亲和素与膜表面模型受体的相互作用

通过构建含有生物素的人工膜,运用3种不同类型的人工膜系统(脂质体、气/液界面LB膜和固体表面支撑单层膜)和相应的观测技术(如椭偏仪、衰减全反射、荧光显微镜膜天平、荧光滴定等)、对亲和素与生物素这一具有极强亲和力的系统之间的特异性相互作用进行了探讨。实验结果表明,膜表面电荷对非特异性吸附的速度有很大的影响,而对最终的蛋白吸附量影响不大;非特异性吸附可以通过二价阳离子的脱附而去除;受体蛋白质与配体间的特异性结合受到侧向空间位阻效应的制约。实验还证明模型受体的柔性是影响其与配体蛋白特异性结合的重要因素。     

生物素化高分子涂层的制备与评价

制备了一种能固载目标蛋白质, 却没有非特异性蛋白质吸附的高分子涂层. 该涂层是可生物降解的油水两亲性的三嵌段聚合物, 即生物素偶联的聚乙二醇-聚丙交酯-聚赖氨酸共聚物. 将高分子溶解于N,N-二甲基甲酰胺中, 并涂布在预先包被了聚赖氨酸的脱脂玻片基质上, 形成高分子涂层, 在其表面包被一层由明胶和聚N-乙烯基吡咯烷酮组成的封闭剂. 使用酶标免疫分析法, 对高分子涂层表面的生物活性进行评价. 依次将辣根过氧化物酶标记的链亲和素和生物素偶联的小鼠球蛋白抗原和碱性磷酸酯酶标记的马抗小鼠抗体固载在高分子涂层表面上, 通过标记酶与底物作用生色. 分析结果表明, 经过封闭以后, 生物素化的高分子涂层表面能够排斥非特异性的蛋白质; 同时特异性蛋白质之间(如生物素和链亲和素之间、抗原和抗体之间)的相互作用依然保留, 并且固定在表面的蛋白质依然保留其生物活性. 因此生物素化的聚乙二醇-聚丙交酯-聚赖氨酸三嵌段高分子可以作为生物活性材料, 用于蛋白质固载和蛋白质分离及分析.     

微悬臂生物传感器检测生物素-亲和素相互作用

将生物素共价键合到微悬臂的硅表面, 利用微悬臂传感技术在流动注样条件下研究了生物素-亲和素的动态作用过程, 同时利用原子力显微镜(AFM)力刻蚀技术验证了生物素化硅表面结合亲和素的有效性. 结果显示亲和素与生物素作用产生较大的表面应力驱动微悬臂偏转, 悬臂偏转的程度和速率与亲和素的浓度相关, 亲和素浓度越大, 微悬臂偏转程度也越大, 同时反应速率也更快. 这项工作为开发新型的无标记检测技术, 研究生物分子之间相互作用提供了有益的探索.     

气相色谱法测定不对称催化合成反应液中手性1,1-二苯基-2-丙醇含量

(S)-(+)-1,1-二苯基-2-丙醇是一种很有用的医药中间体,用它可以合成生物素,此物质广泛应用于医学各领域。近年来大量研究证实,生物素-亲和素(BAS)系统几乎可与目前研究成功的各种标记物结合。在生物素-亲和素(BAS)系统中,借助所形成的生物素-亲和素-酶复合物,追踪生物素标记的抗原或抗体,通过酶催化底物显色,可检出相应的抗体或抗原。由于抗原或抗体分子可偶联多个生物素,     

亲和膜色谱

亲和膜色谱又称亲和膜分离，其在蛋白质的分离纯化中作为一种综合性的技术出现在80年代末。亲和膜色谱主要优点是克服了颗粒状多孔载体扩散传质阻力大的缺点，代之以对流传质，这样就可以在较低的操作压和较高的流速下对目标蛋白进行快速的分离和纯化，从而缩短操作时间、提高纯化效率。本文将就近年来亲和膜色谱及其在蛋白质分离和纯化中的应用作一综述性介绍。     

金标记羟胺放大化学发光检测赭曲霉毒素A

以羧基磁性微球为分离载体,连接氨基捕获探针和适配体,加入生物素化报告序列和赭曲霉毒素A(Ochratoxin A,OTA)竞争结合适体,继续加入链霉亲和素纳米金和羟胺/Au~(3+)以显著提高化学发光检测OTA的灵敏度,从而建立了一种纳米金标记羟胺放大化学发光检测OTA的高灵敏度方法。优化了羧基磁性微球、氨基捕获探针、适配体、生物素化报告序列、链霉亲和素纳米金的用量。优化条件下,在OTA质量浓度0.01~50 ng/m L范围内,化学发光信号值与OTA浓度的对数呈较好的线性关系(r~2=0.992 5),检出限为1.58×10~(-3)ng/mL。对啤酒样品进行OTA加标回收实验,回收率为97.4%~105.4%,相对标准偏差为4.0%~5.5%。     

链亲和素-磁性微粒的制备及其应用

通过物理吸附和共价作用机制, 制备两种链亲和素-磁性微粒, 即链亲和素-金磁微粒和链亲和素-氨基磁粒, 并对其在不同缓冲液中的稳定性进行研究; 采用酶抑制法测定两种链亲和素-磁性微粒对游离生物素的结合能力; 分别以紫外吸收和固相核酸杂交方法, 测定两种链亲和素-磁性微粒对生物素标记寡核苷酸探针的固定化容量及活性, 并与Dynabeads®M-270 Streptavidin进行比较. 结果表明: 通过物理吸附作用制备的链亲和素-金磁微粒, 适用于核酸杂交与检测常用的STE (Tris-NaCl-EDTA) 缓冲系统, 通过共价作用形成的链亲和素-氨基磁粒, 适用于STE和磷酸盐(PBS)缓冲系统; 1 mg链亲和素-金磁微粒和链亲和素-氨基磁粒对游离生物素的最大结合容量分别为4950和5115 pmol; 对生物素标记寡核苷酸探针(24 mer) 的结合容量分别为2839和2978 pmol, 测定结果均是Dynabeads®M-270 Streptavidin的6~7倍; 与FITC-标记互补寡核苷酸的杂交结果表明, 固定于链亲和素-磁性微粒表面的寡核苷酸探针保持了较好的生物学活性.     

生物素-亲和素放大酶联免疫吸附法测定双酚A

建立了可用于快速、灵敏地检测双酚A的生物素-亲和素放大酶联免疫吸附测定法,测得最佳实验条件为包被抗原浓度为13.8mg/L、抗体稀释为2.4×105倍,生物素化二抗和酶标亲和素的最佳稀释倍数分别为2000倍和500倍。在优化条件下,方法的线性范围0.2~1000μg/L;最低检出限0.05μg/L。所建立的方法用于食品和唾液中双酚A含量的测定,样品处理简单,结果满意。     

喷射式流动注射电化学发光免疫检测禽流感H9亚型

利用磁分离和生物素亲和素技术,形成亲和素化磁微球-生物素化抗体-抗原-钌标抗体的免疫夹心复合物,初步建立了体外免疫诊断试剂的制备方法,并利用喷射式流动注射电化学发光体系对制备出的禽流感病毒H9免疫复合物进行检测。实验选择最适的包被抗体,检测抗体和封闭剂,优化Ru标抗体的最佳稀释度。在生物素化的兔抗H9多抗作为亲和素化的磁微球的结合抗体,鼠抗H9单抗作为Ru(bpy)32+标记抗体,2%BSA作为封闭剂,1:50倍稀释的Ru-鼠抗H9单抗条件下,非特异性吸附最低。测定不同浓度的H9抗原,发现抗原浓度在3.125～100μg/mL范围内与电化学发光强度呈较好的线性关系。实验还测定了不同亚型的禽流感病毒、不同来源的毒株和鸡的棉拭子样品。     

金属螯合亲和膜吸附分离与纯化溶菌酶的研究(Ⅰ)──低温氧等离子体改性条件对亲和膜结构与吸附性能的影响

用低温氧等离子体方法对聚丙烯微孔膜表面进行改性.扫描电镜、红外光谱和光电子能谱综合分析结果表明,膜表面上产生了-OH、-COOH和C=O等极性基团,这些基团可被活化和偶联制亲和膜.所制备的Cu²⁺离子金属螯合亲和膜用于对溶菌酶的吸附,在20min和68W的最佳条件下,制成的亲和膜对溶菌酶的吸附量为8μg/cm².     

Highly sensitive biomolecule detection on a quartz crystal microbalance using gold nanoparticles as signal amplification probes.

We report here a novel strategy for the high-sensitive detection of target biomolecules with very low concentrations on a quartz crystal microbalance (QCM) device using gold nanoparticles as signal enhancement probes. By employing a streptavidin-biotin interaction as a model system, we could prepare biotin-conjugated gold nanoparticles maintaining good dispersion and long-term stability by controlling the biotin density on the surface of gold nanoparticles that have been investigated by UV-vis spectra and AFM images. These results showed that 10 microM N-(6-[biotinamido]hexyl)-3'-(2'-pyridyldithio)propionamide (biotin-HPDP) was the critical concentration to prevent the nonspecific aggregation of gold nanoparticles in this system. For sensing streptavidin target molecules by QCM, biotinylated BSA was absorbed on the Au surface of the QCM electrode and subsequent coupling of the target streptavidin to the biotin in the sensing interface followed. Amplification of the sensing process was performed by the interaction of the target streptavidin on the sensing surface with gold nanoparticles modified with 10 microM biotin-HPDP. The biotinylated gold nanoparticles were used as signal amplification probes to improve the detection limit, which was 50 ng/ml, of the streptavidin detection system without signal enhancement, and the calibration curve determined for the net frequency changes showed good linearity over a wide range from 1 ng/ml to 10 microg/ml for the quantitative streptavidin target molecule analysis. In addition, the measured dissipation changes suggested that the layer of biotin-BSA adsorbed on the Au electrode and the streptavidin layer assembled on the biotin-BSA surface were highly compact and rigid. On the other hand, the structure formed by the biotinylated gold nanoparticles on the streptavidin layer was flexible and dissipative, being elongated outward from the sensing surface.     

A case study on biological activity in a surface-bound multicomponent system: the biotin-streptavidin-peroxidase system

The adsorption of multiple protein layers on biotinylated organic surfaces has been characterized using surface plasmon resonance (SPR) and atomic force microscopy (AFM). Diffusion-limited loading of the biotinylated self-assembled monolayers (SAMs) ensures a precise control of the streptavidin surface density. For the subsequent interaction with biotinylated peroxidase, SPR data hint at a streptavidin density dependent orientation during peroxidase adsorption. Microcontact printed well-defined two-dimensional patterned surfaces of biotinylated organothiols and protein-resistant OEG-thiols allow an in-situ differentiation of specific and nonspecific adsorption (e.g., mono- vs multilayer adsorption). Additionally, the very important issue of biological activity of surface-bound enzymes is addressed by comparing the enzyme activities in solution with that for surface-bound species.     

Chemical modifications of Au/SiO2 template substrates for patterned biofunctional surfaces

The aim of this work was to create patterned surfaces for localized and specific biochemical recognition. For this purpose, we have developed a protocol for orthogonal and material-selective surface modifications of microfabricated patterned surfaces composed of SiO(2) areas (100 μm diameter) surrounded by Au. The SiO(2) spots were chemically modified by a sequence of reactions (silanization using an amine-terminated silane (APTES), followed by amine coupling of a biotin analogue and biospecific recognition) to achieve efficient immobilization of streptavidin in a functional form. The surrounding Au was rendered inert to protein adsorption by modification by HS(CH(2))(10)CONH(CH(2))(2)(OCH(2)CH(2))(7)OH (thiol-OEG). The surface modification protocol was developed by testing separately homogeneous SiO(2) and Au surfaces, to obtain the two following results: (i) SiO(2) surfaces which allowed the grafting of streptavidin, and subsequent immobilization of biotinylated antibodies, and (ii) Au surfaces showing almost no affinity for the same streptavidin and antibody solutions. The surface interactions were monitored by quartz crystal microbalance with dissipation monitoring (QCM-D), and chemical analyses were performed by polarization modulation-reflexion absorption infrared spectroscopy (PM-RAIRS) and X-ray photoelectron spectroscopy (XPS) to assess the validity of the initial orthogonal assembly of APTES and thiol-OEG. Eventually, microscopy imaging of the modified Au/SiO(2) patterned substrates validated the specific binding of streptavidin on the SiO(2)/APTES areas, as well as the subsequent binding of biotinylated anti-rIgG and further detection of fluorescent rIgG on the functionalized SiO(2) areas. These results demonstrate a successful protocol for the preparation of patterned biofunctional surfaces, based on microfabricated Au/SiO(2) templates and supported by careful surface analysis. The strong immobilization of the biomolecules resulting from the described protocol is advantageous in particular for micropatterned substrates for cell-surface interactions.     

Steric crowding effects on target detection in an affinity biosensor

This work quantifies the impact of steric crowding on whole antibody (Ab) receptor immobilization and target Ab detection and also demonstrates how the versatile biotin/streptavidin receptor immobilization system must be tuned to optimize target detection in designing biosensors. Results are demonstrated on a label-free optical biosensor fabricated from n-type macroporous porous silicon (PSi) with approximately 88-107 nm diameter pores. We employ a sandwich assay scheme comprising a linking chemistry (biotin/streptavidin) to attach biotinylated anti-rabbit IgG (receptor) to detect rabbit IgG (target). A "bottom-up" approach was taken to investigate each layer of the sandwich assay to optimize target binding. Steric crowding was observed to hinder subsequent layer binding for each layer in the sandwich (biotin, streptavidin, and receptor). Our results give definitive evidence that the onset of steric crowding within the biotin layer occurs at a surface coverage of 57%, which is much higher compared to that from published work on well-ordered self-assembled biotin monolayers on planar gold surfaces. This difference is attributed to the topographical heterogeneity of the PSi substrate. Streptavidin (SA) binding to surface-linked biotin was altered by preblocking the streptavidin binding sites with biotin. Through consistent trends in data, preblocking SA was shown to reduce steric crowding within the SA layer, which translated into increased receptor immobilization. The final detection range of rabbit IgG was 0.07-3 mg mL(-1) (0.4-17 ng mm(-2)), and binding specificity was demonstrated by employing an anti-chicken IgG control receptor. This study underlines the importance of considering binding avidity and surface topography in optimizing chip-based biosensors.     

Preparation and application of streptavidin magnetic particles

Two kinds of streptavidin magnetic particles,namely streptavidin GoldMag particles and streptavidin amino terminal particles were prepared by the methods of physical adsorption and covalent interaction respectively.The streptavidin coated on magnetic particle surface,crucial to many applications,was greatly influenced by the choice of the different buffer.Compared with DynalbeadsM-270 streptavidin, the binding capacity for biotin of different streptavidin magnetic particles was determined by enzyme inhibition method,and the coupling capacity and activity of biotinylated oligonucleotide on their sur- face were also analyzed.The results indicated that the streptavidin GoldMag particle prepared by physical adsorption was stable in STE(NaCl-Tris-EDTA)buffer that was frequently used in nucleic acid hybridization and detection.The streptavidin amino terminal particles prepared by covalent interaction could be used both in STE buffer and PBS(phosphate buffered saline)buffer.The biotin binding ca- pacity for 1 mg of streptavidin GoldMag particles and streptavidin amino terminal particles was 4950 and 5115 pmol respectively.The capacity of biotinylated oligonucleotide(24 bp)coupled on 1 mg of GoldMag and amino terminal magnetic particles was 2839 and 2978 pmol separately.These data were about 6-7 times higher than those of DynabeadsM-270 streptavidin.The hybridization results with FITC-labeled complementary probe on magnetic particle surface demonstrated that the oligonucleotide coupled on streptavidin magnetic particles had high biological activity.     

Surface plasmon resonance spectroscopy and quartz crystal microbalance study of streptavidin film structure effects on biotinylated DNA assembly and target DNA hybridization

Surface plasmon resonance (SPR) spectroscopy is employed for the study of biotinylated DNA assembly on streptavidin modified gold surfaces for target DNA hybridization. Two immobilization strategies are involved for constructing streptavidin films, namely, (1) physical adsorption on biotin-containing thiol treated surfaces through biotin-streptavidin links and (2) covalent attachment to 11-mercaptoundecanoic acid (MUA) treated surfaces through amine coupling. To understand the structural properties of the streptavidin films, a quartz crystal microbalance with energy dissipation monitoring (QCM-D) is used to monitor the streptavidin immobilization procedures. The simultaneously measured frequency (Deltaf) and dissipation factor (DeltaD) changes, together with the SPR angle shifts (Deltatheta), suggest that the streptavidin film assembled on the biotin-containing surface is highly rigid with a well-ordered structure while the streptavidin film formed through amine coupling is highly dissipative and less structured. The subsequent biotinylated DNA (biotin-DNA) assembly and target hybridization results show that the streptavidin film structure has distinct effects on the biotin-DNA binding amount. On the streptavidin matrix, not only the probe DNA density but also the strand orientation mediated by the streptavidin films has distinct effects on hybridization efficiency. Particularly, the molecularly ordered streptavidin films formed on the biotin-containing surfaces ensure a well-ordered DNA assembly, which in turn allows for a higher efficiency in target DNA capture and for a higher sensitivity in the hybridization analysis when compared to the biotin-DNA assembled on the less structured streptavidin films formed through amine coupling.     

Single probe nucleic acid immobilization on chemically modified single protein by controlling ionic strength and pH

In an effort toward determining the feasibility of single molecule analysis, we describe a case whereby the binding of one biotinylated DNA to one streptavidin molecule via electrostatic interactions was controlled by altering in pH 4.0-9.0 and 0.16 of the ion strength. The quantitative analysis of immobilized probe ssDNA was realized in real-time via a quartz crystal microbalance (QCM) and electrochemical (EC) measurement in the range 100 pM to 50 μM of probe oligonucleotide concentration. The variation amount of biotinylated ssDNA immobilized on the streptavidin-modified surface at pH 7.5 was about 0.16 pmol, giving a ratio of streptavidin to biotinylated ssDNA of about 1:1.1. On the other hand, at pH 4.9, it was immobilized about 0.29 pmol. From the shape of the Langmuir plot and QCM, the immobilization efficiency of biotinylated DNA via streptavidin at pH 4.9 was approximately twofold that at pH 7.5. In view points of the reaction velocity, it was increased with decreasing buffer solution pH, indicating a strong interaction of negatively charged probe DNA with the positively charged streptavidin. And also the EC response value of ΔI/I_streptavidin for the immobilized biotinylated ssDNA in pH 4.9 was about 49%, while the corresponding value for the pH 7.5 was approximately 34%. As DNA molecules possess negative charges, electrostatic repulsion occurred between streptavidin and biotinylated ssDNA at pH 7.5. At pH 4.9, the attraction between the biotinylated ssDNA and streptavidin resulted in increased adsorption which has an isoelectric point of about 5.9. It was deduced that the binding of biotinylated ssDNA to one or two of the four binding sites of streptavidin can be controlled by adjusting the pH-controlled electrostatic interaction.     

Adsorption and conformation behavior of biotinylated fibronectin on streptavidin-modified TiO(X) surfaces studied by SPR and AFM

It is well-known that protein-modified implant surfaces such as TiO(2) show a higher bioconductivity. Fibronectin is a glycoprotein from the extracellular matrix (ECM) with a major role in cell adhesion. It can be applied on titanium oxide surfaces to accelerate implant integration. Not only the surface concentration but also the presentation of the protein plays an important role for the cellular response. We were able to show that TiO(X) surfaces modified with biotinylated fibronectin adsorbed on a streptavidin-silane self-assembly multilayer system are more effective regarding osteoblast adhesion than surfaces modified with nonspecifically bound fibronectin. The adsorption and conformation behavior of biotinylated and nonbiotinylated (native) fibronectin was studied by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM). Imaging of the protein modification revealed that fibronectin adopts different conformations on nonmodified compared to streptavidin-modified TiO(X) surfaces. This conformational change of biotinylated fibronectin on the streptavidin monolayer delivers a fibronectin structure similar to the conformation inside the ECM and therefore explains the higher cell affinity for these surfaces.     

Quantum dot-based immunosensor for the detection of prostate-specific antigen using fluorescence microscopy

A sensitive optical method based on quantum dot (QD) technology is demonstrated for the detection of an important cancer marker, total prostate-specific antigen (TPSA) on a disposable carbon substrate surface. Immuno-recognition was carried out on a carbon substrate using a sandwich assay approach, where the primary antibody (Ab)-protein A complex covalently bound to the substrate surface, was allowed to capture TPSA. After the recognition event, the substrate was exposed to the biotinylated secondary Abs. After incubation with the QD streptavidin conjugates, QDs were captured on the substrate surface by the strong biotin-streptavidin affinity. Fluorescence imaging of the substrate surface illuminated the QDs, and provided a very sensitive tool for the detection of TPSA in undiluted human serum samples with a detection limit of 0.25 ng/mL. The potential of this method for application as a simple and efficient diagnostic strategy for immunoassays is discussed.     

A new colorimetric platform for ultrasensitive detection of protein and cancer cells based on the assembly of nucleic acids and proteins

An amplified colorimetric method has been developed for the detection of protein and cancer cells based on the assembly of nucleic acids and proteins for the first time. In this process, the assembly of nucleic acids was triggered by a biotinylated DNA strand after a sandwich immunoreaction. The biotinylated DNA strand and sandwich immunocomplex were connected by streptavidin. Then, the assembly of biotinylated bovine serum albumin (Biotin-BSA) and streptavidin-horseradish peroxidase (SA-HRP) occurred at a node of the assembled products of nucleic acids through the biotin-streptavidin reaction. Under the catalysis of horseradish peroxidase, 3,3′,5,5′-tetramethylbenzidine (TMB) was oxidized by H₂O₂ and the oxidized product was analyzed by its UV–vis absorbance signal and sensitive colorimetric detection. This colorimetric sensor could not only achieve the quantitative determination of protein by UV–vis absorbance but could also be applied for semiquantitative determination by digital visualization. Using alpha-fetoprotein (AFP) as the model target, this proposed colorimetric method showed a wide linear range from 5 pg/mL to 1 ng/mL with a detection limit of 1.95 pg/mL by the instrument, and even 5 pg/mL target protein could be distinguished simply by the naked eye. This approach was then expanded to detect cancer cells based on the recognition of folic acid receptors that were over-expressed on the cancer cells by folic acid-tethered DNA. More importantly, this strategy can be further used as a universal colorimetric method for the determination of viruses or other proteins by changing the corresponding antibodies.