抗生物素蛋白与DNA相互作用的单分子研究

抗生物素蛋白(avidin)在生物单分子实验中被广泛用于DNA与修饰表面的连接,同时avidin也可作为一种DNA载体用于基因治疗中.本文利用原子力显微镜(AFM)、动态光散射(DLS)、单分子磁镊(MT)技术系统地研究了avidin与DNA之间的相互作用,以及avidin引起DNA凝聚的机理.首先通过AFM对avidin-DNA复合体形貌进行观察,发现不但有avidin导致DNA凝聚的环状形貌,同时也存在avidin自身聚集引起的DNA凝聚现象,通过定量分析,发现其凝聚尺寸越来越小,而当avidin浓度大于2 ng·μL~(-1)时,其凝聚尺寸又突然变大.DLS实验结果也显示了同样的规律,伴随着avidin浓度的升高,DNA的粒径大小从大约170 nm减小到125 nm左右,其电泳迁移率由-2.76(10~(-4)cm~2·V~(-1)·s~(-1))变化到-0.1(10~(-4)cm~2·V~(-1)·~(-1)).此外,通过MT技术的力谱曲线变化,发现avidin导致的DNA凝聚与其他多价离子相比,长度的变化曲线几乎呈线性变化,偶尔存在少而小的阶跃,这种变化趋势与组蛋白的变化曲线更相似.因此可以判断,avidin导致DNA凝聚是由avidin与DNA的静电吸引和avidin自身聚集两种相互作用引起的.     

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.     

Immobilization of protein molecules on step-controlled sapphire surfaces

We have studied effects of surface morphology on immobilization of protein molecules using step-controlled sapphire surfaces. Preferential adsorption of avidin molecules on the step edges was observed on the single-stepped sapphire surface. A randomly-stepped sapphire surface was found to be suitable for high-density immobilization of protein molecules. These results indicate atomic scale structures of the substrate surface influence the adsorption efficiency of the proteins. By using an atomic force microscopy (AFM) equipped with a biotin-modified cantilever, we have confirmed that the immobilized avidin molecules on the substrates keep their biological activity. This means that the ligand-receptor interaction can be detected using the phase image mode of a standard AFM.     

Large-scale protein/antibody patterning with limiting unspecific adsorption

A simple synthetic route based on nanosphere lithography has been developed in order to design a large-scale nanoarray for specific control of protein anchoring. This technique based on two-dimensional (2D) colloidal crystals composed of polystyrene spheres allows the easy and inexpensive fabrication of large arrays (up to several centimeters) by reducing the cost. A silicon wafer coated with a thin adhesion layer of chromium (15 nm) and a layer of gold (50 nm) is used as a substrate. PS spheres are deposited on the gold surface using the floating-transferring technique. The PS spheres were then functionalized with PEG-biotin and the defects by self-assembly monolayer (SAM) PEG to prevent unspecific adsorption. Using epifluorescence microscopy, we show that after immersion of sample on target protein (avidin and anti-avidin) solution, the latter are specifically located on polystyrene spheres. Thus, these results are meaningful for exploration of devices based on a large-scale nanoarray of PS spheres and can be used for detection of target proteins or simply to pattern a surface with specific proteins.     

Immobilization of avidin on (001) GaAs surface

To reliably immobilize different biomoieties on surfaces of III-V semiconductors is one of the most critical issues in the development of biodetector devices based on the optical/electronic properties of these materials. Herein we demonstrate the successful immobilization of avidin, a robust and well-studied protein, on a (001) GaAs surface. The immobilization was investigated via specific binding to biotin, which was connected to the GaAs surface through commercially available long- or short-chain amino group terminated alkanethiols (HS(CH₂)₁₁NH₂ or HS(CH₂)₂NH₂), or through a biotinylated thiol synthesized in our laboratory. The immobilization performance was evaluated by photoluminescence and fluorescence microscopy measurements. We found that the biotinylated thiol mixed with a diluent thiol provides the highest avidin immobilization efficiency. PACS 81.05.Ea; 82.65.+r; 87.14.Ee     

The preparation of avidin microspheres using the sonochemical method and the interaction of the microspheres with biotin

Avidin microspheres were prepared using the sonochemical method. It was found that avidin microspheres can bind biotin, but to a lesser degree than the native protein. The binding of the biotin molecules to the avidin microspheres was probed primarily by TPD measurements.     

Organization of Functionalized Gold Nanoparticles by Controlled Protein Interactions

Gold colloidal particles were synthesized and modified by molecular self‐assembly. In addition, the reaction of biotinylated colloids with a tetrameric protein, avidin, was studied by optical absorption spectroscopy and dynamic light scattering. The modification involves the chemisorption of octadecanethiol on the gold, with the further attachment of alkyl biotin and cross‐linking with avidin molecules. The specific interaction of avidin with biotin leads to the controlled cross‐linking of particles. The degree of flocculation was quantified using a semi‐empirical flocculation parameter and its dependence on the biotinylation system was studied. The measured data were in good agreement, showing the possibility of regulating the aggregation rate and size of the aggregates using the experimental time, degree of biotinylation, and avidin concentration. The morphology of the self‐assembled gold monolayers, from the aggregated particles, was imaged by high‐resolution scanning electron microscopy. The structure of the thin particulate film depends on the bulk aggregate size and aggregation rate.     

Toward whole-body optical imaging of rats using single-photon counting fluorescence tomography

We used single-photon counting (SPC) detection for diffuse fluorescence tomography to image nanomolar (nM) concentrations of reporter dyes through a rat. Detailed phantom data are presented to show that every centimeter increase in tissue thickness leads to 1 order of magnitude decrease in the minimum fluorophore concentration detectable for a given detector sensitivity. Specifically, here, detection of Alexa Fluor 647 dyes is shown to be achievable for concentrations as low as 1 nM (<200 fM) through more than 5 cm in tissue phantoms, which indicates that this is feasible in larger rodent models. Because it is possible to detect sub-nM fluorescent inclusions with SPC technology in rats, it follows that it is possible to localize subpicomolar fluorophore concentrations in mice, putting the concentration sensitivity limits on the same order as nuclear medicine methods.     

Optical heterodyne surface-plasmon resonance biosensor

A novel optical heterodyne surface-plasmon resonance (SPR) biosensor with a Zeeman laser is proposed. Two surface plasma waves are excited by two correlated p-polarized waves in a SPR device of the Kretschmann configuration. Two reflected p waves are optically heterodyned such that the magnitude of the heterodyned signal is proportional to the multiplication of two attenuated reflected p waves. Then the detection sensitivity and the dynamic range based on this amplitude-sensitive method are enhanced. In the experiment, the kinetics between mouse immunoglobulin G (IgG) and rabbit antimouse IgG is obtained from sensograms of various concentrations of antimouse IgG. A detection sensitivity of 0.2 nM was achieved. In addition, a concentration of 5 ng/ml of protein G interacting with mouse IgG was measured successfully.     

Solution‐based SERS method to detect dithiocarbamate fungicides in different real‐world matrices

Surface‐enhanced Raman scattering (SERS) has become a valuable tool for the characterization of trace quantities of environmental toxins. Utilizing established wet chemical synthetic protocols, dogbone‐shaped colloidal gold nanoparticle substrates with sharp features were prepared with regions that exhibit significant SERS enhancement due to the lightning rod effect. These highly enhancing substrates were utilized for the quantitative determination of two dithiocarbamate fungicides by SERS in several complex matrices such as tap water, apple juice, and vegetable juice. Limits of detection and quantitation are reported and compared with Environmental Protection Agency mandated maximum allowable concentrations in tap water. In the case of tap water, limits of detection of 13.39 ± 3.89 nM for thiram and 1.78 ± 0.20 nM for ferbam was achieved. The sensitivity of the solution‐based SERS method decreased with increasing complexity of the matrix in which the limit of detection achieved in apple juice is 47.22 nM for thiram and 11.88 ± 1.38 nM for ferbam and that for vegetable juice is 87.01 ± 2.88 nM for thiram and 36.72 ± 2.90 nM for ferbam. It was found that using the solution‐based SERS method results in sensitivities that are greater than that required by Environmental Protection Agency mandated maximum allowable concentrations for complex matrices such as apple and vegetable juice. Copyright © 2013 John Wiley & Sons, Ltd.     

A new imaging method for gold-surface adsorbates based on anomalous reflection

A new imaging method is proposed for ultrathin films with a thickness of a few nanometers, based on the anomalous reflection (AR) of gold. In the AR effect, the reflectivity fairly decreases for blue or purple light (380 nm < λ < 480 nm) with the existence of a transparent dielectric layer at a gold surface. Thus, a thin gold film can be used as an imaging platform. Clear AR images are obtained for a microarray of protein (avidin) spots of diameter 120 μm with gaps of size 50 μm between the spots (36 spots/mm²). The resolution of the AR imaging is governed solely by the illumination spot size. AR imaging is a promising technique for high throughput analysis of biomolecular detection in a microarray format.     

Drop‐coating deposition Raman spectroscopy of porphyrins

We report an improvement of the Raman detection sensitivity of porphyrins by factor of 10⁵ using drop‐coating deposition Raman spectroscopy (DCDR). We were able to obtain Raman spectra from ~20 nM initial (deposited) concentrations of cationic and anionic porphyrin. This detection limit is comparable with or even better than that of surface‐enhanced Raman scattering spectroscopy using Ag nanoparticles. Moreover, DCDR spectrum of protoporphyrin IX, which is known as a marker in clinical diagnostics of cancer, was also obtained from 10 nM deposited concentration. To our knowledge, it is the first demonstration of the resonance DCDR spectroscopy. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Effect of ultrasound irradiation on bacterial internalization and bacteria-mediated gene transfer to cancer cells

The present study demonstrates that ultrasound irradiation can facilitate bacteria-mediated gene delivery (bactofection). Escherichia coli modified with avidin were employed as a vehicle for delivery of the green fluorescent protein (GFP) gene, a model heterologous gene, into the breast cancer cell line MCF-7. Avidin-mediated binding of E. coli to MCF-7 cells enhanced the internalization of E. coli by approximately 17%, irrespective of the use of ultrasound irradiation. Furthermore, the use of ultrasound irradiation increased the internalization by approximately 5%, irrespective of the presence of avidin on the E. coli cell surface. The percentages of GFP-expressing MCF-7 cells at 24 h after bactofection were below 0.5% and 2% for the case with only avidin-modification of E. coli cell surface and only ultrasound irradiation, respectively. However, combining avidin modification with the ultrasound treatment increased this value to 8%. Thus, the use of avidin-modified bacteria in conjunction with ultrasound irradiation has potential as an effective strategy for tumor-targeted bactofection.     

Biological functionality of active enzyme structures immobilized on various solid surfaces

We have investigated biological functionality of immobilized enzyme structures according to the immobilizing routes and the surface properties. Horse radish peroxidase (HRP) was immobilized on various solid surfaces such as gold, SiO₂, sapphire and anodized aluminum oxide (AAO) membrane via non-specific adsorption, avidin-mediated and biotin/avidin-mediated layer-by-layer (LBL) assembly. The catalytic activity as a measure of biological functionality, of the biotin-HRP immobilized by avidin-mediated LBL assembly was found to be better than that of the directly adsorbed HRP on the surfaces of gold, SiO₂, sapphire and AAO due to the easy accessibility of reactants to active sites as well as the retention of three dimensional native structure of enzyme for bioactive functionality. In addition, the catalytic activity of the biotin-HRP in LBL-assembled avidin/biotin-HRP on AAO membrane was found to be highly better than that on other substrates due to the increasing amount of immobilized HRP which can be attributed to the high surface area of the substrate. SEM images show that the functional avidin/biotin-HRP enzyme structures were successfully realized by a sequential process of non-specific adsorption and LBL assembly via biotin–avidin interaction.     

The detection limit of a Gd3+-based T1 agent is substantially reduced when targeted to a protein microdomain

Simple low molecular weight (MW) chelates of Gd(3+) such as those currently used in clinical MRI are considered too insensitive for most molecular imaging applications. Here, we evaluated the detection limit (DL) of a molecularly targeted low MW Gd(3+)-based T(1) agent in a model where the receptor concentration was precisely known. The data demonstrate that receptors clustered together to form a microdomain of high local concentration can be imaged successfully even when the bulk concentration of the receptor is quite low. A GdDO3A-peptide identified by phage display to target the anti-FLAG antibody was synthesized, purified and characterized. T(1-)weighted MR images were compared with the agent bound to antibody in bulk solution and with the agent bound to the antibody localized on agarose beads. Fluorescence competition binding assays show that the agent has a high binding affinity (K(D)=150 nM) for the antibody, while the fully bound relaxivity of the GdDO3A-peptide/anti-FLAG antibody in solution was a relatively modest 17 mM(-1) s(-1). The agent/antibody complex was MR silent at concentrations below approximately 9 microM but was detectable down to 4 microM bulk concentrations when presented to antibody clustered together on the surface of agarose beads. These results provided an estimate of the DLs for other T(1)-based agents with higher fully bound relaxivities or multimeric structures bound to clustered receptor molecules. The results demonstrate that the sensitivity of molecularly targeted contrast agents depends on the local microdomain concentration of the target protein and the molecular relaxivity of the bound complex. A model is presented, which predicts that for a molecularly targeted agent consisting of a single Gd(3+) complex with bound relaxivity of 100 mM(-1) s(-1) or, more reasonably, four tethered Gd(3+) complexes each having a bound relaxivity of 25 mM(-1) s(-1), the DL of a protein microdomain is approximately 690 nM at 9.4 T. These experimental and extrapolated DLs are both well below current literature estimates and suggests that detection of low MW molecularly targeted T(1) agents is not an unrealistic goal.     

Enhanced Fluorescence Anisotropy Assay for Human Cardiac Troponin I and T Detection

Human cardiac troponin I (hcTnI) and troponin T (hcTnT) are the biomarkers of choice for the diagnosis of cardiac diseases. In an effort to improve assay sensitivity, in this study we developed a novel approach to simultaneously detect hcTnI and hcTnT in homogenous solutions by monitoring enhanced-fluorescence-anisotropy changes. Specifically, our design was based on a competition assay by measuring anisotropy change of fluorophore-labeled peptides bound to primary monoclonal antibodies in the presence of nano-gold-modified secondary antibody in response to the presence of target proteins. Enhanced-fluorescence-anisotropy resulted from interaction between the primary antibody and the nano-gold-labeled secondary antibody, which significantly increased the size and decreased tumbling motion of the complex of peptide-antibodies. The measurements were performed to detect hcTnI and hcTnT either individually or simultaneously in a homogenous buffer solution and in the solutions containing human plasma. Our results showed that when fluorescence emission was monitored at a single wavelength selected by a monochromator the assay at all experimental conditions had excellent linear response to the target proteins within the concentration range of 0.5–40 nM. The detection limit is 0.5 nM for both hcTnI and hcTnT in the presence of human plasma. However, when fluorescence emission was monitored using a cutoff filter, the linear response of the assay to the target proteins is within 15–500 pM. The detection limit is 15 pM which is close to the recommended 99th percentile cutoff point for concentrations of hcTnI and hcTnT tests to discriminate healthy and diseased conditions. Homogenous nature, rapid response time, and easy implementation of our assay design make it a useful tool for disease biomarker and protein sensing.     

Rapid detection of 2,2′,4,4′‐tetrabromodiphenyl ether (BDE‐47) using a portable Au‐colloid SERS sensor

In situ rapid detection and identification of polybrominated diphenyl ethers, a group of well‐known persistent organic pollutants, present a great challenge. To develop a portable and sensitive surface‐enhanced Raman scattering (SERS) sensor for rapid 2,2′,4,4′‐tetrabromodiphenyl ether (BDE‐47) detection, we adopted the most commonly used Au nanoparticles, which are effective in the analysis of hydrophobic BDE‐47 with a simple optimization in citrate content and sampling technique. Qualitative and quantitative determination of BDE‐47 was achieved using a portable Raman spectrometer. The SERS response exhibited a linear dependence on the BDE‐47 concentration up to 1000 nM with a detection limit of 75 nM. The density function theory‐calculated Raman spectra agreed well with the experimental observations, and the results justified the existence of electromagnetic enhancement and charge transfer mechanism. This in situ SERS platform allows easy and reliable detection of hydrophobic molecules such as BDE‐47 in complex matrices. Copyright © 2014 John Wiley & Sons, Ltd.     

Targeted and ultrasound-triggered cancer cell injury using perfluorocarbon emulsion-loaded liposomes endowed with cancer cell-targeting and fusogenic capabilities

This study investigated the targeting and ultrasound-triggered injury of cancer cells using anticancer drug-free liposomes that contained an emulsion of perfluoropentane (ePFC5) and were co-modified with avidin as a targeting ligand for cancer cells and the hemagglutinating virus of Japan (HVJ) envelope to promote liposome fusion with the cells. These liposomes are designated as ePFC5-loaded avidin/HVJ liposomes. ePFC5-loaded liposomes were sensitized to ultrasound irradiation. Liposomes modified with avidin alone (avidin liposomes) showed binding to MCF-7 human breast cancer cells, and liposomes modified with HVJ envelope alone (HVJ liposomes) were found to fuse with MCF-7 cells. The irradiation of MCF-7 cells with 1 MHz ultrasound (30 s, 1.2 W/cm², duty ratio 30%) combined with ePFC5-loaded avidin/HVJ liposomes resulted in a decrease in cell viability at 1 h after irradiation to 43% of that of controls without ultrasound irradiation or liposomes. The cell viability was lower than that of cells treated with ultrasound irradiation with ePFC5-loaded avidin liposomes or ePFC5-loaded HVJ liposomes. This indicates that co-modification of liposome with avidin and HVJ envelope could enhance ultrasound-induced cell injury in the presence of ePFC5-loaded liposomes.     

Bioimaging of Peroxynitrite in MCF-7 Cells by a New Fluorescent Probe Rhodamine B Phenyl Hydrazide

Peroxynitrite is a potent oxidizing and nitrating agent which has detrimental effects on cells by altering the structure and function of biomolecules present within. A fluorescent probe rhodamine B phenyl hydrazide (RBPH) has been proposed for peroxynitrite (ONOO^?) imaging in MCF-7 cells based on its oxidation property, which converts RBPH to pink colored and highly fluorescent rhodamine B. The fluorescence emission intensity of the rhodamine B produced in the above process is linearly related to the concentration of peroxynitrite. The method obeys Beer’s law in the concentration range 2–20 nM and the detection limit has been found to be 1.4 nM. The possible reaction mechanism of peroxynitrite with RBPH to produce rhodamine B has been discussed with spectroscopic evidence. The Probe is selective to the peroxynitrite in the pH range 6–8 which is near physiological pH. Fluorescence microscopic studies suggest that the probe is cell permeable and hence peroxynitrite was imaged in MCF-7 cells.