基于巯基化合物和纳米金自组装技术固定化方法的研究

该文提出了一种压电免疫传感器新的蛋白的固定化方法.先在压电石英晶体电极表面自组装半胱氨酸,再通过纳米金与氨基的共价键合作用,在巯基自组装膜的氨基表面形成一均匀的纳米金单层膜,实现蛋白质分子(抗IgG)在传感器表面的固定.探讨了半胱氨酸自组装及IgG抗体固定等一系列实验条件及参数对传感器性能的影响.传感器的频率响应和IgG的浓度在0.33～98.91μg/mL范围内具有良好的线性关系.在0.5 mol/LNaCl+0.01 mol/L NaOH溶液中,蛋白质层可容易地被洗脱,使免疫传感器获得再生.     

基于电聚合硫堇膜与纳米复合材料界面的直接电化学免疫传感器的构建

本工作采用电聚合手段在玻碳电极表面制备聚硫堇基质膜,借硫堇膜上富含的氨基基团实现对碳纳米管与纳米金颗粒二元纳米复合材料的化学组装,制备稳定的碳纳米管/纳米金/聚硫堇传感界面, 以此传感界面固定免疫活性物质,发展了一种无电子媒介的酶免疫传感器.结果表明,电极采用碳纳米管/纳米金颗粒界面固定免疫活性物质,其传感响应性能明显优于单独采用碳纳米管的情况.以人IgG免疫体系为例,电极以此界面固定人IgG抗体,并通过夹心方式直接响应酶催化H2O2的还原电流信号,实现了对人IgG浓度的灵敏测定.此外,采用甘氨酸-HCl缓冲液洗涤使用后的传感器,可有效洗脱其表面的免疫复合物,实现传感器的重复使用.     

丙烯腈-丙烯酸共聚物多孔膜的硫化物传感器

采用自由基聚合方法在金电极表面合成丙烯腈-丙烯酸的共聚物多孔膜并用作固定酶的载体.再通过戊二醛将辣根过氧化物酶交联固定在丙烯腈-丙烯酸的共聚物多孔膜上制得过氧化氢生物传感器.硫化物抑制酶的活性使还原电流减小,根据电流域小的程度实现了对硫化物的测定.丙烯腈-丙烯酸共聚物多孔膜具有大的表面积和高的吸附能力.有利于改进传感器的检测下限.实验结果表明:将辣根过氧化物酶固定在丙烯腈-丙烯酸的共聚物多孔膜金电极上,作为一种检测硫化物的抑制型传感器,分析响应性能良好.     

基于蛋白膜伏安技术的第三代生物传感器

蛋白膜伏安技术,就是使用伏安法研究固定在电极表面的一层或多层蛋白质分子形成的膜.蛋白质在生物体内广泛存在于荷电界面上,如生物膜就是一种荷电界面,电极作为一种研究荷电界面的模型系统有助于人们深入了解蛋白质在生物膜中发生电子传递的分子机制.因此,蛋白膜伏安法对于研究蛋白质(酶)电子传递的机制,揭示相关的生物学过程具有独特的优势.     

IgG蛋白镶嵌L-B膜的特性研究

通过蛋白质镶嵌技术，可以有控制地将具有特殊功能的IgG蛋白嵌入框架单分子膜中，并沉积到光学器件表面，以形成生物传感器的表面敏感器。本文通过对表面压的测定，研究了亚相pH值，IgG蛋白的浓度变化对亚相表面磷脂分子与蛋白相互作用的影响；通过石英表面的荧光发射，测量了固相蛋白混合膜中吸附与穿插蛋白质的相对含量及稳定性。     

碳纳米管组装电化学免疫传感器测定IgG抗体的研究

应用吸附法将IgG抗原固定于多壁碳纳米管修饰的玻碳电极表面,制备用于IgG抗体检测的电化学免疫传感器。以辣根过氧化物酶为标记物,对苯二酚为底物,利用辣根过氧化物酶标记IgG抗体与待测IgG抗体竞争电极表面固定的IgG抗原,建立了免疫竞争法检测IgG抗体的高灵敏度电化学分析方法。碳纳米管的大比表面积和电化学催化作用,提高了分子识别物质的固定量和电化学检测的灵敏度。工作电位为 0.030 V(vs.SCE)时,响应电流与IgG抗体浓度在0.30~10μg/mL范围内呈良好的线性关系,检出限为0.11μg/mL。     

结合PAH聚电解质和纳米金界面的压电免疫传感器研究

提出了一种聚丙烯氯化铵(PAH)-纳米金固定抗体的压电免疫传感器界面的构建方法.先在压电石英晶振的金电极表面自组装一层半胱氨酸单层膜,通过戊二醛交联带大量NH2基的聚电解质PAH,随后在PAH膜表面自组装一层纳米金粒子,以静电吸附作用固定IgG抗体,研制成一种新的压电免疫传感器的界面,用于对相应抗原的检测.研究了PAH浓度及抗体固定化等实验条件的影响,探讨了传感器的主要响应特性与再生性能,并与戊二醛直接固定的传感器的性能进行了比较.结果表明,前者固定的抗体的活性较高,响应频率较大,检测的线性范围较宽,非特异性吸附小,能有效地改善传感器的灵敏度和检测限,而且容易进行传感器的再生.     

自组装纳米金内毒素压电传感器的研究

利用纳米金颗粒自组装于金片表面上,用于固定多粘菌素B(PMB),研制了一种内毒素压电传感器。先在石英晶体电极表面自组装1,6—己二硫醇,再通过纳米金与巯基的共价键合作用,在巯基自组装膜的另一端的巯基表面形成一均匀的纳米金单层膜,实现PMB在传感器表面的固定。实验探讨了影响纳米金自组装和PMB固定等主要实验参数和条件;考核了采用此固定化方法传感器的响应性能。结果表明:与单纯采用自组装方法比,传感器在灵敏度和分辨力方面均有较大的改善。     

一种基于纳米金／石墨烯／普鲁士蓝（PB）修饰玻碳电极非标记免疫传感器

采用电聚合的方法将普鲁士蓝（PBl聚合在玻碳电极表面,再将石墨烯修饰在PB上面,然后再采用电沉积的方法将HAuCL直接还原成纳米金粒子,沉积在石墨烯表面,最后将羊抗人IgG抗体直接固定于该修饰的玻碳电极表面,制备了用于人IgG抗原检测的非标记电化学免疫传感器。利用循环伏安法和交流阻抗研究了修饰电极表面的电化学特性,用差分脉冲伏安法对人IgG抗原进行了测定。实验表明,此免疫传感器在含不同浓度人IgG的PBS溶液（pH6．98）中测定,响应电流与人IgG浓度在5．55～455．5ng／mL范围内有良好的线性关系,其相关系数r=0．9926,检测限为0．015ng／mL（S／N=3）。该免疫传感器具有制备简单、响应时间快（5min）、稳定性好等特点。     

电化学极化玻碳电极构建的葡萄糖生物传感器

在硝酸溶液中电化学极化处理玻碳电极(GCE),以硫堇(TH)为电子介体,通过金-硫、金-氮共价键作用和静电吸附作用将纳米金(GNPs)、葡萄糖氧化酶(GOD)和辣根过氧化物酶(HRP)修饰于电极上,最后在电极上滴涂Nafion水溶液制备抗干扰膜并固定酶电极,构建了一种新型双酶葡萄糖生物传感器.实验结果表明:电化学极化处...     

溅射二氧化钛电极对过氧化氢电化学检测的研究

介绍一种使用微电子技术制造的二氧化钛电极,并且研究了该电极在磷酸缓冲液中的电化学特性。循环伏安法测量的结果显示,过氧化氢和氧气都能在该电极上还原,而且过氧化氢的还原更为明显。在磷酸盐缓冲液中,以Ag/AgCl为参考电极,当所施加电压为-300V时,安培法显示二氧化钛电极对过氧化氢有很快的响应速度,其对于0.1mmol/L过氧化氢的响应电流是0.4μA,而由于溶解氧所带来的背景还原电流仅为14nA。为了研究这种电极在生物传感器中的应用可能性,实验中把葡萄糖氧化酶固定在钛氧化物电极薄膜上并测试了相应特性,发现存在的主要问题为酶在氧化钛表面的固定较为困难。     

A reusable capacitive immunosensor with a novel immobilization procedure based on 1,6-hexanedithiol and nano-Au self-assembled layers

A novel immobilization procedure of antibody proteins for capacitive immunosensing, based on thiolor sulfur compound (1,6-hexanedithiol, HDT) and colloid Au layers is proposed. The insulating organic monolayer film was first formed by the spontaneous assembly of HDT from solution onto gold. When these thiol-rich surfaces are exposed to Au colloid, the sulfurs form strong bonds to gold nanoparticles, anchoring the clusters to the electrode substrate. After the assembly of gold nanoparticles layer, the original formed organic thiols surface was restored, and a new nano-Au surface was obtained. Thus, the antibody could be immobilized through electrostatic adsorption between nano-Au and the antibody proteins. After use, the formed immunocomplex layer can be rinsed out, via a saline solution with extreme pH. Therefore, the immunosensor can be regenerated repeatedly, highlighting a clear advantage of this new approach with respect to classical immunoassays employing covalent immobilization.     

A label-free amperometric immunosensor based on horseradish peroxidase functionalized carbon nanotubes and bilayer gold nanoparticles

In this work, a novel label-free amperometric immunosensor has been constructed for detecting α-1-fetoprotein (AFP) based on nanocomposite of horseradish peroxidase (HRP) labeled carbon nanotubes (CNTs). First, the gold nanoparticles (AuNPs) were electrodeposited on the surface of the glass carbon electrode by electrochemical reduction of gold chloride tetrahydrate (HAuCl₄) to immobilize horseradish peroxidase labeled carbon nanotubes (HRP-CNTs). Then HRP-CNTs bioconjugate was immobilized on the surface of the electrodeposited AuNPs layer by the combination of forces (coordination and electrostatic force). Subsequently, it was immersed into gold colloidal nanoparticles (GNPs) solution, which was used to immobilize antibody biomolecules (anti-AFP). Enhanced sensitivity was obtained by using bioconjugates featuring HRP labeled (HRP-CNTs), which had lager specific surface area and good electronic catalysis (current response signal) compared to carbon nanotubes. Under optimized conditions, the linear ranges were from 0.2 to 200 ng mL⁻¹ with a detection limit of 0.067 ng mL⁻¹ (at an S/N of 3). The proposed immunosenor showed good precision, acceptable stability and reproducibility and could be used for the detection AFP in normal human serum, which provided a potential alternative tool for the detection of protein in clinical diagnosis.     

Development of a screen-printed cholesterol biosensor: Comparing the performance of gold and platinum as the working electrode material and fabrication using a self-assembly approach

Gold (Au) and platinum (Pt) were used as the working electrode material to detect cholesterol in solution through enzymatically generated hydrogen peroxide (H₂O₂). Both gold and platinum were capable of detecting cholesterol through the electrochemical oxidation of H₂O₂, and could be used as the working electrode material. By comparison, however, Au was preferable over Pt in terms of higher response current and better sensitivity. Therefore, Au was chosen as the working electrode material for the fabrication of a thick-film screen-printed cholesterol biosensor consisting of three electrodes on an alumina substrate (working: Au, reference: Ag/AgCl, and counter: Au). The immobilization of the enzyme cholesterol oxidase (ChOx, E.C. 1.1.3.6) on the Au working electrode was achieved using a self-assembly approach. A thiol, 3-mercaptopropionic acid (MPA), was self-assembled onto the gold working electrode forming a thin organic layer that served as the anchor for the enzyme immobilization. 1-Ethyl-3(3-dimethylamino propyl)carbodiimide methiodide (EDC) was then used to immobilize the enzyme ChOx covalently on the gold working electrode through the carbodiimide coupling between the carboxyl (–COOH) groups of the self-assembled MPA layer and the amino (–NH₂) groups of the enzyme. Electrochemical measurements showed that this biosensor responded well to cholesterol, confirming that the self-assembly immobilization method was effective. The reproducibility, the interference, and the storage stability of the biosensor were studied and assessed.     

基于蛋白A-纳米金-丝网印刷电极的雌二醇免疫传感器

将蛋白A固定在纳米金修饰的丝网印刷电极表面,纳米金大的比表面积以及较高的表面自由能使蛋白A比较牢固地固定在其上;蛋白A分子可以定向结合17β-雌二醇抗体的Fc片段,使抗体在电极表面有序固定化。利用17β-雌二醇和酶标17β-雌二醇的酶免疫竞争反应,研制了快速测定17β-雌二醇的电化学传感器。用循环伏安法和扫描电子显微镜对修饰过程进行了表征。优化测定条件后,传感器呈现了很高的测定灵敏度,电流响应信号与17β-雌二醇浓度在0.1μg/L～20μg/L范围内呈良好的负线性相关,检出限为0.035μg/L(S/N=3)。应用该免疫传感器测定了正常人群尿样中17β-雌二醇含量,测定范围与报道的高效液相色谱法测定结果相一致,样品加标回收率为96%～114%。     

Hybrid phenol biosensor based on modified phenoloxidase electrode

Electrolytic deposition has been widely used to immobilize biomacromolecules, and it is always the most important factor to preserve or even increase an activity of the immobilized protein. We report here simple and rather universal method for the highly efficient immobilization of laccase for amperometric biosensing. Laccase from Cerrena unicolor has been successfully immobilized (electrolytic deposition) on the surface of thin, ordered polythiophene films (3-methylthiophene/3-thiopheneacetic acid/N-heptyl-3,6-bis(2-thiophene)carbazole). Two different compounds capable of mediating laccase-catalyzed reactions have been tested by cyclic voltammetry. They exhibited quasi-reversible electrodic behaviour with formal redox potentials ranging from 68 and 918 mV (E⁰vs. SCE). The immersion of the laccase-coated electrode in solution with substrate generated large catalytic currents easily recorded by cyclic voltammetry at low potential scan rates. Considering the fact, that immobilization strategy showed high efficiency, obtained results suggest that method for phenoloxidase immobilization has a great potential of enabling high throughput fabrication of bioelectronics’ devices.     

Direct electron transfer of glucose oxidase at glassy carbon electrode modified with functionalized carbon nanotubes within a dihexadecylphosphate film

A glassy carbon electrode modified with functionalized multiwalled carbon nanotubes (CNTs) immobilized by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) in a dihexadecylphosphate film was prepared and characterized by cyclic voltammetry and scanning electron microscopy. It was used as a support for FAD or glucose oxidase (GOx) immobilization with EDC/NHS crosslinking agents. Cyclic voltammetry of GOx immobilized onto the surface of CNTs showed a pair of well-defined redox peaks, which correspond to the direct electron transfer of GOx, with a formal potential of −0.418 V vs. Ag/AgCl (3 M KCl) in 0.1 M phosphate buffer solution (pH 7.0). An apparent heterogeneous electron transfer rate constant of 1.69 s⁻¹ was obtained. The dependence of half wave potential on pH indicated that the direct electron transfer reaction of GOx involves a two-electron, two-proton transfer. The determination of glucose was carried out by square wave voltammetry and the developed biosensor showed good reproducibility and stability. The proposed method could be easily extended to immobilize and evaluate the direct electron transfer of other redox enzymes or proteins.     

基于二氧化锆纳米粒子固定乙酰胆碱酯酶的甲基对硫磷传感器

先在金电极表面电沉积二氧化锆纳米粒子并固定乙酰胆碱酯酶(AChE),将此电极浸入含有不同浓度的有机磷溶液中,根据电极在底物氯化乙酰巯基胆碱中电化学信号强度的大小来实现溶液中有机磷的定量检测.以甲基对硫磷为分析目标物,研究了传感器的主要响应特性、选择性及再生性能,考察了底物浓度、工作电位及溶液pH值对分析性能的影响.结果表明,该有机磷传感器在5.0×10-7～5.0×10-4 g/L浓度范围内对目标分析物有线性响应,检出限为1.0×10-7 g/L.该传感器灵敏度高,非特异性吸附小,再生性好,所用的二氧化锆纳米粒子层制备简单、操作方便,具有较大的应用潜力.     

Characterization of a novel antibody immobilization combining protein G with parylene-H for surface plasmon resonance immunosensors

Here we present a method for selectively and efficiently immobilizing antibodies to enhance the detection performance of surface plasmon resonance immune-sensors (SPRIs) for diagnostic applications. To improve the performance of antibody arrays, protein G was used as antibody-selective linkage layer with aldehyde functionalized poly-(para-xylylene) film. To estimate the efficiency of antibody immobilization, immunoglobulin G (IgG) was measured using the anti-IgG immobilized SPRIs. To demonstrate the proof-of-concept validation, the signal detected from the IgG using parylene-H film was compared with that of a combination of parylene-H and protein G in SPRIs. The results showed that the detection of IgG on the immobilized anti-IgG layer using the combination of parylene-H and protein G has a larger change of signal than that of using parylene-H layer. These results also imply that the anti-IgG was densely and efficiently immobilized on the modified surface with the linkage layer in a combination with parylene-H and protein G. Therefore, we believe that this combinatorial approach could selectively immobilize the antibodies, and also be applied for detection and diagnosis of immune diseases in the field of many SPRIs applications.     

Development of enzyme immobilized monolith micro-reactors integrated with microfluidic electrochemical cell for the evaluation of enzyme kinetics

This paper describes a simple and efficient method for producing an on-chip enzyme immobilized monolith micro-reactor that integrates a microfluidic electrochemical cell for rapid characterization of enzymatic kinetics. The monolith was generated using a sol–gel method, followed by PEI functionalization and enzyme immobilization via electrostatic attraction between electronegative enzymes and electropositive PEI polymers. Using the proposed immobilization strategy, a glucose oxidase (GOD) immobilized monolith micro-reactor has been produced with the controllable porosity that gives better enzyme kinetics compared to previously reported devices. This can be attributed to a favourable enzyme-substrate affinity in which more than 98% of the immobilized enzyme remains in an active conformation. The kinetic studies conducted have identified that a similar value of the k _cat is obtained for immobilized GOD (13.4 s⁻¹) and GOD free in solution (14 s⁻¹) whilst the immobilized Michaelis constant K _m(app) (7.2 mM) is ~4 times lower than GOD in solution (25 mM). In addition, the immobilized GOD exhibits increased stability, retaining at least 95% of the initial activity when stored of 30 days at 4°C, compared to only 60% for GOD in solution. Furthermore, the same enzyme immobilization strategy has been used for choline oxidase immobilization and similar kinetics to choline oxidase in solution were observed, once again indicating better maintenance of the enzyme conformation provided by the proposed method.