Immunofluorescent Labeling of Human HepG2 Cells with CdTe Quantum Dot Probe Conjugated with Anti-pan CK MAb

A relatively sensitive, specific, and photostable method for the detection of cytokeratin of cancer cells via conjugation with cadmium telluride quantum dots（CdTe QDs） was described. Water soluble CdTe QDs were conjugated to anti-pan-cytokeratin（CK） monoclonal antibody（MAb） through coupling reagent [1-ethyl-3-（3-dimethyla- mino propyl）carbodiimide, EDC] and the conjugates were purified by dialysis. The expression of pan CK protein in HepG2 cells was observed by immunocytochemistry and direct immunofluorescence via QDs-Ab conjugates respectively. Fluorescence intensity and photostability of QDs were compared with those of FITC（fluorescein isothiocyanate）. The results show that the QDs-Ab conjugates recognized specifically pan CK protein in HepG2 cells. Compared with FITC, CdTe QDs had higher brightness and photostability without obvious photobleaching under continuous exciting light illumination for 30 min and after the placement at room temperature for 3 d. The results indicate that conjugates of CdTe quantum dot with anti-pan CK MAb can be used for labeling cancer cells derived from epithelial tissues, which provides the basis for the detection of circulating tumor cells（CTCs）.

A Convenient Fluorescent-labeled Assay for in vitro Measurement of DNA Mismatch Repair Activity

Objective The assay of DNA mismatch repair （MMR） activity can be used as a biomarker for environmental condition detection and human disease diagnosis. Radioactive 32p-endlabeled DNA containing mismatch is extensively used as the substrate for MMR activity analyses. The aim of the present study is to develop a simple non-radioactive, but equally specific and sensitive method for the MMR activity assay. Methods A fluorescent label was chosen to replace the radioactive isotope label. Sensitive evaluation of the fluorescent label was carried out for the first time, and then the fluorescent label was compared with the isotope label in the MMR activity and DNA binding assays. Result LOD （limit of detection） of the fluorescent label was about 0.1 fmol and the relative signal strength displayed a pretty good linear relationship. Moreover, the fluorescent label method has equivalent sensitivity and performance as compared with the classical radioactive method in experiments. Conclusion In light of the sensitivity, reproducibility, safety, rapidity and long lifespan of the fluorescent label, this improved method can be applied to evaluation of biologic and toxic effects of environmental pollutants on man and other forms of life.

Preparation and Performance of Fluorescein Isothiocyanate⁃Labeled Fluorescent Starch and Polyvinyl Alcohol for Warp Sizing

In order to solve the problems of low accuracy and poor variety adaptability of the current measurement method of the permeability and coating property of sizing paste in textile industry, taking starch and polyvinyl alcohol(PVA) as the representatives of the most commonly used textile sizes, various concentrations of fluorescent molecules fluorescein isothiocyanate(FITC) were used to label starch and PVA to prepare fluorescein(F)-starch and F-PVA fluorescent sizes with different degrees of labeling(DLs) for the first time, respectively. Then the starch and PVA derivatives were employed to size pure cotton warp yarns. After preparing the sections of the sized yarns, the permeability and coating percentage of starch and PVA paste to the yarns were calculated by using a fluorescence microscope and the Photoshop software, respectively. The results demonstrate that F-starch and F-PVA with appropriate DL of 0.791% and 0.161%, respectively, exhibit good fluorescence property and similar sizing performance to the sizing performance of unlabeled starch and PVA. It is considered that fluorescence labeling of sizing agents with fluorescein units can provide an innovative way for the accurate determination of the permeability and coating property of sizing paste to warp yarns.

Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector

Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase

The LB Films of Dansyl Chloride Labeled Octadecylamine and Its Fluorescence Lifetime

Octadecylamine was derivatized with dansyl chloride (5-dimethylaminonaphthalene-1-sulfonyl chloride) In order to simplify and understand the LB films of fluorescent probe labeling proteins. its monolayer and multilayers in the absence and presence of stearic acid were deposited by LB technique. Fluorescence spectra and lifetimes of the fluorescent products were studied to elucidate the microenvironment of molecules in the LB films.

Novel Water Soluble Fluorescent Trimethine Indocyanines Containing N-ρ-Carboxybenzyl Group

Two fluorescent indocyanine dyes containing at least one p-carboxybenzyl group on the nitrogen atoms in the heterocyclic rings were designed and synthesized. Their absorption maxima were 549 nm and 551 nm in water respectively. They had good water solubility and photostability.

Synthesis of New Near-infrared Fluorescent Dyes

A new near-infrared fluorescent dye, 9-N-(2-hydroxyethyl)-N-methylamino-6-carbethoxy-5H-benzo[a]phenoxazin-5-one 1, was prepared from the reaction of N-(2-hydroxyethyl)-N-methyl-4-nitrosoaniline hydrochloride and ethyl 1,3-dihydroxynaphthoate. Five more fluorescent compounds were synthesized by the reaction of the resulting dye 1 with appropriate amino acid or carboxylic acids.

Preparation, Characterization and Pharmacokinetics of Fluorescence Labeled Propylene Glycol Alginate Sodium Sulfate

A rapid and sensitive fluorescence labeling method was developed and validated for the microanalysis of a sulfated polysaccharide drug,namely propylene glycol alginate sodium sulfate(PSS), in rat plasma. Fluorescein isothiocyanate(FITC) was selected to label PSS, and 1, 6-diaminohexane was used to link PSS and FITC in order to prepare FITC-labeled PSS(F-PSS) through a reductive amination reaction. F-PSS was identified by UV-Vis, FT-IR and 1H-NMR spectrum. The cell stability and cytotoxicity of F-PSS were tested in Madin-Darby canine kidney(MDCK) cells. The results indicated that the labeling efficiency of F-PSS was 0.522% ± 0.0248% and the absolute bioavailability was 8.39%. F-PSS was stable in MDCK cells without obvious cytotoxicity. The method was sensitive and reliable; it showed a good linearity, precision, recovery and stability. The FITC labeling method can be applied to investigating the absorption and metabolism of PSS and other polysaccharides in biological samples.

The Preliminary Report on Rumen Protozoa Grazing Rate on Bacteria with a Fluorescence-Labeled Technique

Studies on the bacterial predation rate by rumen protozoa were carried out under laboratory conditions using a technique of fluorescence-labeled bacteria （FLB）. Four Xuhuai goats were used in this experiment to obtain rumen protozoa and bacteria. Two groups were designed as follows： One group was the whole bacteria which were labeled using fluorescence through removing free bacteria from rumen fluid （WFLB）; the other group was the bacteria which were labeled using fluorescence without removing free bacteria from rumen fluid （FLB）. The result indicated that the bacterial predation rates of rumen protozoa was 398.4 cells/（cell h） for the group WFLB, 230.4 cells/（cell h） for the group FLB, when the corresponding values expressed as bacteria-N, they were 2.15 pg N/（cell h） for the group WFLB, and 1.24 pg N/（cell h） for the group FLB, respectively. Extrapolating the assimilation quantity of nitrogen by ciliates on bacteria of Xuhuai goat, there were 103.2 mg N/（d capita） for the group WFLB, and 59.5 mg N/（d capita） for the group FLB, respectively. It was estimated that protein losses due to microbial recycling were 0.645 g pro/（d capita） for the group WFLB and 0.372 g pro/（d capita） for the group FLB, respectively. In addition, the fluorescence-labeled technique would be a potential assay for the determination of bacterial predation rate by rumen protozoa.

Studies on the Sructure, Mechanism of Green Fluorescent Protein and Its Application

The green fluorescent protein (GFP) from jellyfishAequorea victoria has unique superiority as a kind of biological label. GFP has been widely used in all fields of biology based on the recent studies on its structure, characteristic and mechanism of bioluninescence.

Primed In Situ Labeling Technique for Subtelomeric Rearrangements in 70 Children with Idiopathic Mental Retardation

Subtelomeric rearrangements contribute to idiopathic mental retardation （MR）, but most children with idiopathic MR do not show any chromosome abnormalities with standard cytogenetic analysis. The primed in situ labeling （PRINS） technique, using an oligonucleotide primer complementary to the telemetric repeat sequences （TTAGGG）, can identify chromosome telomeric abnormality （deletion） in idiopathic MR children. In this study, seventy children with idiopathic MR were enrolled and subjected to PR1NS. The results showed normal karyotype in all the children, subtelomeric rearrangements （lq del and 4q del） in 2 cases, which was confirmed by fluorescence in situ hybridization （FISH）. It was concluded that PRINS is effective for the detection of subtelomeric rearrangements and may become a routine technique for cytogenetical abnormality screening.

Obtaining the Fluorescent Chitosan for Investigations in the Analytical Ultracentrifuge

1) In order to achieve the visibility of the chitosan macromolecule for the UV optical system of the analytical ultracentrifuge on investigation of the molecular characteristics and polymers interactions, the labeling of chitosan by a new fluorophore of fluorescein-5-isothiocyanat was carried out. 2) Samples of fluorescent chitosan with two different degrees of fluorophore substitution and various degrees of acetylation were obtained. 3) The labeled chitosans with the fluorescein-5-isothiocyanat allowed estimating the sedimentation coefficient and the molecular characteristic in the analytical ultracentrifuge. 4) The sensitivity of the UV-optical system of the analytical ultracentrifuge for the obtained fluorescent samples of chitosan relatively to the fixation of the meniscus and the influence of the wavelength and rotation speed were estimated.

Chemical tags and beyond:Live-cell protein labeling technologies for modern optical imaging

Imaging proteins with high resolution is crucial for studying cellular physiology and pathology.Fluorescence imaging is a privileged method to visualize proteins with subcellular precision in live cells.In recent years,there has been a tremendous advance in the field of fluorescent dyes that are optically more sophisticated than genetically-encodable fluorescent proteins.In this review,we aim to discuss modern bioconjugation methods to specifically incorporate these dyes into protein-of-interests.We focus on advances in live-cell labeling strategies and fluorescent probes,especially the HaloTag,SNAP-tag,TMP-tag,and unnatural amino acid systems and their applications.These protein labeling methods,along with cutting-edge dyes and novel microscopy methods,have become the infrastructure for biological research in the era of super-resolution imaging.

An ultrasensitive time-resolved fluorescent immunoassay method for determination aflatoxins B1 in edible oil

Edible oil is one major nutritional ingredient to human and widely consumed directly. The contamination of aflatoxin B1 （AFB1） in edible oils has been attracted exten-sive efforts due to its hazard to human health and life. To avoid the digestion of edible oils contaminated by AFB1 the development of rapid and sensitive sensing method for AFB1 is required. Herein, a quantitative, sensitive and rapid method for AFB1 detection in edible oils was proposed by using ultrasensitive time-resolved fluorescent immunosensing （TRFIS） method. This method poses unique advantages from both time-resolved fluorescent sens-ing method and immunochromatographic assay format. The nanospheres were modified with fluorescent europium and then captured the home-made monoclonal antibody against AFB1 （3G1）. After optimization, by using a competitive immunosensing manner, this TRFIS method has a detectable linear range of 0.54-20.0 μg/kg with minimum detectable concen-tration of 0.18μg/kg. It can be completed merely within 10 min with recovery from 87.0% to 121.9%. The agreement was observed between the results by TRFIS and high perfor-mance liquid chromatography （HPLC） methods. This research provides a promising sens-ing method for sensitive and rapid determining AFB1 in edible oils.

Kinetics of Coil-to-Globule Transition of DansyI-Labeled Poly（N-isopropyl- acrylamide） Chains in Aqueous Solution

The coil-to-globule transition of thermally sensitive linear poly（N-isopropylacrylamide） （PNIPAM） labeled with dansyl group is induced by 1.54 μm laser pulses （widths10 ns）. The dansyl group is used to follow the transition kinetics because its fluorescence intensity is very sensitive to its micro-environment. As the molar ratio of NIPAM monomer to dansyl group increases from 110 to 300, the effect of covalently attached dansyl fluorophores on the transition decreases. In agreement with our previous study in which we used 8-anilino- l-naphthalensulfonic acid ammonium salt free in water as a fluorescent probe, the current study reveals that the transition has two distinct stages with two characteristic times, namely, Tfast≈0.1 ms, which can be attributed to the nucleation and formation of some ＂pearls＂ （locally contracting segments） on the chain, and tslow≈0.5 ms, which is related to the merging and coarsening of the ＂pearls＂.Tfast is independent of the PNIPAM chain length over a wide range （Mw=2.8× 10^6-4.2 × 10^7 g/mol）. On the other hand, Tslow only slightly increases with the chain length.

Visualization of integrin molecules by fluorescence imaging and techniques

Integrin molecules are transmembraneαβheterodimers involved in cell adhesion,trafficking,and signaling.Upon activation,integrins undergo dynamic conformational changes that regulate their affinity to ligands.The physiological functions and activation mechanisms of integrins have been heavily discussed in previous studies and reviews,but the fluorescence imaging techniques-which are powerful tools for biological studies-have not.Here we review the fluorescence labeling methods,imaging techniques,as well as Förster resonance energy transfer assays used to study integrin expression,localization,activation,and functions.

Application and development of fluorescence probes in MINFLUX nanoscopy(invited paper)

The MINimal emission FLUXes(MINFLUX)technique in optical microscopy,widely recognized as the next innovative fluorescence microscopy method,claims a spatial resolution of 1-3 nm in both dead and living cells.To make use of the full resolution of the MINFLUX microscope,it is important to select appropriate fluorescence probes and labeling strategies,especially in living-cell imaging.This paper mainly focuses on recent applications and developments of fluorescence probes and the relevant labeling strategy for MINFLUX microscopy.Moreover,we discuss the deficiencies that need to be addressed in the future and a plan for the possible progression of MINFLUX to help investigators who have been involved in or are just starting in the field of super-resolution imaging microscopy with theoretical support.

Fluorescence Tracking of Exogenous DNA in Genetic Transformation of the Chinese Oak Silkmoth Antheraea Pernyi via Sperm-Mediated Gene Transfer

Exogenous DNA expressing green fluorescent protein( GFP) and labeled with fluorescein isothiocyanate( FITC) was used to transform the Chinese oak silkmoth Antheraea pernyi( A. pernyi)via sperm-mediated gene transfer( SMGT). Sperms entry into the female reproductive system and eggs were observed using fluorescence microscopy. The ability of A. pernyi sperms to uptake exogenous DNA was confirmed,and transfer of the exogenous DNA was shown by GFP expression in the transgenic eggs. Our result suggested that SMGT could also be used to directly generate transgenic A. pernyi expressing functional genes of interest.

Effects of Fluorescent Pair on the Kinetics of DNA Strand Displacement Reaction

Fluorescent labels are widely used in the characterizations of DNA-based reaction network operations.We systematically studied the effects of commonly used fluorescent pairs on thermal stabilities of signal-substrate duplex and the strand displacement kinetics.It is demonstrated that the modifications of duplex with fluorescent pairs stabilize DNA duplex by up to 3.5℃,and the kinetics of DNA strand displacement circuit is also evidently slowed down.These results highlight the importance of fluorescent pairs towards the kinetic modulation in designing nucleic acid probes and complex DNA dynamic circuits.

RGDC Peptide Modified Quantum Dots Labelling and Imaging of Tumor Cells

The labelling and imaging of tumor cells were investigated via arginine-glycine-aspartic acidcysteine（RGDC） peptide-labelled quantum dots（QDs）. The results show that RGDC modified QDs can label SMMC-7721 tumor cells and adhere to cellular membrane. In constrast, the unmodified QDs are mainly dispersed around the cell. We also found that the RGDC-QDs can penetrate into the cell at 2 h of incubation. After 6 h of incubation, RGDC-QDs can accumulate in a unique intracellular region.