采用抗体包被纳米金多层自组装膜的HCG安培免疫传感器

研制了一种采用人绒毛膜促性腺激素抗体(anti-HCG)包被巯基丁二酰胺(HL)/纳米金(AuNPs)多层膜修饰金电极的免疫传感器(AU |{HL/AuNPs}n-anti-HCG),用于检测人唾液中的HCG.通过层层自组装技术在Au电极表面交替修饰多层{HL/AuNPs}n膜,再通过膜内的AuNPs吸附anti-HCG,构筑了HCG安培免疫传感器.以K3Fe(CN)6为探针,在pH 7.0的PBS缓冲液中加入HCG,随着HCG与电极表面anti-HCG发生特异性免疫反应,形成的抗原/抗体免疫复合物使得K3Fe(CN)6在电极表面的响应电流减小,减小量(Ip)与膜表面的免疫反应程度相关,以此作为HCG定量的依据.对各修饰电极表面进行了表征和定量分析.实验表明:该免疫传感电极中{HL/AuNPs}n膜内存在的大量AuNPs显著增加了anti-HCG吸附量,从而使得其HCG检测范围变宽(0.1～100 mg/L),检出下限为0.056 mg/L(3σ).     

基于银钯铂复合纳米材料标记的癌胚抗原免疫传感器的研制

合成了中空囊状银钯铂复合纳米材料,并以此为标记物,研制了一种标记型癌胚抗原(CEA, Carcinoembryonic Antigen)免疫传感器用于CEA的快速检测.首先利用Au-SH键将CEA抗体(anti-CEA)固定在金电极表面,与CEA抗原免疫结合后,再与银钯铂复合纳米材料标记过的anti-CEA结合,制成夹心型的免疫传感器.考察了免疫传感器的电化学特性,同时研究了培育时间和抗体固定浓度等实验条件对传感器性能的影响.该传感器在CEA抗原质量浓度为0.1～40 ng/mL的范围内有良好的线性关系,检测下限为0.03 ng/mL.实验结果表明: 该免疫传感器操作简便、灵敏度高、选择性好,具有良好的重现性和稳定性.     

基于酶生物催化沉积放大的日本血吸虫压电免疫传感器

提出了一种基于酶生物催化沉积放大的日本血吸虫压电免疫传感器.采用混合自组装单层膜技术在石英晶振金电极表面制备巯基丙酸(MPA)和巯基乙醇(ME)混合功能基底膜,通过偶联剂盐酸1 - 乙基 - 3 - (3 - 二甲基氨基丙基)碳二亚胺(EDC)及N - 羟基琥珀酰亚胺(NHS)活化混合膜上富含的羧基基团用于日本血吸虫抗原(SjAg)的高效共价固定.通过夹心式免疫反应,将辣根过氧化酶(HRP)标记的羊抗兔二抗固定到晶体表面,利用HRP催化H2O2 氧化底物4 - 氯 - 1 - 萘酚在晶振表面形成不溶性沉积物,使免疫检测信号得以显著放大.在优化的实验条件下,该传感器可灵敏检测感染兔血清样中日本血吸虫抗体(SjAb)浓度(稀释比)的线性范围是1∶10 000～1∶100,可望用于日本血吸虫病的早期临床诊断.     

基于甲胎蛋白抗体和巯基丁二酰胺铜(II)共固定修饰电极的人血清中甲胎蛋白免疫传感器

研制基于甲胎蛋白抗体（Ab-AFP）和巯基丁二酰胺铜(II)(CuL)共固定修饰玻碳电极(GCE| Ab-AFP/TiO2/CuL)的免疫传感器,用于测定人血清中AFP抗原水平。该免疫传感器是利用溶胶-凝胶气相沉积技术,将AFP抗体分子固定在CuL修饰玻碳电极表面制备而成。电极表面的CuL具有电活性,对H2O2有良好的电化学还原催化。当该免疫传感器在含AFP样品的溶液中于25℃温育30min后,AFP抗原与Ab-AFP抗体分子的免疫结合物导致CuL的电子传递被部分阻碍,使CuL对H2O2 电催化还原的效率降低,电流值的下降量和AFP浓度成正比,可用于AFP定量测定。在 pH=6 的PBS中, 25℃温育30min的优化条件下,该传感器对AFP的检测线性范围为0.5~3.9和3.9~100ng/mL,检出下限为0.081 ng/mL。该免疫传感器制备过程比基于酶标抗体修饰传感器简单,尤其是对H2O2催化响应迅速,稳定性好,可以在磷酸盐缓冲溶液中稳定存放15 天。对于构建无试剂的免疫传感器和临床免疫分析提供了一种新颖的方法。     

基于胶体金标记银增强的日本血吸虫免疫传感器

 提出一种高灵敏的、基于银增强放大和免疫胶体金技术压电石英晶体免疫传感器。采用夹心法模式,在电极表面固定日本血吸虫抗原,用以捕获血清中的日本血吸虫兔抗血吸虫抗体,再与胶体金标记的羊抗兔IgG二抗形成免疫复合物,加入银增强溶液,通过胶体金催化银离子还原,使大量银沉积在纳米金周围,提高压电石英晶体免疫传感器的灵敏度。结果表明,基于胶体金标记银增强的日本血吸虫压电石英晶体免疫传感器,对兔抗血吸虫抗体具有较高的识别能力,其操作简单、灵敏度高、线性范围宽、检测下限低。将该方法应用于兔血清中日本血吸虫抗体的测定,取得了满意结果。     

基于G-四链体-氯血红素DNA酶的传感器对血清中甲胎蛋白的检测

建立了一种夹心式用于检测血清中甲胎蛋白(alpha-fetoprotein,AFP)的新型传感器。利用微孔板表面羧基与抗体表面的氨基之间的相互作用,将AFP的单克隆抗体(monoclonal antibodies,McAbs)修饰到微孔板的表面;再利用抗体抗原的定向固定效应,将AFP键合到McAbs的表面,接着将生物素标记的AFP单克隆抗体(biotinylated McAbs)与AFP结合;最后,利用链霉亲和素(streptavidin,SA)与生物素的特异性结合作用,将G-四链体-氯血红素DNA酶键合到生物素标记的单克隆抗体上,从而制得高灵度、高特异性的AFP传感器。用2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)-H2O2体系检测AFP的浓度,信号值与AFP的浓度在0.05 ng·mL-1至20 ng·mL-1之间成线性关系,检测限为0.02 ng·mL-1。此传感器可应用于血清中AFP的检测。     

一种适用于教学的酶联免疫吸附试验的抗原抗体系统制备

目的:探讨设计一种教学成本低、便于教学使用、有助于培养医学生综合实验设计能力和提高实验基本操作技能的酶联免疫吸附实验抗原抗体系统.方法:以提纯的人IgG免疫家兔获取兔抗人IgG抗体,再用其包被酶标反应板,以人血清(人IgG)作抗原,用酶标记羊抗人IgG抗体结合抗原及催化底物显色反应.结果:该双抗体夹心法,底物显色反应清楚肉眼即可作出判定,非特异干扰极小.结论:我们设计的酶联免疫吸附实验-双抗体夹心法原理清晰、便于理解和掌握、教学成本较低、结果易于观察、便于教学大量使用.     

基于磁性微球HCG化学发光免疫检测方法的研究

以本实验室制备的表面富含羧基的磁性高分子微球作为免疫检测固相载体,将磁性微球与抗体进行偶联,通过优化偶联条件制备得到免疫磁珠,建立基于磁性微球的化学发光免疫检测方法,用来检测HCG.并研究影响化学发光强度的各项因素,绘制了HCG标准曲线,在HCG抗原浓度为0.5~300IU/L范围内线性关系良好,相关系数R=0.990.     

免疫蛋白分子与纤维膜的相互作用研究——酶斑板的设计原理

本文研究了免疫蛋白大分子(抗体、抗原)与另一些蛋白大分子(不同种类的抗体、抗原)之间以及它们与纤维膜之间的相互作用,也研究了表面改性对这些相互作用的影响,设计并研制出了酶斑板,经多家医院用于临床诊断的结果表明,应用界面化学的原理和技术,设计研制酶斑板是可行的。     

免疫蛋白分子与纤维膜的相互作用研究——酶斑板的设计原理

本文研究了免疫蛋白大分子(抗体、抗原)与另一些蛋白大分子(不同种类的抗体、抗原)之间以及它们与纤维膜之间的相互作用,也研究了表面改性对这些相互作用的影响,设计并研制出了酶斑板,经多家医院用于临床诊断的结果表明,应用界面化学的原理和技术,设计研制酶斑板是可行的.     

一种求得纳滤膜荷电密度的快速方法

基于荷电纳滤膜中同离子浓度显著低于反离子浓度的特点,将Donnan-Steric Pore Model(DSPM)模型中的荷电密度近似表达为膜中反离子浓度与其化合价乘积,进而将其用于DSPM模型的简化,并通过与Spiegler-Kedem方程关联建立了纳滤膜荷电密度的计算方程.采用荷负电的NF270纳滤膜,实验研究了膜对NaCl溶液的截留特性,利用建立的荷电密度计算方程求得了NF270膜在NaCl浓度分别为2,8,21,50mol/m3时的荷电密度,并建立了该膜荷电密度与NaCl浓度间的关系式.将求得的荷电密度应用于DSPM模型,理论计算了膜对NaCl的截留性能,计算结果与实验结果相一致,表明所建立的荷电密度计算方程可作为一种求取纳滤膜荷电密度的快速方法.     

共价固定乙肝表面抗体电容型免疫传感器研究

利用硫脲中的硫原子和金之间强的共价效应,以及氨基与醛基的键合作用,将乙肝表面抗体(HBsAb)牢牢的固定在金电极表面,同时通过十二硫醇封闭电极,从而制得高灵敏、高稳定电容型乙肝表面抗原(HBsAg)免疫传感器。通过交流阻抗技术考察了溶液pH值和离子强度对电极性能的影响。结果表明:该免疫传感器操作简便,易于再生,电容响应与乙肝表面抗原浓度在7.5~750 ng.mL-1范围内呈线性关系,检出限为1.9 ng/mL。     

石墨烯修饰离子液体电极制备癌胚抗原免疫传感器

以离子液体1-丁基-3甲基咪唑六氟磷酸盐为黏合剂制备了碳糊电极.利用石墨烯和金纳米修饰碳糊电极,通过金纳米的吸附能力将癌胚抗体修饰到电极表面制成免疫传感器,用于癌胚抗原的灵敏测定.利用循环伏安法对传感器进行了表征.结果表明,修饰电极显著地促进了电子在电极表面的传递速度,提高了方法的检测灵敏度.在优化条件下,峰电流变化值与癌胚抗原的浓度在0.5～5.0μg/L和5.0～120.0μg/L范围内呈良好的线性关系.信噪比等于3时,癌胚抗原的检出限为0.05μg/L.新方法被应用于人血清样品分析.     

电化学共沉积法制备金-壳聚糖纳米复合膜修饰电极及其在免疫传感器中的应用

采用电化学法,在低电位下壳聚糖与氯金酸混合为电解液,直接共沉积,制备出金-壳聚糖纳米复合膜修饰的电极.实验考查了壳聚糖与氯金酸质量比对金-壳聚糖纳米复合膜形貌的影响、不同沉积时间对其形貌的影响及不同沉积电位对复合膜的活性表面积的影响.并用此复合膜成功固定了人类绒毛膜促性腺激素抗体,研制成无电子媒介的人类绒毛膜促性腺激素免疫传感器.通过电化学交流阻抗、循环伏安法和计时电流法研究免疫传感器的制作过程和电化学特性.考察了电极修饰过程中的抗原、抗体培育时间、pH值、温度等条件对传感器性能的影响.该传感器在人类绒毛膜促性腺激素为0.2～100mIU/mL的范围内有良好的线性关系,检测下限为0.1mIU/mL.     

Fluorescent immunosensor based on fluorescence resonance energy transfer between CdSe/ZnS quantum dots and Au nanorods for PRRSV detection

An ultrasensitive and selective sandwich-type fluorescent immunosensor based on fluorescence resonance energy transfer (FRET) between CdSe/ZnS quantum dots (QDs) and Au nanorods (AuNRs) was developed for the rapid detection of porcine reproductive and respiratory syndrome virus (PRRSV). Modified CdSe/ZnS QDs and AuNRs were bioconjugated with primary antibodies M and GP5, respectively. When the target antigen was present, a well-known sandwich-type form CdSe/ZnS QDs-M antibody-antigen-GP5 antibody-AuNRs was produced, and thus reducing the distance between the QDs and NRs. As a result, FRET occurred and the fluorescence intensity of CdSe/ZnS QDs decreased, and this decrease was used to determine the antigen concentration. Ultrasensitive detection of PRRSV at low concentrations in swine serum was achieved with a limit of detection of 0.55 TCID₅₀/mL and a linear detection range of 10¹–3.5 ?× ?10⁴ TCID₅₀/mL. Therefore, the fluorescent immunosensor is selective and highly sensitive; specifically, it can detect PRRSV at low concentrations in swine serum over a large concentration range. This proposed detection method can facilitate the development of high-performance fluorescent immunosensors for the detection of PRRSV and other antigens.     

Voltage-induced membrane movement

Thermodynamics predicts that transmembrane voltage modulates membrane tension and that this will cause movement. The magnitude and polarity of movement is governed by cell stiffness and surface potentials. Here we confirm these predictions using the atomic force microscope to dynamically follow the movement of voltage-clamped HEK293 cells in different ionic-strength solutions. In normal saline, depolarization caused an outward movement, and at low ionic strength an inward movement. The amplitude was proportional to voltage (about 1 nm per 100 mV) and increased with indentation depth. A simple physical model of the membrane and tip provided an estimate of the external and internal surface charge densities (-5 x 10(-3) C x m(-2) and -18 x 10(-3) C x m(-2), respectively). Salicylate (a negative amphiphile) inhibited electromotility by increasing the external charge density by -15 x 10(-3) C x m(-2). As salicylate blocks electromotility in cochlear outer hair cells at the same concentration, the role of prestin as a motor protein may need to be reassessed.     

Muscarinic modulation of a transient K+ conductance in rat neostriatal neurons

Neurons of the neostriatum are richly innervated by cholinergic neurons of intrinsic origin. Both pre- and post-synaptic muscarinic receptors mediate the effects of acetylcholine (ACh). Activation of these receptors is functionally significant, particularly in Parkinson's disease. Current-clamp studies indicate that muscarinic receptors serve to decrease the responsiveness of neostriatal neurons to excitatory inputs. Here we present evidence that this effect is caused, in part, by the muscarinic modulation of the A-current, a transient outward potassium current. The voltage dependence of this current suggests that normally it enhances spike repolarization and slows discharge rate, but does not affect 'synaptic integration'. We find that under the influence of muscarinic agonists, the voltage dependence of A-current activation and inactivation is shifted towards more negative membrane potentials and the peak conductance is increased. Therefore, at relatively hyperpolarized resting potentials, ACh transiently alters the functional role of the A-current, allowing it to suppress excitatory inputs and further slow the discharge rate. But at relatively depolarized resting potentials, ACh increases excitability by removing the A-current through inactivation.     

X-ray structure of a voltage-dependent K+ channel

Voltage-dependent K+ channels are members of the family of voltage-dependent cation (K+, Na+ and Ca2+) channels that open and allow ion conduction in response to changes in cell membrane voltage. This form of gating underlies the generation of nerve and muscle action potentials, among other processes. Here we present the structure of KvAP, a voltage-dependent K+ channel from Aeropyrum pernix. We have determined a crystal structure of the full-length channel at a resolution of 3.2 A, and of the isolated voltage-sensor domain at 1.9 A, both in complex with monoclonal Fab fragments. The channel contains a central ion-conduction pore surrounded by voltage sensors, which form what we call 'voltage-sensor paddles'-hydrophobic, cationic, helix-turn-helix structures on the channel's outer perimeter. Flexible hinges suggest that the voltage-sensor paddles move in response to membrane voltage changes, carrying their positive charge across the membrane.     

高灵敏电位型风疹疫苗免疫传感器的研究

通过纳米金双层膜将抗风疹血清共价固定在铂电极上,从而制成高灵敏电位型风疹疫苗免疫传感器.通过抗原-抗体反应前后的电位差值考察了电极表面的电化学特性,并对该免疫传感器的性能进行了详细的研究.该传感器对风疹疫苗的检测的线性范围是3.6×10-8～1.0×10-6g/mL,检出限为1.8×10-8g/mL,线性相关系数为0.996 1.