有机玻璃微流控芯片表面的蛋白质固定化及应用研究

高分子聚合物由于具有种类繁多、价格低廉、加工方法多种多样以及容易实现批量生产等优势,已越来越多地成为微芯片材料的首选^[1];将生物分子固定于固相载体上,与底物发生异相生化反应,从而使得产物易与反应体系分离,并可多次重复使用,无污染且大大降低消耗等特点而备受关注^[2].但是,由于聚合物材料表面较强的疏水性和化学惰性,无法直接通过如吸附、包埋、共价键合等常规的实验手段完成生物分子的有效固定.     

蛋白质DNA混合微点阵和微流控芯片的整合

在活化的石英片上制作蛋白质和DNA微点阵, 并可逆地将其与含有通道的多聚二甲基硅氧烷弹性橡胶封接在一起, 使蛋白质和DNA微点阵组装在微通道列阵内; 实现在微通道列阵内同时检测和分析蛋白质与DNA的功能. 为了降低多聚二甲基硅氧烷弹性橡胶的疏水性, 增强其生物相容性, 实验通过多聚赖氨酸对多聚二甲基硅氧烷弹性橡胶的修饰, 提高了它的亲水性, 使溶液能够在微通道内顺畅地流通. 实验表明, 这种混合芯片能够提高检测速度和增加检测的信息量.     

应用双槽电化学腐蚀法制备用于蛋白质芯片构建的多孔硅基底

通过双槽电化学腐蚀法制备大面积(12 mm×58 mm)均匀的多孔硅片,以小鼠免疫球蛋白G(IgG)与兔抗小鼠IgG抗体的相互作用为模型,证明其表面修饰环氧基团后能作为一种基底材料用于蛋白质微阵列芯片的构建。结果表明,兔抗小鼠IgG抗体检测的灵敏度与多孔硅基底制备时所采用的腐蚀电流密度、腐蚀时间、氢氟酸浓度有关。当电流密度为500 mA/cm2,腐蚀时间为450 s,HF浓度为25%时,IgG在多孔硅基底上的固定量最大,IgG芯片对兔抗小鼠IgG抗体的检出限为10μg/L,检测线性范围为0.32～10.0 mg/L。本方法制备的大面积均匀的多孔硅基底能够应用于蛋白质芯片的制作,并具有制备工艺简单,蛋白质固定量大等优点。     

用于识别不同细胞蛋白质组的噬菌体抗体芯片

将4个鼠源噬菌体抗体克隆和1个人源噬菌体抗体克隆偶联到羧基终止的硅片表面,制成分析型模型芯片.挑选健康人体淋巴细胞为正常细胞的代表, HeLa细胞为肿瘤细胞的代表,提取细胞的全部蛋白质并用荧光染料Cy3标记,与制成的分析芯片反应,得到了不同的结合图谱.实验结果表明,以噬菌体抗体为分子感受器的分析芯片可用于识别不同细胞的蛋白质组.     

对克伦特罗具有高特异性的酶联免疫吸附分析法研究

合成了克伦特罗(CL)半抗原2-(1-(4-氨基-3,5-二氯苯基)-2-(叔丁胺基)乙氧基)乙酸,采用活性酯法将半抗原与牛血清白蛋白偶联合成免疫原,经免疫、分离、纯化获得抗CL多克隆抗体,建立了对CL具高特异性的直接竞争酶联免疫吸附分析(dc-ELISA)法。本方法对CL检测的线性浓度范围为1.0×10!4～1.0 mg/L,定量检测下限为0.13μg/L。在尿样中添加CL标准至含量分别为10.0和1.0μg/L,dc-ELISA法检测的回收率分别为90.0%～115.9%和80.5%～112.4%;相对标准偏差(RSD)分别为7.1%和12.6%(n=10)。特异性实验结果表明:抗体与CL结构类似物沙丁胺醇(SAL)的交叉反应率<0.3%,因此本研究所制备的抗体可有效避免现有克伦特罗ELISA试剂盒、胶体金检测试纸假阳性率高的问题。     

高密度噬菌体抗体芯片对细胞表面蛋白的识别

采用正常人和白血病患者的白细胞对人源噬菌体抗体库进行淘选, 以获得对两种细胞表面蛋白特异的抗体. 通过pVIII展示系统, 使抗体以多价展示于重组噬菌体颗粒表面, 从上述两组中各挑选出48个克隆分别固定于环氧基片上, 并以空白噬菌体和牛血清白蛋白作为对照, 制成高密度噬菌体抗体芯片. 取来自3名正常人和3名白血病患者的白细胞裂解物样品, 用荧光染料Cy3标记, 与噬菌体抗体芯片反应, 对微阵共聚焦扫描得到的荧光图谱进行分析. 在白血病白细胞表面蛋白的识别图谱中有8组斑点显著不同于正常图谱. 由此表明, 噬菌体抗体芯片可用于识别细胞表面蛋白.     

芯片式流通池顺序注射可更新表面反射光谱法用于酶反应检测

将芯片式流通池顺序注射可更新表面反射光谱法用于酶反应检测。HRP催化H2O2氧化BPR底物的反应用于对H2O2的检测。此反应体系与葡萄糖氧化酶联用，用于对血清中葡萄糖的检测。     

多重反应监测(MRM):靶标蛋白质定量的新方法

多重反应监测(multiple reaction monitoring,MRM)是针对靶标分子的一种质谱分析技术.该技术采用三重四级杆质谱仪,检测靶标分子的母离子和子离子的质谱响应信号,从而获取较灵敏和高重现性的定性和定量信息,近年来在蛋白质组学领域得到了广泛应用.与全谱性的蛋白质组学分析不同,MRM注重有限目标的蛋白质定量测定,因此,它在蛋白质分析检测领域中的应用极有发展潜力.在临床检验中,酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)是蛋白质定量分析的常规技术,但是ELISA在多重蛋白质生物标志物的测定方面具有一定限制.随着蛋白质组学的深入进行,MRM的定量分析优势可否应用于临床检测已提至日程,世界范围内多个研究团队一直致力于推动这一领域的发展,也取得了令人瞩目的成就.本文简单介绍了MRM技术的原理、优势及发展前景等,同时,对其在蛋白质组学研究及临床应用中的潜力进行了讨论.     

化学生物学中光敏分子微阵列的表面构建与应用

分子微阵列是有机合成（特别是组合化学合成）方法应用于生物和医学研究而发展起来的高科技集成技术,通过把微电子、微加工技术和有机化学合成反应相结合,在固体基质（如硅片、玻片、瓷片等）表面构建微型的生物有机化学分子系统,以实现对细胞、蛋白质、核酸及其他生物组分进行快速、敏感、高效地处理．近年来,随着表面化学构建策略研究的不断深入和迅猛发展,分子微阵列技术的应用领域不断拓展,已从最初用于核酸分子的杂交测序延伸到基因组功能研究的各个方面．本文着重综述了光敏分子微阵列的表面化学构建策略研究及其在化学生物学分析中应用的最新进展,并展望了其发展的未来趋势．内容主要包括：小分子与多肽分子微阵列、蛋白质分子微阵列、核酸分子微阵列和糖分子微阵列等．     

Comparative Assessment of Oriented Antibody Immobilization on Surface Plasmon Resonance Biosensing

Protein A and protein G are extremely useful molecules for the immobilization of antibodies. However, there are limited comparative reports available to evaluate their immobilization performance for use as biosensors. In this study, a comparative analysis was made of approaches that use protein A and protein G for avian leukosis virus detection. The antibody‐protein binding affinities were determined using surface plasmon resonance (SPR) analysis. The immobilization efficiency was obtained by calculating the number of the protein molecular binding sites. The positive influence of sensor response on antigen detection indicates that the amount of immobilized antibody plays a major role in the extent of immobilization. Moreover, the biosensors constructed using both proteins were found to be regenerative. The SPR results from this study suggest that the surfaces of protein G provide a better equilibrium constant and binding efficacy for immobilized antibodies, resulting in enhanced antigen detection.     

Effect of Reaction Format on a McAb-Based Heterologous ELISA for the Insecticide Parathion

Abstract

Based on the selected monoclonal antibody, the effect of three reaction formats on sensitivity, specificity, and determination ability for real samples were investigated. The results showed the antibody-coated format was the optimal assay for parathion determination. The sensitivity of the assay was 3.20 ng/ml, with a detection limit of 0.40 ng/ml, and the assay time was 1.5 h. The average recoveries of parathion in river water, rice, cucumber, green soy bean, and cabbage were 98.56%, 89.46%, 99.25%, 118.57%, and 101.39%, respectively. In addition, when rice and cabbage extracts were analyzed by the assay and HPLC, the correlation was greater than 0.9.     

A comparative analysis of polyurethane hydrogel for immobilization of IgG on chips

Hydrogels are considered an optimum material for protein chip surfaces, since they provide a quasi-liquid environment which allows protein activity to be maintained and shows good spot morphology as well as excellent immobilization capacity. In the following, we present a polyurethane (PU) chip that electrostatically binds IgG. The PU surface is optimized with regard to layer thickness (∼200 nm), hydrogel (2%) and immobilized antibody concentration (0.5 mg mL⁻¹; 0.3 ng spot⁻¹), pH and ionic strength of the print buffer as well as to blocking solution. Evaluation is done in a direct IgG immunoassay using the Nexterion slide H as a reference. It is shown that higher IgG loading is achieved on the PU chip than on slide H, no matter whether 1× PBS (pH 7.2), Sörensen (pH 5.8) or Nexterion buffer was used as a spotting solution. Moreover, the crossreactivity with goat IgG, human IgG and monoclonal anti-CRP spotted in Nexterion buffer was as low as ≤0.74% (slide H: ≤3.34%).     

Surface Activation of Poly(Methyl methacrylate) by Plasma Treatment: Stable Antibody Immobilization for Microfluidic Enzyme-Linked Immunosorbent Assay

With the aim of obtaining stable antibody immobilization on the poly(methyl methacrylate), PMMA channel surface, PMMA substrates were activated with O₂ plasma treatment to introduce surface polar groups on it. The plasma-treated PMMA surfaces were characterized using water contact angle measurement, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). It was observed that plasma treatment significantly improved the surface wettability with changing surface chemistry and topography. The strategy of immobilization of a model antibody, anti-goat IgG on plasma-treated PMMA involved two steps. First the plasma-treated PMMA was functionalized with (3-aminopropyl)thriethoxy silane, APTES off-chip which facilitated covalent capturing of antibody via a crosslinking agent in the inner surface of PMMA channel in the second step. The antibody immobilization on plasma-treated PMMA was also confirmed using AFM, XPS, and fluorescence microscopy. The anti-IgG covalently captured on channel surface was evaluated with sandwich ELISA protocol on-chip using fluorescence microscopy. The observed results demonstrate that this technique could be extended to integrate the current diagnostic techniques into the plastic chip for important biomarker diagnosis.     

交联聚苯乙烯微球的表面化学改性及吡啶基卟啉的固载化

以无致癌毒性的1,4-二氯甲氧基丁烷为氯甲基化试剂,先将交联聚苯乙烯(CPS)微球氯甲基化,制得氯甲基化微球CMCPS。 然后使微球CMCPS与4-吡啶甲醛进行季铵化反应,将4-吡啶甲醛键合到CPS微球表面,制得键合吡啶甲醛(PyAL)的改性微球PyAL-CPS。 考察了主要反应条件对季铵化反应的影响,最终确定季铵化反应的优化条件:溶剂CCl4,反应温度60 ℃,最后使改性微球PyAL-CPS、4-吡啶甲醛及吡咯在固-液界面上进行Adler反应,形成固载吡啶基卟啉(PyP)的微球PyP-CPS。 用IR和UV-Vis以及Zn2+配合物生成法表征了微球PyP-CPS的形成及负载量。     

Quartz‐crystal Microbalance Measurements of CD19 Antibody Immobilization on Gold Surface and Capturing B Lymphoblast Cells: Effect of Surface Functionalization

We have investigated different surface functionalization methods to immobilize CD19 antibody on gold surface to capture B lymphoblast cells associated with the acute lymphoblastic leukemia disease. Quartz Crystal Microbalance measurements were performed to analyze the binding kinetics of each layer and determine the optimum method, which results in higher cell capture rates. The random orientation of antibody and oriented antibody through protein G was investigated and protein G presence resulted in 15,2 Hz frequency shift for 10⁴ cells/mL. The 3‐mercaptopropyltrimethoxysilane (MPS) and 11‐Mercaptoundecanoic acid (MUA) coatings of gold surface together with 4‐(N‐Maleimidomethyl)cyclohexane‐1‐carboxylic acid 3‐sulfo‐N‐hydroxysuccinimide ester sodium salt (Sulfo‐SMCC) and N‐Ethyl‐N’‐(3‐dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N‐hydroxysulfosuccinimide (NHS) linker layers were tested on QCM for protein G and antibody binding. The results indicate that MUA, EDC/NHS, protein G, antibody CD19 is the optimum surface modification among the tested combinations. By using the optimum surface functionalization method, minimum 10³ cell per mL was measured as 1.9 Hz frequency shift.     

单晶硅作为新型的基因芯片基底的探索

成功地将单晶Si(111)表面光化学引入官能团的技术与水溶性碳二亚胺催化多肽合成技术结合起来,制备并表征了ss-DNA 共价修饰的Si(111)表面.进一步证明了该表面具有特异性杂交的活性和反复使用的稳定性.为制备出可靠的、可反复使用的基因芯片提供了一种新的方法.     

Preparation of a Novel Polymer‐Modified Si Surface for DNA Immobilization

The design of a novel polymer‐modified overlayer composed of PPAPE and GPMS on a silicon wafer for immobilization of DNA molecules is described. After hydroxylation of Si(100) surfaces, GPMS molecules were self‐assembled onto these surfaces. PPAPE molecules were then covalently attached to the epoxy‐terminated surfaces. The incubation time and concentration of PPAPE was found to effect both layer thickness and water CA. The type of organic solvent and the pH were found to change the nature of the PPAPE‐modified surface for DNA immobilization. It is concluded that PPAPE‐modified surfaces show advantages for DNA immobilization by electrostatic interactions between DNA molecules and positively charged free amino groups of the PPAPE‐modified surfaces at the appropriate pH values.

     

Comparison of Physical Adsorption and Covalent Coupling Methods for Surface Density-Dependent Orientation of Antibody on Silicon

The orientation of antibodies, employed as capture molecules on biosensors, determines biorecognition efficiency and bioassay performance. In a previous publication we demonstrated for antibodies attached covalently to silicon that an increase in their surface amount Γ, evaluated with ellipsometry, induces changes in their orientation, which is traced directly using Time-of-Flight Secondary Ion Mass Spectroscopy combined with Principal Component Analysis. Here, we extend the above studies to antibodies adsorbed physically on a 3-aminopropyltriethoxysilane (APTES) monolayer. Antibodies physisorbed on APTES (0 ≤ Γ ≤ 3.5 mg/m²) reveal the Γ ranges for flat-on, side-on, and vertical orientation consistent with random molecular packing. The relation between orientation and Γ is juxtaposed for silicon functionalized with APTES, APTES modified with glutaraldehyde (APTES/GA) and N-hydroxysuccinimide-silane (NHS-silane). Antibody reorientation occurs at lower Γ values when physisorption (APTES) is involved rather than chemisorption (APTES/GA, NHS-silane). At high Γ values, comparable proportions of molecules adapting head-on and tail-on vertical alignment are concluded for APTES and the NHS-silane monolayer, and they are related to intermolecular dipole–dipole interactions. Intermolecular forces seem to be less decisive than covalent binding for antibodies on the APTES/GA surface, with dominant head-on orientation. Independently, the impact of glutaraldehyde activation of APTES on vertical orientation is confirmed by separate TOF-SIMS measurements.     

Detection of antinuclear antibodies by a colloidal gold modified optical fiber: comparison with ELISA

A fiberoptic evanescent-wave sensor has been developed for the measurement of antinuclear antibodies in sera from patients and healthy individuals. The sensor was constructed on the basis of modification of the unclad portion of an optical fiber with self-assembled gold colloids, where the colloidal gold surface was further functionalized with extractable nuclear antigens. Results show that detection of antinuclear antibodies by this sensor agrees quantitatively with the clinically accepted enzyme-linked immunosorbent assay (ELISA) method. This sensing platform has the following advantages: label-free and real-time detection capability, simple to construct and use, highly sensitive, and does not require a secondary antibody. The sensitivity of this platform is at least an order of magnitude higher than that of the ELISA method and thus may lead to a new direction in recognition of immune response. Biomolecular binding of antinuclear antibodies (ANA) with extractable nuclear antigens (ENA)-functionalized gold nanoparticles results in a change of surface plasmon absorption. When light propagates in an optical fiber by multiple total internal reflection, such a change in signal can be significantly enhanced.