以伴刀豆球蛋白为固定基质的脲酶生物传感器

本文制备了一种以溶剂聚合膜pH电极作原电极,利用伴刀豆球蛋白(Con A)与糖蛋白间的特异性识别作用,将Con A和脲酶表面的麦芽糖残基结合,采用交替沉积Con A和脲酶,进行多层脲酶膜组装的脲酶生物传感器。研究了酶固定化条件的影响,优化了实验条件,测试了传感器对尿素的生物电化学响应。在6.9×10-5~1.0×10-3mol.L-1的浓度范围内传感器响应的电极电位与尿素浓度的对数成正比,检出限为4.5×10-5mol.L-1。将传感器用于牛奶样品中回收率的测定,结果满意。     

辣根过氧化物酶电极在H2O2分析中的应用

辣根过氧化物酶(HRP)能催化过氧化氢与氢供体间的氧化还原反应,是当今生物传感器研究的热点之一.HRP分子内含有α-D-葡萄糖和α-D-甘露糖,是一种糖蛋白,在pH 7.0下,能与具有识别α-D-葡萄糖和α-D-甘露糖功能的外源植物凝集素伴刀豆球蛋白(Con A)结合.通过Con A与HRP之间的识别作用在半胱氨酸修饰的金表面构造HRP多层自组装膜电极,以亚甲蓝(MB)溶液为介体,对电极进行了电化学表征,并用该酶电极测定了过氧化氢浓度.     

光学活性偶氮苯自组装膜的制备及其蛋白吸附行为

研究了在紫外光作用下, 牛血清白蛋白(BSA)在偶氮苯自组装膜上光控可逆的吸附行为. 首先合成羧基偶氮苯衍生物, 并在金膜表面制备偶氮苯自组装膜, 采用紫外吸收光谱(UV)、原子力显微镜(AFM)观察偶氮苯衍生物的光学顺反异构现象以及偶氮苯自组装膜表面形貌的变化. 同时利用等离子体表面谐振仪(SPR)考察偶氮苯光学异构对牛血清白蛋白(BSA)在自组装膜表面上的吸附行为的影响. 结果表明, BSA在偶氮苯自组装膜表面的吸附作用主要来自于BSA分子与自组装膜之间的静电作用及亲疏水作用. 在紫外光作用下, 偶氮苯自组装膜可以实现光控可逆的牛血清白蛋白分子吸附行为.     

刀豆球蛋白A诱导的葡聚糖纳米凝胶高层级自组装

基于葡聚糖这类结构明确的水溶性多糖,以接枝聚合诱导自组装法(GISA)制备得到具有交联结构的葡聚糖纳米凝胶。在此基础上,利用刀豆球蛋白A(Con A)与葡聚糖中葡萄糖单元之间的特异性识别作用,诱导葡聚糖纳米凝胶的高层级自组装,从而制备尺度可控的Con A-葡聚糖纳米凝胶高层级自组装体。通过透射电镜、红外光谱、核磁共振光谱、等温滴定量热法等手段对高层级自组装体的粒径、结构和形貌进行表征,探讨了其高层级自组装的机理。此外,还探究了Con A以及Con A-葡聚糖纳米凝胶高层级自组装体对人肺癌细胞A549的细胞毒性。结果表明:该自组装体的尺寸同葡聚糖与Con A的质量比直接相关;游离的Con A对A549细胞具有抑制作用,且Con A在参与高层级自组装的过程中生物活性保持不变。     

阻抗滴定法测定3-巯基丙酸自组装膜的表面酸度

利用交流阻抗和循环伏安法研究了巯基丙酸自组装膜的组装过程及表面羧基的解离性质.研究表明,由于巯基丙酸的链长较短,自组装膜的组装过程表现为快吸附,然后表现为缓慢组装的过程.利用阻抗值随溶液pH的变化绘制出阻抗滴定曲线,得出了自组装膜表面巯基丙酸的表面酸度,研究了饱和吸附与不饱和吸附对表面酸度的影响.利用氢键作用和静电相互作用对实验结果进行了解释.     

糖蛋白-凝集素自组装构筑有序膜及在酶电极的应用

利用糖蛋白-凝集素的识别作用交替沉积伴刀豆球蛋白（Con A）与辣根过氧化物酶(HRP)制备酶自组装多层膜，用原子力显微镜（AFM）观测了自组装膜的表面形貌、表面粗糙度； AFM和椭圆偏振研究测定了自组装膜的厚度.结果表明, Con A和HRP膜厚分别为9.0和4.6 nm，与两者的晶体衍射结果一致，说明生物识别自组装方法能很好地保持分子的原有形态.以亚甲蓝（MB）溶液为介体，用循环伏安法测定了表面修饰了三层（Con A/HRP）自组装膜的金电极对H2O2的电化学催化还原作用，在H2O2浓度为0.2～1.0 mmol•L－1时，响应电流对H2O2浓度变化成线性，酶电极灵敏度为24.0 mA•mol－1•L，表观米氏常数为4.2 mmol•L－1.     

静电组装金纳米粒子制备局域表面等离子体共振传感膜

采用聚电解质自组装技术制备局域表面等离子体共振(LSPR)传感膜的方法, 在玻璃基片上依次沉积聚电解质PDDA, PSS和PVTC, 并通过静电吸附构建胶体金纳米粒子自组装膜形成LSPR传感膜. 利用扫描电镜对LSPR传感膜表面形貌以及膜中金纳米粒子的粒径进行了表征, 同时通过紫外-可见消光光谱对其灵敏度和渗透深度等重要参数进行检测. 研究结果表明, 所制备的LSPR传感膜粒子分布均匀、单分散性好、稳定性高、重现性好; 消光峰位对样品溶液折射率的检测灵敏度为71 nm/RIU, 相应的峰强检测灵敏度为0.21 AU/RIU, 对表面吸附层的渗透深度约为16 nm.     

电流滴定法对自组装膜表面酸碱性的研究

近年来 ,有序分子自组装体系在基础研究和应用领域均得到了较大的发展 .选择末端可以解离的自组装分子而制成的自组装膜 ,可以方便地通过调节底液的 p H值来控制膜体系的荷电状况 [1] ,这在利用静电作用吸附蛋白质 [2 ] 、多肽 [3] 、 DNA[4 ] 、聚电解质 [5] 、金属离子及其它物种 [6] 等方面具有重要作用 .精确测定表面解离常数 (即表面 p Ka)和控制自组装膜表面的荷电状况 ,无疑在理论研究和实践中均占有重要地位 .目前 ,一些方法如接触角滴定 [7] 、微分界面电容 [8] 、电流滴定法 [9,10 ] 以及 AFM的力曲线滴定 [11,12 ]被用于表面…     

Au单晶表面氟表面活性剂FSN自组装膜的电化学行为

研究Au(111)和Au(100)表面非离子型氟表面活性剂FSN自组装膜的电化学行为.电化学扫描隧道显微术和循环伏安法测试表明,在0～0.8 V电位区间,FSN自组装膜未发生氧化还原,均一性好,可稳定地存在于电极表面,并显著抑制硫酸根离子在电极表面的吸附和Au单晶表面的重构.在FSN自组装膜Au单晶电极的初始氧化阶段,Au(111)表面有少量突起,而Au(100)表面呈现台阶剧烈变化,但FSN自组装膜的吸附结构没有改变.与Au(100)表面相比,Au(111)表面形成的FSN自组装膜可更有效地抑制Au表面的氧化.     

基于多壁碳纳米管和聚丙烯胺层层自组装的葡萄糖生物传感器

经混酸处理后的多壁碳纳米管(MWCNTs)末端及侧壁带有含氧基团,能与阳离子聚电解质通过静电作用结合,也能与酶蛋白非特异性结合。利用层层自组装法在铂(Pt)电极表面构建了聚丙烯胺(PAA)-MWC-NTs-葡萄糖氧化酶(GOx)膜,研究了自组装薄膜的表面微观形貌和电化学性质。组装层数为6层时最优,对葡萄糖响应线性范围为5.0×10-4~2.10×10-2mol/L;检出限为1.0×10-4mol/L(S/N=3);灵敏度为4.95μA/(mmolcm2),响应时间3.80s;GOx表观米氏常数为17.79mmol。对抗坏血酸等具有较强的抗干扰能力,10天后电极响应电流保持最初的91.79%。3次平行实验的RSD为4.85%。     

Binding of monomeric and dimeric Concanavalin A to mannose-functionalized dendrimers

Because of the central role of Concanavalin A (Con A) in the study of protein-carbohydrate interactions, a thorough understanding of the multivalent functions of Con A is imperative. Here, the association of monomeric and dimeric derivatives of Con A with mannose-functionalized generation two through six PAMAM dendrimers is reported. Hemagglutination assay results indicate relatively low activity of the dendrimers for monomeric Con A, with small increases as the dendrimer generation increases. Isothermal titration microcalorimetry experiments indicate monovalent binding by the dendrimers with monomeric Con A and divalent binding by the dendrimers with dimeric Con A. Continuous (and comparable) but narrowing increases in enthalpy and entropy and the slight increase in association constants with monomeric Con A as the dendrimer generation increases suggest favorable proximity effects on binding. Both the hemagglutination assay and the calorimetry experiments suggest that statistical binding enhancements can be observed with monomeric Con A. The results described here should allow for a more quantitative evaluation of the enhancements that are often observed in protein-carbohydrate interactions for glycosylated frameworks binding to Con A.     

The effects of aggregation, protein binding and cellular incorporation on the photophysical properties of benzoporphyrin derivative monoacid ring A (BPDMA)

Absorption, fluorescence and laser flash photolysis spectroscopies were used to investigate the effects of self-aggregation, binding to human serum albumin and incorporation in cancer cells on the photophysics of benzoporphyrin derivative monoacid ring A (BPDMA). Aggregation of BPDMA has been studied in mixtures of methanol and phosphate-buffered saline (PBS). The extent of aggregation was dependent on dye concentration and solvent composition, becoming particularly marked in mixtures containing less than 30% methanol. A dimerization constant K_d or 9 × 10⁶ M⁻¹ was determined by fluorescence experiments for BPDMA in pure PBS. In addition to spectral modifications, aggregation induces a lowering of the fluorescence and intersystem crossing quantum yields. Human serum albumin binds BPDMA with an association constant K_b of 5.2 × 10⁵ M⁻¹ in PBS. When bound to HSA, BPDMA displays photophysical properties very similar to the monomer in organic solvents. The molar ratio [HSA]/[BPDMA] corresponding to complete binding of the dye was determined to be approximately 10. Efficient generation of the triplet state of BPDMA was also observed from aqueous cellular suspensions containing incorporated photosensitizer.     

Kinetic studies on the interactions between glycolipid biosurfactant assembled monolayers and various classes of immunoglobulins using surface plasmon resonance

Kinetic studies on the interactions between self-assembled monolayers of mannosylerythritol lipids (MELs), which are glycolipid biosurfactants abundantly produced by microorganisms, and various classes of immunoglobulins including human IgG, IgA, and IgM were performed using surface plasmon resonance (SPR). The effect of the MEL structure on the binding behavior of HIgG was examined. Assembled monolayers of MEL-A having two acetyl groups on the headgroup gave a high affinity (K_d = 1.7 × 10⁻⁶ M) toward HIgG, while those of MEL-B or MEL-C having only one acetyl group at C-6′ or C-4′ position gave little affinity. Our kinetic analysis revealed that the binding manner of HIgG, HIgA (K_d = 2.4 × 10⁻⁷ M), and HIgM (K_d = 2.2 × 10⁻⁷ M) to the assembled monolayers of MEL-A is not the monovalent mode but the bivalent mode, and both the first and second rate association constants (k_a1, k_a2) increase with an increase in the number of antibody binding sites, while those for dissociation (k_d1, k_d2) changed little. Moreover, we succeeded in directly observing great amounts of HIgG, HIgA, and HIgM bound to MEL-A monolayers using atomic force microscopy (AFM). Finally, we found that MEL-A assembled monolayer binds toward various IgG derived from mouse, pig, rabbit, horse, goat, rat, and bovine as well as human IgG (HIgG), and the only exception was sheep IgG. These results clearly demonstrate that MEL-A assembled monolayers would be useful as noble affinity ligand system for various immunoglobulins.     

Electrochemical piezoelectric quartz crystal impedance study on the interaction between concanavalin A and glycogen at Au electrodes

The carbohydrate research has emerged as a "new frontier" in chemical/biological field. The binding of lectin with carbohydrate is one of the important courses of life activities. The report studies concanavalin A (Con A)-glycogen interaction on gold electrode surfaces by electrochemical piezoelectric quartz crystal impedance (EPQCI) method. The piezoelectric quartz crystal (PQC) parameters, resonant frequency shift (Deltaf(0)) and the motional resistance change (DeltaR(1)), and the electrochemical impedance (EI) parameters, electrolyte resistance change (DeltaR(s)) and the double layer capacitance change (DeltaC(s)), were measured and discussed simultaneously. Two methods were adopted for measuring the Con A-glycogen association. Based on EPQCI measurement during Con A reaction with glycogen adsorbed on Au electrode, association constant K(a) and the amount of the binding sites s calculated are 1.48 x 10(6) M(-1) and 4.09, respectively. Based on single PQC measurement of glycogen reaction with Con A assembled on Au electrode, K(a) was estimated to be 1.26 x 10(6) M(-1).     

Carbohydrate analysis prepares to enter the "Omics" era

In this issue, Houseman and Mrksich describe a carbohydrate array preparation method that can be used to analyze protein-carbohydrate interactions and to characterize the substrate specificity of a carbohydrate-modifying enzyme. Carbohydrate chips were prepared by a novel procedure that allows the covalent attachment of carbohydrate-diene conjugates to a specially engineered monolayer surface. The surface presents a precisely controllable ratio of reactive benzoquinone and inert ethylene glycol groups. Nonspecific adsorption of proteins to the surface is extremely low, and the surface is compatible with popular detection techniques. The immobilization technique was demonstrated to be compatible with recently developed automated solid phase carbohydrate synthesis methods, paving the way for the development of highly complex carbohydrate arrays.     

高覆盖率氟代癸基三氯硅烷自组装单分子膜的制备

通过液相沉积在云母表面制备1H, 1H, 2H, 2H-全氟癸基三氯硅烷(FDTS)自组装单分子膜(SAMs)。室温下,将1.0 mmol·L^-1的FDTS溶液静置水解15 min,再把云母浸入自组装30 min,原子力显微镜(AFM)表征发现,液相沉积过程中FDTS的团聚现象得到有效解决。该方法制备出了高覆盖率(85% ± 2%)和低均方根粗糙度(0.58 nm)的FDTS SAMs,且单分子膜的生长过程符合Langmuir一级动力学吸附模型。在液相沉积过程中,若水解和组装同时进行,过长的水解时间(大于30 min)或组装时间(大于30 min)均会导致FDTS的团聚,进而极大降低SAMs的质量。     

电势诱导的N-异丁酰基-L-半胱氨酸分子在金(111)表面的相转变

Functional solid substrates modified by self-assembled monolayers (SAMs) have potential applications in biosensors, chromatography, and biocompatible materials. The potential-induced phase transition of N-isobutyryl-L-cysteine (L-NIBC) SAMs on Au (111) surfaces was investigated by in-situ electrochemical scanning tunneling microscopy (EC-STM) in 0.1 mol·L^-1 H₂SO₄ solution. The NIBC SAMs with two distinct structures (α phase and β phase) can be prepared by immersing the Au (111) substrate in pure NIBC aqueous solution and NIBC solution controlled by phosphate buffer at pH 7, respectively. The as-prepared α phase and β phase of NIBC SAMs show various structural changes under the control of electrochemical potentials of the Au (111) in H₂SO₄ solution. The α phase NIBC SAMs exhibit structural changes from ordered to disordered structures with potential changes from 0.7 V (vs saturated calomel electrode, SCE) to 0.2 V. However, the β phase NIBC SAMs undergo structural changes from disordered structures (E < 0.3 V) to γ phase (0.4 V < E < 0.5 V) and finally to the β phase (0.5 V < E < 0.7 V). EC-STM images also indicate that the phase transition from the β phase NIBC SAMs to the α phase occurs at positive potential. Combined with density functional theory (DFT) calculations, the phase transition from the β phase to the α phase is explained by the potential-induced break of bonding interactions between ——COO^- and the negatively charged gold surfaces.     

基于聚酪氨酸/铋修饰玻碳电极的Pb2+传感器研究

采用循环伏安法及原位镀铋制备了聚酪氨酸/Bi复合膜修饰玻碳电极(p-Tyr/Bi/GC),并用交流阻抗谱及扫描电镜表征了复合膜电极的电子传递阻抗及表面形态。发现聚酪氨酸膜能促进电极表面电子交换,有利于高灵敏Pb²⁺传感器的研制。以复合膜电极对Pb2+的方波阳极溶出伏安响应电流探讨了聚酪氨酸修饰玻碳电极的最佳制备及测试条件。结果表明电极制备液中酪氨酸的最佳浓度为1.5 mmol·L^-1,聚合圈数为15;测试液中Bi3+的最佳浓度为1.0μmol·L-1,pH值为6.0,富集电位为-1.20 V。在最佳条件下,复合膜电极对Pb2+的响应线性方程为:Ip(μA)=0.5032+54.68c(μmol·L-1)(r=0.9947),线性范围为0.01~0.16μmol·L^-1,检出限(3S/k)为0.8 nmol·L^-1。复合膜电极具有灵敏度高、稳定性好、抗干扰能力强的特点,用于茶叶样品中铅的测定,回收率为90.6%~96.3%,相对标准偏差不大于3.9%。     

对乙酰氨基酚在石墨烯/壳聚糖复合膜修饰电极上的电化学行为研究

制备了石墨烯-壳聚糖复合物修饰玻碳电极(GO/CS-GCE),考察了对乙酰氨基酚(APAP)在修饰电极上的电化学行为,发现石墨烯-壳聚糖复合物能较好改善玻碳电极对APAP的电化学性能,APAP在修饰电极上的电化学反应过程是受吸附控制的2电子,2质子反应过程;进一步研究发现在pH=9.16的碳酸钠-碳酸氢钠缓冲体系中,对...     

Studying protein-carbohydrate interactions by amide hydrogen/deuterium exchange mass spectrometry

Protein-carbohydrate interactions play a significant role in biological processes. Presented here is the novel application of amide hydrogen/deuterium exchange mass spectrometry (amide exchange-MS) to the study of the interaction between a protein and its carbohydrate substrate. The degree of deuterium incorporation into hen egg lysozyme was monitored with and without substrate to verify that a carbohydrate can provide sufficiently stable protection of the amide hydrogen atoms in a protein's backbone from exchange with deuterated solvent. The substrate protected a number of amide hydrogens from exchange, implying that protein-carbohydrate binding systems will be compatible with amide exchange-MS. Endopolygalacturonase-II (EPG-II) from Aspergillus niger, a pectin-degrading enzyme, was chosen as the first carbohydrate-binding system to be extensively studied using quenched amide exchange-MS. Monitoring the changes in deuterium incorporation of EPG-II in the presence and absence of an oligomer of galacturonic acid implied the location of substrate binding. This study demonstrates the ability of amide exchange-MS to investigate protein-carbohydrate interactions.