosomal cleavage of DNA, resulting cell death. In this study, rat heart microvascular endothelial cells (MVEC) were treated with different doses of Hcy at different time intervals. Apoptosis was measured by DNA laddering and transferase-mediated dUTP nick-end labeling (TUNEL) assay. ROS production and MP were determined using fluorescent probes (2,7-dichlorofluorescein (DCFH-DA) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzamidazolocarbocyanin iodide (JC-1), respectively, by confocal microscopy. Differential gene expression for apoptosis was analyzed by cDNA array. The results showed that Hcy-mediated ROS production preceded the loss of MP, the release of cytochrome-c, and the activation of caspase-9 and -3. Moreover the Hcy treatment resulted in a decrease in Bcl(2)/Bax ratio, evaluated by mRNA levels. Caspase-9 and -3 were activated, causing cleavage of PARP, a hallmark of apoptosis and internucleosomal DNA fragmentation. The cytotoxic effect of Hcy was blocked by using small interfering RNA (siRNA)-mediated suppression of caspase-9 in MVEC. Suppressing the activation of caspase-9 inhibited the activation of caspase -3 and enhanced the cell viability and MP. Our data suggested that Hcy-mediated ROS production promotes endothelial cell death in part by disturbing MP, which results in subsequent release of cytochrome-c and activation of caspase-9 and 3, leading to cell death.

Free-volume distributions of polystyrene probed by positron annihilation: comparison with free-volume theories

The free-volume hole distribution in polystyrene (T g = 95°C, molar weight 106000) has been investigated as a function of temperature. The hole volume distributions are determined from orthopositronium distributions. The hole volumes are distributed between 35 and 200 A 3 . The fractions of free volumes are distributed from 2.0% to 11% of total volume. The obtained free-volume distributions are compared and discussed with the experimental distributions obtained by photochromic and florescent probes and with the Turnbull-Cohen free-volume theory. The distributions of free-volume fractions fit well with the theoretical function according to the Simha-Somcynsky lattice model