Application of organic fluorescent probe-assisted near infrared fluorescence imaging in cervical cancer diagnosis / 生物工程学报

Fluorescence imaging has been widely used in the fields of biomedicine and clinical diagnosis. Compared with traditional fluorescence imaging in the visible spectral region (400-760 nm), near-infrared (NIR, 700-1 700 nm) fluorescence imaging is more helpful to improve the signal-to-noise ratio and the sensitivity of imaging. Highly-sensitive fluorescent probes are required for high-quality fluorescence imaging, and the rapid development of nanotechnology has led to the emergence of organic dyes with excellent fluorescent properties. Among them, organic fluorescent probes with the advantages of high safety, good biocompatibility, and high optical stability, are more favorable than inorganic fluorescent probes. Therefore, NIR fluorescence imaging assisted with organic fluorescent probes can provide more structural and dynamic information of biological samples to the researchers, which becomes a hot spot in the interdisciplinary research field of optics, chemistry and biomedicine. This review summarizes the application of NIR organic fluorescent probes in cervical cancer imaging. Several typical organic fluorescent probes (such as indocyanine green, heptamethine cyanine dye, rhodamine and polymer fluorescent nanoparticles) assisted NIR fluorescence imaging and their applications in cervical cancer diagnosis were introduced, and the future development and application of these techniques were discussed.

Fluorescent probes for the detection of reactive oxygen species in human spermatozoa / 亚洲男科学杂志(英文版)

Reactive oxygen species (ROS) production is a by-product of mitochondrial activity and is necessary for the acquisition of the capacitated state, a requirement for functional spermatozoa. However, an increase in oxidative stress, due to an abnormal production of ROS, has been shown to be related to loss of sperm function, highlighting the importance of an accurate detection of sperm ROS, given the specific nature of this cell. In this work, we tested a variety of commercially available fluorescent probes to detect ROS and reactive nitrogen species (RNS) in human sperm, to define their specificity. Using both flow cytometry (FC) and fluorescence microscopy (FM), we confirmed that MitoSOX™ Red and dihydroethidium (DHE) detect superoxide anion (as determined using antimycin A as a positive control), while DAF-2A detects reactive nitrogen species (namely, nitric oxide). For the first time, we also report that RedoxSensor™ Red CC-1, CellROX®Orange Reagent, and MitoPY1 seem to be mostly sensitive to hydrogen peroxide, but not superoxide. Furthermore, mean fluorescence intensity (and not percentage of labeled cells) is the main parameter that can be reproducibly monitored using this type of methodology.

Advances in second near-infrared fluorescent nanoprobes-based in vivo imaging for biomedical applications / 上海交通大学学报（医学版）

Besides the advantages of high sensitivity, non-invasive, and real-time imaging that the general optical imaging technologies have, the second near-infrared (1 000-1 700 nm) in vivo imaging is regarded as one of the most promising optical imaging technologies in clinical applica-tion at present due to its advantages such as low autofluorescence background, deep tissue penetration and clear imaging. In the second near-infrared imaging system, the fluorescence probe is the most critical technical difficulty and the current research hotspot. Thereinto, due to the unique physical and chemical properties of nanomaterials, the second near-infrared fluorescent nanoprobe is an ideal contrast agent for the current second near-infrared optical imaging technology. The reported inorganic materials that can be used as the second near-infrared fluores-cent nanoprobes include quantum dots, rare earth nanoparticles, and single-walled carbon nanotubes, and the reported organic materials are mainly conjugated polymers. In this paper, the research progresses of the second near-infrared fluorescence nanoprobes mentioned above are re-viewed in order to better understand their application to the field of biomedicine, and promote the further research and clinical application of the second near-infrared in vivo imaging to the biomedical field.

Monitoring Glutathione Dynamics and Heterogeneity in Living Stem Cells

Glutathione (GSH) is a major antioxidant in cells, and plays vital roles in the cellular defense against oxidants and in the regulation of redox signals. In a previous report, we demonstrated that stem cell function is critically affected by heterogeneity and dynamic changes in cellular GSH concentration. Here, we present a detailed protocol for the monitoring of GSH concentration in living stem cells using FreSHtracer, a real-time GSH probe. We describe the steps involved in monitoring GSH concentration in single living stem cells using confocal microscopy and flow cytometry. These methods are simple, rapid, and quantitative, and able to demonstrate intracellular GSH concentration changes in real time. We also describe the application of FreSHtracer to the sorting of stem cells according to their GSH content using flow cytometry. Typically, microscopic or flow cytometric analyses of FreSHtracer and MitoFreSHtracer signals in living stem cells take ~2~3 h, and the fractionation of stem cells into subpopulations on the basis of cellular GSH levels takes 3~4.5 h. This method could be applied to almost every kind of mammalian cell with minor modifications to the protocol described here.

Current development of fluorescent probes for cellular reactive oxygen species / 军事医学

Reactive oxygen species(ROS)plays an important role in physiological and pathological processes in cells. Different effects can be produced depending on the type and subcellular localization of ROS.Therefore,various fluorescent probes have been developed to quantify and localize ROS specifically.However,due to technical limitations,these probes have defects in specificity and stability.In this paper, we review existing cellular and mitochondrial ROS probes, and summarize their characteristics.

A Novel Fluorescence Sensor for Highly Sensitive Detection of Glucose / 分析化学

A fluorescence nanosensor based on an easily prepared fluorescent molecule, 1-oxo-1H-phenalene-2,3-dicarbonitrile (OPD), was developed for highly sensitive detection of glucose.Under the catalysis of horseradish peroxidase (HRP), 3,3′,5,5′-tetramethylbenzidine (TMB) was oxidized into oxidized TMB (oxTMB) by H2O2.And the fluorescence of OPD was quenched by the intense absorption of the formed oxTMB, thus realizing effective quantitative detection of H2O2.The linear range was 0.05-0.8 μmol/L and 1-10 μmol/L respectively, with limit of detection of 0.02 μmol/L.Besides, on the basis of transformation of glucose into H2O2 through the catalysis of glucose oxidase, this nanosensor could be further exploited for highly sensitive detection of glucose.The TMB-HRP-OPD sensor exhibited linear range of 0.1-3.0 μmol/L and 4.0-30 μmol/L respectively for detection of glucose, with limit of detection of 0.02 μmol/L.Furthermore, it was successfully applied to the determination of glucose in real human serum and the results were in good agreement with the clinical data.

Preparation of Coal-based C-dots and Their Application in Trace CuⅡ Detection / 分析化学

One of the hot spots of nanomaterials research is the preparation of carbon-dots ( C-dots) by simple steps with cheap raw materials and looking for its potential application. In this study, coal-based C-dots was prepared from coal mined of Wucaiwan in Xinjiang by mixed acids ( H2 SO4+HNO3 )/ultrasound treatment, and at the same time of structural characterization, the coal-based C-dots were used as fluorescent probe to detect metal ions in water. It was found that the coal-based C-dots were polycyclic aromatic hydrocarbons with particle size of (8±4) nm linked with oxygen-containing groups such as nitro group, possessing the annulus wall of multilayer graphene fragment structures built up by sp2 carbons. This endowed the coal-based C-dots with good dispersity in water, high absorbance and strong fluorescence. The coal-based C-dots were used as viable probes in that their fluorescence was selectively quenching by CuⅡ. The finding was exploited to design a fluorometric assay for CuⅡ with a detection limit of 9. 6 nmol/L.

Preparation of Coal-based C-dots and Their Application in Trace CuⅡ Detection / 分析化学

One of the hot spots of nanomaterials research is the preparation of carbon-dots ( C-dots) by simple steps with cheap raw materials and looking for its potential application. In this study, coal-based C-dots was prepared from coal mined of Wucaiwan in Xinjiang by mixed acids ( H2 SO4+HNO3 )/ultrasound treatment, and at the same time of structural characterization, the coal-based C-dots were used as fluorescent probe to detect metal ions in water. It was found that the coal-based C-dots were polycyclic aromatic hydrocarbons with particle size of (8±4) nm linked with oxygen-containing groups such as nitro group, possessing the annulus wall of multilayer graphene fragment structures built up by sp2 carbons. This endowed the coal-based C-dots with good dispersity in water, high absorbance and strong fluorescence. The coal-based C-dots were used as viable probes in that their fluorescence was selectively quenching by CuⅡ. The finding was exploited to design a fluorometric assay for CuⅡ with a detection limit of 9. 6 nmol/L.

Advances of validation of bioactive small molecule targets / 药学实践杂志

With the development of science and the progress of age,medicinal chemistry is not only limited to the lead discovery and the structure-activity relationship studies,but also finding the target of bioactive small molecule drugs has be-come a tough issue to be solved.The identification and validation of bioactive small molecule targets has become the most criti-cal and difficult task,which plays a decisive role in academic and pharmaceutical research.Herein we summarize the current methods for target identification of small molecules,and mainly discuss about the target identification method by the chemical probes.Recent cases of successful application were also introduced to demonstrate the strategy of probe synthesis and design.

Characterizing the conformational change process of bovine serum albumin solution by fluorescence parameters / 中国药学杂志

OBJECTIVE: To study the conformational change process of bovine serum albumin (BSA) in guanidinium chloride through evaluating the fluorescence parameters, thus to elucidate the phenomenon from molecular level and to establish the relationship between the conformation of protein and the environment. METHODS: Intrinsic tryptophan fluorescence, fluorescence quenching and fluorescence probes spectrophotometry were selected to study the denaturation process of BSA in guanidinium chloride. RESULTS: An attenuation of intensity was observed both in BSA and ANS-BSA conjugates with the increasing concentration of guanidium chloride. A red shift on fluorescence emission peak occurred in the ANS-BSA conjugates, while the same result appeared in BSA only after a blue shift. The fluorescence quenching constant Ksv reduced to its minimum 3.469 × 10 L · mol-1 · s-1 in 0.5 mol · L-1 guanidium. CONCLUSION: It was shown that the denaturation process of BSA in guanidinium chloride was consistent with a three-state model, and the conformational change of the binding site on BSA of ANS was much more sensitive than that of tryptophane residue.

Antioxidant activity of natural compounds of Stachytarpheta cayennensis (Rich.) Vahl, Verbenaceae, by scavenger of mitochondrial reactive oxygen species

The etiology of a range of diseases is associated with an excessive generation of reactive oxygen species (ROS). Exacerbated ROS production leads to mitochondrial dysfunction, cellular damage and apoptosis. Nowadays, many strategies are being developed for the targeted delivery of antioxidants compounds to mitochondria, trying to minimize the damages. Production of ROS was investigated by the molecular probes CM-H2DCFDA and Amplex Red. The purpose of this work is to evaluate the antioxidant capacity of verbascoside, martinoside, betulinic acid from the Stachytarpheta cayennensis and quercetin by an in vitro assay with isolated mitochondria from mice's brain The results showed that all compounds tested exhibited a scavenger effect on the ROS generated by the isolated mitochondria, which displayed a dependent dose increase.

Damage assessment of the equine sperm membranes by fluorimetric technique

To validate a practical technique of simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in equine spermatozoa three fluorescent probes (PI, FITC-PSA and MITO) were associated. Four ejaculates from three stallions (n=12) were diluted in TALP medium and split into 2 aliquots, 1 aliquot was flash frozen in liquid nitrogen to induce damage in cellular membranes. Three treatments were prepared with the following fixed ratios of fresh semen: flash frozen semen: 100:0 (T100), 50:50 (T50), and 0:100 (T0). A 150-µL aliquot of diluted semen of each treatment was added of 2 µL of PI, 2 µL of MITO and 80 µL of FITC-PSA; incubated at 38.5ºC/8 min, and sperm cells were evaluated by epifluorescent microscopy. Based in regression analysis, this could be an efficient and practical technique to assess damage in equine spermatozoa, as it was able to determine the sperm percentage more representative of the potential to fertilize the oocyte.

Para validar uma técnica prática de avaliação simultânea das membranas plasmática, acrossomal e mitocondrial em espermatozóides eqüinos três sondas fluorescentes (PI, FITC-PSA e MITO) foram associadas. Quatro ejaculados de três garanhões (n=12) foram diluídos em meio TALP e divididos em duas alíquotas, uma alíquota foi submetida a flash frozen em nitrogênio líquido para induzir danos nas membranas celulares. Três tratamentos foram preparados com as seguintes proporções de sêmen fresco: sêmen flash frozen: 100:0 (T100), 50:50 (T50), e 0:100 (T0). Uma amostra de 150 µL de sêmen diluído de cada tratamento foi adicionada de 2 µL de PI, 2 µL de MITO e 80 µL de FITC-PSA; incubadas à 38,5ºC/8 min, e as células espermáticas foram avaliadas por microscopia de epifluorescência. Baseados na análise de regressão esta é uma técnica eficiente e prática para determinar danos em espermatozóides eqüinos, capaz de determinar a porcentagem de espermatozóides mais representativa do potencial fertilizante do ovócito.

Rapid prenatal diagnosis of spinocerebellar ataxia type 3 by using fluorescent PCR

Spinocerebellar Ataxia Type 3 (SCA3) is a rare autosomal dominative disorder in which one of the neurodegenerative disorders is caused by a CAG repeat expansion on chromosome 14q32.1. The age at onset of disease is related to the size of the expanded CAG repeat. We present the prenatal diagnosis of SCA3 in a woman whose husband was known to carry an unstable CAG repeat expansion in the MJD gene. The diagnosis was made using PCR with a fluorescent probe for an expanded MJD allele. The normal ranges of (CAG)n of SCA3 are 14~38 repeats. The husband, who had a family history of SCA3, has an expanded allele of 69 CAG repeats with a normal allele of 27 repeats. His wife had two normal alleles with 26 and 32 CAG repeats. The fetus had two normal alleles with 26 and 27 CAG repeats; consequently, the baby was healthy. We report a case of prenatal diagnosis of SCA3 using a fluorescent PCR which is rapid and accurate.

Viability evaluation of engineered tissues

Biohybrid artificial organs encompass all devices capable of substituting for an organ or tissue function and are fabricated from both synthetic materials and living cells. The viability of engineered tissue could be related to the viability of implanted cells. The system of viability assay for mammalian cell culture can be applied to the determination of cell viability for engineered tissue. This review explores various methods of cell viability assay which can be applied to the viability evaluation of engineered tissue. The major criteria employed in viability assays include survival and growth in tissue culture, functional assay, metabolite incorporation, structural altercation, and membrane integrity. Each viability assay method is based on different definitions of cell viability, and has inherent advantages and disadvantages. In order to be able to assess the viability of cells with one assay method, it is desirable to compare the viability measurements from various assays derived from different criteria.