蛋白质微阵列芯片技术及其在抗体筛选中的应用

以兔IgG为模式蛋白质,对其在醛基修饰玻片表面的固定浓度、固定时间和温度等条件进行了优化,结果表明：在室温下,当固定蛋白质的浓度为1g／L、固定时间为4h时,可获得理想的蛋白质固定效果;蛋白质的定量检测范围为1μg／L～10mg／L。按优化的蛋白质微阵列芯片制作条件将规模化制备的抗体制作成抗体微阵列芯片,通过与荧光标记的人球蛋白和人白蛋白的相互作用,实现了对不同抗体株抗球蛋白和抗白蛋白活性的快速筛选与比较。     

一种孔雀石绿免疫亲和柱及其制备方法和用途

<正>申请公布号:CN104174185A申请公布日:2014.12.03申请人:北京美正生物科技有限公司摘要本发明涉及一种孔雀石绿免疫亲和柱制备方法及其用途。该免疫亲和纯化柱利用蛋白G偶联到琼脂糖凝胶上载体,然后用抗孔雀石绿的抗体与琼脂糖上的蛋白G偶联。再利用交联剂对结合孔雀石绿抗体的蛋白G–琼脂糖凝胶载体进行交联。用交联后的载体制备免疫亲和柱。该纯化柱     

血红素蛋白质-琼脂糖膜修饰电极的表征和电催化特性

利用琼脂糖(agarose)水凝胶将肌红蛋白(Mb)、血红蛋白(Hb)、辣根过氧化物酶(HRP)和过氧化氢酶(Cat)4种血红素蛋白质固定在裂解石墨电极表面,形成稳定的血红素蛋白质-agarose膜修饰电极.用紫外-可见和红外光谱及原子力显微法对血红素蛋白质-琼脂糖膜修饰电极进行了表征.紫外-可见和红外光谱显示,在琼脂糖凝胶中,血红素蛋白质保持原始构象.溶液的pH(3.0～10.0)可逆地改变血红素蛋白质的构象,从而影响其光谱性质.原子力显微图象表明血红素蛋白质与agarose水凝胶之间存在较强的作用.研究了血红素蛋白质催化还原O2、H2O2的机理.稳定的血红素蛋白质-agarose修饰电极能运用于H2O2的定量测定.     

基于室温离子液体和有序大孔金膜的酶传感器

将室温离子液体1-丁基-3-甲基咪唑四氟硼酸盐([BMIM]BF4)、N,N-二甲基甲酰胺(DMF)、及不同种类的膜材料与各种酶一起通过混合涂布、逐层修饰的方式固定于三维有序大孔金膜电极表面,构建一种新型的生物酶传感器。血红蛋白(Hb)、肌红蛋白(Mb)和辣根过氧化物酶(HRP)在该修饰电极上呈现出明显的还原峰,这归因于三种酶活性中心的直接电化学行为。分别构建四种不同膜材料的修饰电极,研究了硅凝胶、壳聚糖、Nafion膜和琼脂糖水凝胶四种材料对于L-乳酸脱氢酶(LDH)修饰电极催化响应的影响,结果表明选择壳聚糖作为膜材料是最优选择。将壳聚糖修饰的LDH金膜电极用做检测乳酸浓度的生物传感器,该传感器表现出良好的催化性能,线性响应范围为10~250nmol/L,检测限(S/N=3)为3.3nmol/L。     

基于碳纳米管的脂质体电化学发光免疫传感器检测人免疫球蛋白G

将人免疫球蛋白G(hIgG)抗体固定在多壁碳纳米管修饰的玻碳电极表面,制备了一种电化学发光(ECL)免疫传感器.以hIgG抗体标记的联吡啶钌脂质体为标记物,采用三明治型检测方式,成功建立了hIgG的ECL免疫检测技术.电化学发光强度与hIgG的浓度在0.01～0.8 μg/L范围内呈良好的线性关系; 线性回归方程为y=618.7x(μg/L)-23.9(n=6,r=0.995); 检出限为0.004 μg/L.用于人血清中hIgG的检测,结果令人满意.     

免标记电化学免疫传感器用于微囊藻毒素-LR的灵敏检测

采用纳米金作为抗体的固定基质,同时以硫堇为电活性指示剂,构建了一种免标记的电化学免疫传感器用于微囊藻毒素-LR(MC-LR)的灵敏检测。将萘酚修饰到金电极表面,通过静电作用将硫堇捕获。加入纳米金与硫堇的氨基结合,抗体将通过与纳米金作用而固定到电极表面。通过MC-LR与其相应抗体的特异性结合作用阻碍硫堇的电子传递,实现MC-LR的电化学检测。在最优实验条件下,MC-LR的质量浓度与电信号在5~100μg/L范围内呈良好的线性关系,检出限为0.71μg/L,可满足饮用水检测需求,能够用于实际水样中M C-LR的测定。     

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。     

碳纳米管/纳米金复合膜电化学免疫传感器用于微囊藻毒素的检测研究

在玻碳电极表面修饰碳纳米管,并用多电位阶跃法在碳纳米管表面沉积纳米金制得碳纳米管/纳米金复合膜。通过纳米金和微囊藻毒素-(亮氨酸-精氨酸)抗体之间的吸附作用,将抗微囊藻单克隆抗体固定于电极表面,以牛血清白蛋白封闭非特异性吸附位点,研制了检测微囊藻毒素的电化学免疫传感器。利用微囊藻毒素与其抗体之间的特异性识别作用构建"三明治"夹心结构的免疫分析模式,以辣根过氧化物酶标记抗体为二抗,利用微分脉冲伏安法实现了对微囊藻毒素的检测。在优化条件下,此传感器的响应电流与微囊藻毒素浓度在0.50~12.0μg/L范围内呈良好的线性关系,检出限为0.30μg/L(S/N=3)。对实际水样进行了微囊藻毒素的加标回收实验,回收率在93.0%~108.5%之间,相对标准偏差为3.8%~5.0%。     

金纳米簇信号放大的电化学免疫传感器

通过抗原抗体的特异性识别作用以及金纳米簇(AuNCs)探针和金标银染的双重信号放大作用,构建了一种新的电化学免疫传感器,对人的免疫球蛋白(IgG)进行了检测。受贻贝分泌的黏附蛋白启示,首先将聚多巴胺薄膜修饰在铟锡氧化物电极(ITO)上,并对一抗抗体进行固定,通过观察电化学阻抗的变化来监控免疫传感器的构建过程。将待检测的IgG抗原组装在该电极上并与AuNCs标记的二抗反应,最后经银染反应,用溶出伏安法对IgG的含量进行定量检测,其灵敏度达到0.5 ng/L。该方法可应用于实际血清样品中IgG含量的测定。     

血红素-琼脂糖修饰电极的电化学和电催化性质研究

用循环伏安法研究了血红素-琼脂糖凝胶修饰电极在水和有机溶剂/水混合溶液中的电化学和催化还原过氧化物的性质。包埋在琼脂糖水凝胶膜中的血红素能与电极之间直接传递电子,且具有过氧化物模拟酶的特性,能快速地催化还原过氧化物,可用于这些物质的定量检测。     

二氧化碳和丙烯直接合成甲基丙烯酸的NiPMo/TiO2催化剂的研究

用溶胶-凝胶法以磷钼酸(MPA)的镍盐溶液水解钛酸四丁酯制备了NiPMo/TiO2催化剂.使用ICP、 XRD、 TG-DTA、 IR、 TPD-MS和微反应技术研究了催化剂的化学组成、热稳定性、化学吸附性质和催化反应性能.杂多钼酸盐与TiO2通过O2-在TiO2表面发生了键合.在623 K下,杂多阴离子仍保持原有的Keggin结构.CO2在Lewis酸位Ni(Ⅱ)和Lewis碱位Ni-O-Mo的桥氧协同作用下生成CO2卧式吸附态Ni(Ⅱ)←O-(CO)←(O--Ni).丙烯有多种吸附态在催化剂上吸附.在563 K、 1 MPa和空速1500 h-1的反应条件下,丙烯的摩尔转化率为3.2%,产物MAA选择性为95%.     

Synthesis of 1-benzyloxypyrazin-2(1H)-one derivatives

Different approaches for the synthesis of 1-benzyloxypyrazin-2(1H)-one derivatives from simple amino acids have been investigated. A library of 33 precursors for the preparation of N-hydroxy pyrazinones was obtained in moderate to good yields.     

Toward new camptothecins. Part 6: Synthesis of crucial ketones and their use in Friedländer reaction

In the context of the preparation of camptothecin and luotonin A analogs, the synthesis of some key keto-precursors and their use in Friedländer condensation are described. This paper also focuses on the stability of these keto intermediates and emphasizes the major differences between indolizinones and pyrroloquinazolinones series. Noteworthy is also the report of some original structures isolated as by-products of some experiments.     

CoFe2O4自形成磁性液体场致结构化对磁化的影响

The Langevin paramagnetic theory can’t describe the relation between magnetization of ferrofluids and applied magnetic field. The structuralization of ferrofluids, which is considered the main influence factor of the magnetization, is regarded. The part of magnetization works is deposited when the structure is forming. This action influences the magnetization of ferrofluids directly or indirectly. On the base of the “compressing” model, the Langevin function that usually describes the magnetization of ferrofluid is modified, and a well-fitted curve is obtained. An equation of the relation between the equivalent volume fraction after being “compressed” and the intensity of magnetic field is discovered, which approximately describes the process of magnetization. The relation between the approximate initial susceptibility and the volume fraction can be obtained from modified formula.     

Highly regioselective Buchwald–Hartwig amination at C-2 of 2,4-dichloropyridine enabling a novel approach to 2,4-bisanilinopyridine (BAPyd) libraries

The highly regioselective Buchwald–Hartwig amination at C-2 of the cheap and readily accessible reagent, 2,4-dichloropyridine with a range of anilines and heterocyclic amines is described. This new methodology is robust and provides a facile access to 4-chloro-N-phenylpyridin-2-amines on 0.25 mol scale. These intermediates undergo a further Buchwald–Hartwig amination at higher temperature to enable rapid exploration of the chemical space at C-4 and to provide a library of 2,4-bisaminopyridines.     

KMnO4-mediated oxidative CN bond cleavage of tertiary amines: Synthesis of amides and sulfonamides

KMnO₄-mediated oxidative CN bond cleavage of tertiary amines producing secondary amine was introduced, which was trapped by electrophiles (acyl chloride and sulfonyl chloride) to form amides and sulfonamides. The reaction could take place at mild condition, tolerating a wide range of function groups and affording products in moderate to excellent yields.     

Chemistry of heterocyclic compounds at the A. E. Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of Sciences, over 50 years (Review)

The review contains a concise historical account and information on the most significant researches undertaken by the staff at the A. E. Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of Sciences on the Chemistry of Heterocyclic Compounds. Dedicated to Academician of the Russian Academy of Sciences B. A. Trofimov on his 70th jubilee. Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 10, pp. 1443–1502, October, 2008.     

A general and convenient synthesis of 4-(tosylmethyl)semicarbazones and their use in amidoalkylation of hydrogen,heteroatom, and carbon nucleophiles

A general synthesis of previously unknown semicarbazone-based α-amidoalkylating reagents, 4-(tosylmethyl)semicarbazones, has been developed. The synthesis involved three-component condensation of semicarbazones of aliphatic or aromatic aldehydes with the same or other aldehydes and p-toluenesulfinic acid. The scope and limitations of this reaction were investigated. The compounds obtained were demonstrated to be an efficient α-(4-semicarbazono)alkylating agents. They were reacted with H- (sodium borohydride), O- (sodium methylate), S- (sodium phenylthiolate), N- (pyrrolidine, sodium succinimide), P- (trialkyl phosphites), and C-nucleophiles (sodium diethyl malonate) to give the corresponding products of the tosyl group substitution, 4-substituted semicarbazones, including analogues of nitrofurazone. Among the prepared compounds tested in vitro for antibacterial and antifungal activity, three nitrofuryl-containing semicarbazones exhibited high biological activities with minimum inhibitory concentration (MIC) values of 8–32 μg/mL.     

Sulfur-directed metal-free and regiospecific methyl C(sp3)–H imidation of thioanisoles

Regiospecific and direct imidation of the methyl C(sp³)–H bond of thioanisoles is realized under mild and metal-free conditions with N-fluorobis(benzenesulfonyl)imide as an oxidant and nitrogen source. Proposed mechanism suggests that thionium ion intermediates and a Pummerer-type reaction are involved. The imidation has advantages such as high step-economy, excellent functionality tolerance, and regiospecificity, giving structurally diverse imidation products.     

Synthesis of novel chiral bidentate hydroxyalkyl-N-heterocyclic carbene ligands and their application in palladium-catalyzed Mizoroki–Heck couplings and asymmetric addition of diethylzinc to benzaldehyde

A small library of new chiral bidentate hydroxyalkyl-imidazolium salts 1 is conveniently synthesized on multi-gram scale from inexpensive and commercially available chiral pool amino acids. The corresponding carbenes, generated by deprotonation of imidazolium salts 1, in combination with palladium(II) chloride were tested in the Mizoroki–Heck coupling reaction. The most significant results in terms of yields and reactivities were achieved with low catalyst loading. The catalytic activities of these imidazolium salts were also investigated in the asymmetric addition of diethylzinc to benzaldehyde. The use of MgO nanoparticles as an additive in conjunction with these ligands played a crucial role in increasing the efficiency of these reactions.