Connecting Effective Immune Response,Fluorescent Granzyme B-like Peptide,Specific Peptide Binding Patterns,Patients with Cancer and Viral Infection,in Remission,Clinical Significance,and Liquid Biopsy

Functional cytotoxic-T-lymphocytes (CTL) with granzyme B play an important role in an effective immune response to tumor growth and infection progression. Tumor cells and platelets in peripheral whole blood smears of cancer patients have shown the presence of innate binding targets for GP1R, a fluorescent synthetic Granzyme B-like peptide. It is not known if similar GP1R-binding targets and specific binding patterns are detectable in peripheral blood of patients with viral infection. It is also not known if a specific binding pattern may be associated with an effective immune response to indicate a favorable prognosis. We reviewed the GP1R-binding patterns in the peripheral blood smears of 5 patients in remission at the time of sampling (3 with cancer and 2 with flu-like symptoms) and a negative control. We show with fluoroscopic images that there are: 1) fluorescent GP1R-binding targets mostly in the cytoplasmic areas of nucleated cells in patients with breast and lung cancer who have longer survival, 2) intense fluorescent deposits mostly in the nuclear areas of segmented neutrophils in patients recovered from severe to mild flu-like symptoms, 3) discernible fluorescent deposits in the cytoplasmic areas of small lymphocyte-like elements and overall intense fluorescent stain in large cells in the patient with advanced pancreatic cancer who had shorter survival, 4) GP1R-binding targets in numerous platelet-like elements in all 5 patients. The control sample did not show similar binding patterns. The potential association between specific GP1R-binding patterns in peripheral blood samples and prognostic significance, and its use as liquid biopsy are discussed.     

Dendrimer Interactions with Hydrophobic Fluorescent Probes and Human Serum Albumin

The interactions between three types of polyamidoamine dendrimers (with anionic, cationic, and neutral charge on a surface) and fluorescent dye 1-anilinonaphthalene-8-sulfonate (ANS) were studied. Double fluorimetric titration method was employed to estimate a binding constant and the number of binding centers. As fluorescent probes can serve as models of toxin molecules, dendrimers, and human serum albumin (HSA) abilities to bind ANS were compared. In the presence of HSA and dendrimers, ANS located both in HSA and in dendrimers, but the interactions between ANS and HSA were stronger.     

Synthesis of a New NIR Fluorescent Nd Complex Labeling Agent

Fluorescent analysis has been widely used in biological, chemical and analytical research. A useful fluorescent labeling agent should include NIR emission, a large Stoke’s shift, and good labeling ability without interfering with the pharmacological profile of the labeled compound. Thus, we planned to develop an M-AMF-DOTA(Nd) derivative composed of an NIR fluorescent moiety and a maleimide conjugating moiety as a new NIR fluorescent labeling agent which fulfills these requirements. M-AMF-DOTA(Nd) was synthesized from 4-amino-fluorescein and was conjugated with an avidin molecule (Avidin-AMF-DOTA(Nd)) through Lys-side chains by reaction with 2-iminothiolane. The fluorescent features of M-AMF-DOTA(Nd) and Avidin-AMF-DOTA(Nd) were comparatively evaluated. A binding assay of Avidin-AMF-DOTA(Nd) with D-biotin and a tumor cell-uptake study were performed to estimate the effects of conjugation on the biological and physicochemical features of the protein. M-AMF-DOTA(Nd) was obtained in 22% overall yield. M-AMF-DOTA(Nd) had a typical NIR fluorescence from the Nd ion (880 nm and 900 nm from 488 nm excitation). Avidin-AMF-DOTA(Nd) was easily synthesized and also had typical NIR fluorescence from the Nd ion without loss of fluorescent intensity. The binding affinity of Avidin-AMF-DOTA(Nd) to D-biotin was equivalent to naive avidin. Avidin-AMF-DOTA(Nd) was taken up by tumor cells in the same manner as avidin conjugated with fluorescein isothiocyanate, an established, widely used fluorescent avidin. Results from this study indicate that M-AMF-DOTA(Nd) is a potential labeling agent for routine NIR fluorescent analysis.     

EHPG与铝(Ⅲ)结合的光谱研究

在 pH 7 4 ,0 1mol·L- 1 N 2 羟乙基哌嗪 N′ 2 乙磺酸 (Hepes)及室温条件下 ,使用紫外吸收差光谱和荧光光谱进行了铝 (Ⅲ )对N ,N′ 乙烯 二 [2 (2 羟基苯基 )苷氨酸 ](EHPG)的滴定。结果表明铝 (Ⅲ )与EH PG形成 1∶1的配合物。铝 (Ⅲ )与EHPG结合后其紫外差光谱在 2 35和 2 91nm处出现吸收峰 ;配合物在 2 35nm的摩尔吸光系数是 1 2 7× 10 4 cm- 1 ·mol- 1 ·L ;条件稳定常数是lgK =14 2 0± 0 0 3;铝 (Ⅲ )对EHPG的荧光猝灭是由于EHPG的酚羟基质子解离并与铝 (Ⅲ )配合而引起的。     

Iminobiotin Binding Induces Large Fluorescent Enhancements in Avidin and Streptavidin Fluorescent Conjugates and Exhibits Diverging pH-Dependent Binding Affinities

The pH-dependent binding affinity of either avidin or streptavidin for iminobiotin has been utilized in studies ranging from affinity binding chromatography to dynamic force spectroscopy. Regardless of which protein is used, the logarithmic dependence of the equilibrium dissociation constant (K_d) on pH is assumed conserved. However a discrepancy has emerged from a number of studies which have shown the binding affinity of streptavidin for iminobiotin in solution to be unexpectedly low, with the K_d at values usually associated with non-specific binding even at strongly basic pH levels. In this work we have utilized a Bodipy fluorescent conjugate of avidin and an Oregon Green fluorescent conjugate of streptavidin to determine the K_d of the complexes in solution in the pH range of 7.0 to 10.7. The study was made possible by the remarkable fluorescent enhancement of the two fluorescent conjugates (greater than 10 fold) upon saturation with iminobiotin. The streptavidin-iminobiotin interaction exhibited almost no pH dependence over the range studied, with K_d consistently on the order of 10⁻⁵ M. In contrast, under identical experimental conditions the avidin-iminobiotin interaction exhibited the expected logarithmic dependence on pH. We discuss the possible origins for why these two closely related proteins would diverge in their binding affinities for iminobiotin as a function of pH.     

A Simple Model-Free Method for Direct Assessment of Fluorescent Ligand Binding by Linear Spectral Summation

Fluorescent tagged ligands are commonly used to determine binding to proteins. However, bound and free ligand concentrations are not directly determined. Instead the response in a fluorescent ligand titration experiment is considered to be proportional to the extent of binding and, therefore, the maximum value of binding is scaled to the total protein concentration. Here, a simple model-free method is presented to be performed in two steps. In the first step, normalized bound and free spectra of the ligand are determined. In the second step, these spectra are used to fit composite spectra as the sum of individual components or linear spectral summation. Using linear spectral summation, free and bound 1-Anilinonaphthalene-8-Sulfonic Acid (ANS) fluorescent ligand concentrations are directly calculated to determine ANS binding to tear lipocalin (TL), an archetypical ligand binding protein. Error analysis shows that the parameters that determine bound and free ligand concentrations were recovered with high certainty. The linear spectral summation method is feasible when fluorescence intensity is accompanied by a spectral shift upon protein binding. Computer simulations of the experiments of ANS binding to TL indicate that the method is feasible when the fluorescence spectral shift between bound and free forms of the ligand is just 8 nm. Ligands tagged with environmentally sensitive fluorescent dyes, e.g., dansyl chromophore, are particularly suitable for this method.

Study on the spectral behavior of four fluorescent dyes and their interactions with nucleic acid by the luminescence method

The spectral characteristics of four fluorescent dyes: tetrabromofluorescein (TBF), tetrachlorotetrabromofluorescein (TTF), lissamine rhodamine RB 200 (LSR) and methylene violet (MV) were studied by fluorescence and paper substrate room temperature phosphorescence (PS-RTP) methods. The factors affected the luminescence were investigated including pH value, paper substrate and drying conditions, etc. The fluorescence polarizations and PS-RTP lifetimes of these compounds were obtained. The intensity of fluorescence and phosphorescence of TBF and TTF increased in the presence of DNA. Contrarily, that of LSR and MV decreased. The fluorescence quenching and polarization studies indicate that TTF, LSR and MV intercalate into DNA base pairs. In addition, the interaction of TBF with DNA is the comprehensive interaction of two modes: intercalation and groove binding. It was also found that ionic strength could affect the binding of fluorescent dyes and DNA.     

Effect of ultrasonic treatment on the structure and functional properties of mantle proteins from scallops (Patinopecten yessoensis)

In this study, scallop mantle protein was treated by ultrasound at different powers, and then analyzed by ANS fluorescent probes, circular dichroism spectroscopy, endogenous fluorescence spectrum, DNTB colorimetry and in-vitro digestion model to elucidate the structure–function relationship. The results indicated that ultrasound can significantly affect the secondary structure of scallop mantle protein like enhancing hydrophobicity, lowering the particle size, increasing the relative contents of α-helix and decreasing contents of β-pleated sheet, β-turn and random coil, as well as altering intrinsic fluorescence intensity with blue shift of maximum fluorescence peak. But ultrasound had no effect on its primary structure. Moreover, the functions of scallop mantle protein were regulated by modifying its structures by ultrasound. Specifically, the protein had the highest performance in foaming property and in-vitro digestibility under ultrasonic power of 100 W, oil binding capacity under 100 W, water binding capacity under 300 W, solubility and emulsification capacity under 400 W, and emulsion stability under 600 W. These results prove ultrasonic treatment has the potential to effectively improve functional properties and quality of scallop mantle protein, benefiting in comprehensive utilization of scallop mantles.     

Fluorescence Investigation on the Interaction of a Prevalent Competitive Fluorescent Probe with Entomic Odorant Binding Protein

In recent years, one prevalent competitive fluorescent probe, N-phenyl-1-naphthylamine (1-NPN), was frequently utilized to measure the binding affinity of entomic odorant binding proteins (OBPs) with diverse plant volatiles or pheromones. Nevertheless, the details and model of the binding interaction are still largely unknown, although it is vital to investigate the physiological function of OBPs. Here we studied the binding interaction between 1-NPN and OBP2, a recombinant OBP from eastern honeybee, Apis cerana, by the combination of fluorescence quenching spectra, synchronous fluorescence spectra, ultraviolet spectra, circular dichroism spectra, and molecular docking. The Stern–Volmer curve of the fluorescence quenching of OBP2 by 1-NPN indicated it was a static quenching mechanism, and the binding constants and binding number were determined, respectively. Based on the Förster theory of nonradiation energy transfer (FRET), the binding distance was calculated, and the intrinsic fluorescent energy was predicted to transfer from the donor OBP2 to the acceptor 1-NPN. Synchronous fluorescence spectra and circular dichroism spectra were used to investigate the conformational change in binding progress. The thermodynamic parameters showed that the interaction was mainly driven by hydrophobic force, which was validated by the molecular docking; meanwhile, the binding mode was revealed and one hydrogen bond was found between the nitrogen atom of 1-NPN and Glu29 of OBP2.     

Fluorescent analysis of interaction of flavonols with hemoglobin and bovine serum albumin

We have studied the fluorescent properties of flavonols (quercetin, fisetin, morin, rutin) with the aim of studying possible interaction with hemoglobin and bovine serum albumin (BSA). We observed an increase in the intensity of intrinsic fluorescence for all the flavonols except rutin in the presence of BSA. From the changes in the fluorescence spectra, we concluded that tautomeric forms are formed on interaction with hemoglobin. We determined the interconnection between the structure of related flavonols and their fluorescent properties on interaction with proteins, and we determined the binding constants for binding with BSA and hemoglobin. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 74, No. 5, pp. 659–664, September–October, 2007.     

Quantitative optical imaging of the pharmacokinetics of fluorescent-specific antibodies to tumor markers through tissuelike turbid media

Fluorescent optical imaging of tumors deep within tissue depends on specific binding of antibodies to the tumors' surface markers. These fluorescent antibodies propagating in the vicinity of the tumor can be attached to and (or) diffused away from it. We illustrate application of a new tool, based on the random-walk theory in turbid media, for extracting the pharmacokinetics of these fluorescent antibodies by data deconvolution, excluding the effect of upper turbid tissue layers.     

Interaction of the Iron(II) Cage Complexes With Proteins: Protein Fluorescence Quenching Study

Interaction of the iron(II) mono- and bis-clathrochelates with bovine serum albumin (BSA), β-lactoglobulin, lysozyme and insulin was studied by the steady-state and time-resolved fluorescent spectroscopies. These cage complexes do not make significant impact on fluorescent properties of β-lactoglobulin, lysozyme and insulin. At the same time, the monoclathrochelates strongly quench a fluorescence intensity of BSA and substantially decrease its excited state lifetime due to their binding to this protein. This occurs due to the excitation energy transfer from a tryptophan residue to a cage molecule or/and to the change of the tryptophan nearest environment caused by either clathrochelate binding or an alteration of the BSA conformation. The effect of the iron(II) bis-clathrochelate on BSA fluorescence is much weaker as compared to its monomacrobicyclic analogs as a result of an increase in its size.     

Improving colloidal properties of quantum dots with combined silica and polymer coatings for in vitro immuofluorenscence assay

Semiconductor quantum dots (QDs) are promising fluorescence probes for immuofluorescence assay in the biological applications. However, water solubilization and non-specific binding are two critical issues to be addressed for the practical uses. Here, we reported a new type of QDs with combined silica and polymer coating. QDs with excellent colloidal properties were prepared via carboxylation of the amino groups on the surface of silica-coated QDs by reacting with multi-carboxyl poly (acrylic acid) (PAA). Hydrodynamic size of PAA-functionalized silica-coated QDs was around 40 nm. They were highly fluorescent (about 47.8% quantum yield). No precipitate of QDs was observed after 3 month storage at 4 °C. When cancer cells (HeLa) were used, the functionalized QDs exhibited little or no non-specific cellular binding. The results from in vitro experiments indicated that PAA-functionalized silica-coated QDs-antibody bioconjugates had excellent antigen-capture ability and exhibited little or no non-specific binding to polystyrene spheres which were used to immobilize the antigen for immuoflurescence assay. The PAA-functionalized silica-coated QDs with improved colloidal properties could serve as excellent alternative fluorescent probes for biodetection.     

Synthesis of Novel Fluorescent Cyclohexenone Derivatives and their Partitioning Study in Ionic Micellar Media

An approach is demonstrated toward the synthesis of four novel cyclohexenone derivatives (CDs) via a convenient route of Michael addition of ethyl acetoacetate. The molecular structures of CDs were confirmed by means of FT-IR, ¹H NMR, EIMS, UV and also by X-ray single crystal structure analysis. CDs are strongly fluorescent compounds and their fluorescent spectra exhibits intense violet fluorescence. To model the binding to biological membranes the behavior of CDs in micellar solutions of a cationic surfactant, cetyltrimethylammonium bromide (CTAB) and an anionic surfactant, sodium dodecylsulfate (SDS) has also been examined. The characteristics of partition and binding interactions of CDs with CTAB and SDS were investigated by UV-Visible and fluorescence spectroscopic techniques. Higher values of all mentioned interactions in case of CTAB, compared to SDS, indicate that there are greater interactions between the CDs and CTAB than with SDS.     

A Spectroscopic Study of 2-[4′-(Dimethylamino)phenyl]-benzothiazole Binding to Insulin Amyloid Fibrils

The spectroscopic properties of 2-[4′-(dimethylamino)phenyl]-benzothiazole (BTA-2) in solution and in the presence of amyloid fibrils were investigated using absorption and fluorescence spectroscopy. Solution studies show that BTA-2 forms micelles in aqueous solutions, but that the dye can be solvated upon the addition of acetonitrile (CH₃CN). BTA-2 binds to amyloid fibrils in solution leading to a characteristic blue-shift in the emission spectrum and an increase in fluorescence intensity. However, in solutions with increasing CH₃CN concentration, there was a marked decrease in binding of the BTA-2 to fibrils. Studies demonstrating the effect of BTA-2 concentration on binding were performed. A comparison with the standard amyloid fluorescent marker, thioflavin T (ThT), showed that BTA-2 is more fluorescent, making it an excellent dye to label amyloid samples.     

番红花红T与牛血清白蛋白作用的光谱特性

用荧光光谱法研究了番红花红T与牛血清白蛋白(BSA)的结合反应.结果显示番红花红T对牛血清白蛋白的荧光有猝灭作用,其猝灭类型属于静态猝灭;得到了不同温度下的结合常数和结合位点数;利用Van't Hoff方程计算得到该猝灭反应的热力学参数,结果表明番红花红T主要以静电作用力与BSA相互作用;同步荧光光谱显示番红花红T对B...     

Bioconjugated Fluorescent Polymeric Nanoparticles for Imaging and Targeted Therapy of HER2-Overexpressing Cancer Cells

We report the development of Herceptin-conjugated fluorescent polymeric nanoparticles (PNp) probes. Synthesis of fluorescent conjugated polymer as the core, preparation of the core/shell PNp, the ability of immobilizing Herceptin on PNp, targeting and imaging of bioconjugated PNp toward HER2-overexpressing cancer cells, and therapeutic effect on cell cycle, together with the expression of apoptosis related proteins, were investigated. We have achieved active tumor targeting by rapid PNp-antibody binding to tumor-specific antigens. Besides, Herceptin-conjugated PNp can suppress the growth of HER2-overexpressing cancer cells.     

Performance of Fluorescent Labels in Sedimentation Bead Arrays—A Comparison Study

An extensive study is described to identify the most suitable fluorescent label in magnetic microsphere sedimentation arrays. The investigated fluorescent labels, commonly used in multiplex analysis, include organic dyes, (fluorescein, Alexa488, Cy5) fluorescent proteins (R-Phycoerythrin, Allophycocyanine, PBXL-3) polymer nanoparticles (FluoSpheres, PD-Pt) and semiconductor nanocrystals (Quantum dots). DNA hybridization assays on magnetic microspheres were applied as model systems to reveal label performance. The fluorescent labels were characterized under optimized conditions regarding signal intensity, non-specific binding and photo-stability. The advantages and drawbacks of individual labels are discussed. The limit of detection and dynamic ranges are determined to compare the performance of selected labels. Detection limits of 2 × 10⁻¹⁰ mol/L are found for the determination of oligonucleotides using PBXl-3 as label, which is comparable with typical flow cytometer systems. The results and protocols are highly valuable for any type of bead based assays and can be easily transferred.     

Translational and Rotational Motions of Albumin Sensed by a Non-Covalent Associated Porphyrin Under Physiological and Acidic Conditions: A Fluorescence Correlation Spectroscopy and Time Resolved Anisotropy Study

The interaction between a free-base, anionic water-soluble porphyrin, TSPP, and the drug carrier protein, bovine serum albumin (BSA) has been studied by time-resolved fluorescence anisotropy (TRFA) and fluorescence correlation spectroscopy (FCS) at two different pH-values. Both rotational correlation times and translational diffusion times of the fluorescent species indicate that TSPP binding to albumin induces very little conformational changes in the protein under physiological conditions. By contrast, at low pH, a bi-exponential decay is obtained where a short rotational correlation time (phi (int) = 1.2 ns) is obtained, which is likely associated to wobbling movement of the porphyrin in the protein binding site. These physical changes are corroborated by circular dichroism (CD) data which show a 37% loss in the protein helicity upon acidification of the medium. In the presence of excess porphyrin formation of porphyrin J-aggregates is induced, which can be detected by time-resolved fluorescence with short characteristic times. This is also reflected in FCS data by an increase in molecular brightness together with a decrease in the number of fluorescent molecules passing through the detection volume of the sample.     

Fluorescent Biological Label for Recognizing Human Ovarian Tumor Cells Based on Fluorescent Nanoparticles

In this paper, we report a method for recognizing human ovarian tumor(HOT) cells using fluorescent biological label based on core-shell nanoparticles. The luminescent nanoparticles were synthesized with a water-in-oil(W/O)micromulsion technique. The fluorescent silica core-shell nanoparticles modified with anti-HER2 antibody using bifunctional cross-linker glutaraldedhyde targeted the corresponding tumor antigen in the cell surface of the SKOV3 ovarian cancer cells. The specific immunoreactivity of antibody-nanoparticles with cells was characterized by laser scanning microscopy (LSM) and scanning electron microscope (SEM). The results showed that the method offered potential advantages of sensitivity and simplicity due to high binding efficiency between nanoparticles and cells and provided an alternative method for the detection of HOT.