基于微/纳米加工技术的微型电化学免疫传感器研究

生物传感器的研制越来越趋向于微型化、集成化、智能化以及无创伤的方向发展.研制基于微/纳米加工技术的电化学免疫传感器顺应了这一趋势,利用微电子机械系统(MEMS)技术在硅基芯片上制备微型三电极系统和SU-8微型池,并采用自组装单层膜和纳米金修饰微型电极表面用于抗体的固定化,研制出新型的电化学免疫传感器.研究表明,这种微型电化学免疫传感器易于实现批量生产,便于集成,检测过程只需要少量的样品,大大降低有毒试剂的消耗,减少环境污染,同时具有分析成本低,响应时间快,检测下限低和适用于现场快速检测等优点.     

基于对羟基苯硫酚自组膜免疫传感器的研究

利用自组装技术将对羟基苯硫酚(4—HTH)固定在金片电极上,以1—乙基—3—(3—二甲氨基)碳二亚胺盐酸盐(EDC)和N—羟基硫代琥珀亚胺(NHS)作为活化剂,将羊抗—小鼠IgG以共价键形式固定在对羟基苯硫酚自组装单分子膜修饰的金片电极上,制备了一种免疫传感器。根据抗体与抗原之间特异反应前后引起的电极电位变化,对小鼠IgG进行定量测定。利用循环伏安法分别对裸金片电极、对羟基苯硫酚自组装膜、抗体膜、抗原膜进行了表征。结果表明:该免疫传感器灵敏度高、响应速度快、检测范围宽,其检测下限为0.2μg/L,检测范围为0.2~30μg/L,并可以再生、重复利用,寿命可达10 d左右     

Alpha-1-fetoprotein antibody functionalized Au nanoparticles: Catalytic labels for the electrochemical detection of α-1-fetoprotein based on TiO2 nanoparticles synthesized with ionic liquid

A novel strategy for the preparation of amperometric immunosensor for rapid determination of α-1-fetoprotein (AFP) in human serum has been developed. TiO₂ nanoparticles (NPs) were prepared by solvothermal reaction using TiCl₄ as raw materials and the mixture of ionic liquids and doubly distilled water as solvent. α-1-fetoprotein antibody (AFP Ab) was mixed with TiO₂ NPs/chitsotan (CHIT) solution and immobilized onto the surface of a glassy carbon electrode. AFP (Ab) functionalized Au NPs were used as catalytic labels for the amperometric detection of AFP by means of the electrocatalyzed reduction of Au NPs to H₂O₂. The electrochemical behavior of the immunosensor was studied. Other experimental conditions such as pH, immunoreactions temperature and time were also studied. The prepared immunosensor offers an excellent amperometric response for AFP ranging from 1.0 to 160.0 ng/mL with a detection limit of 0.1 ng/mL. The result shows that the immunosensor displays rapid response, high sensitivity, good reproducibility and favorable stability.     

基于抗体包被金磁纳米微粒修饰的磁性安培免疫传感器研制及对人血清癌抗原19-9的检测

在金电极表面修饰壳聚糖(CS)膜,并在酸性pH下利用CS上形成的-NH3+静电吸引Fe(CN)]3-6-(FeCN)到电极表面,制成了在pH 6.5磷酸盐缓冲液(PBS)中具有电活性的Au | CS-FeCN电极;再通过外加磁场吸引,在Au | CS-FeCN表面修饰一层血清癌抗原19-9单克隆抗体(anti CA19-9)包被的金磁纳米微粒(Fe3O4(核)／Au(壳),简称GMP),由此构建了一类快速检测CA19-9的无试剂安培免疫传感器(Au| CS-FeCN／GMP-anti CA19-9).用扫描电镜(SEM),X-射线荧光光谱(XFS)和X-射线光电子能谱(XPS)对电极表面进行了表征;并采用循环伏安法(CV),示差脉冲伏安法(DPV)、交流阻抗法(E1S)分 别研究了该传感器的电化学性质和对CAl9-9的检测性能.实验表明:Au I CS-FeCN／GMP-anti CAl9-9电极在含CAl9-9的pH 6.5 PBS中于35℃下温育25 min,其DPV还原电流下降值(AI)与CA19-9在0.1～10 U／mL和10~50 U／mL范围内成正比,检出下限为0.056 U／mL(3d).用于血清样本检测并和标准ELISA方法对照,结果一致.只需移去外加磁场,用PBS清洗电极表面即可实现电极更新.该磁性免疫传感器集分离、富集和测定于一身,灵敏度高、稳定性好、表面易更新,有望用于人血清中痕量CA19-9的快速检测.     

Ionic liquid and nanogold-modified immunosensing interface for electrochemical immunoassay of hepatitis B surface antigen in human serum

A mediator-free electrochemical immunoassay protocol based on a disposable immunosensor for the detection of hepatitis B surface antigen (HBsAg) in human serum was developed. To fabricate such an immunosensor, a layer of sol–gel composite film containing room temperature ionic liquid and chitosan was initially formed on a glassy carbon electrode. Nanogold particles were then adsorbed onto the membrane via the amine groups of chitosan molecules, and then horseradish peroxidase (HRP)-labeled hepatitis B surface antibodies (HRP-anti-HBs) were immobilized onto the nanogold surface. With a non-competitive immunoassay format, the antibody–antigen complex could be formed by a simple one-step immunoreaction between the immobilized HRP-anti-HBs and HBsAg in sample solution. The formed immunocomplex inhibited partly the active center of the HRP, which decreased the immobilized HRP toward the reduction of H₂O₂. The performance and factors influencing the performance of the immunosensor were evaluated. Under optimal conditions, the current change obtained from the carried HRP relative to H₂O₂ system was proportional to HBsAg concentration in the range of 1.5–400 ng/mL with a detection limit of 0.5 ng/mL (at 3δ). The reproducibility, selectivity, and stability of the proposed immunosensor were acceptable. Moreover, the proposed immunosensors were used to analyze HBsAg in human serum specimens. Analytical results of clinical samples suggested that the developed immunosensor has a promising alternative approach for detecting HBsAg in the clinical diagnosis.     

可再生使用的磁性纳米修饰C反应蛋白电流型免疫传感器

利用Fe3O4(核)/Au(壳)(简称GMPs)标记C反应蛋白酶标抗体(HRP-anti CRP),构建了一类新型的磁性纳米探针(HRP-anti CRP/GMPs),将其修饰在丝网印刷电极(SPCE)表面构建了可再生使用的CRP安培型酶联免疫传感器.首先将多壁碳纳米管(MCNTs) -硫堇(Thi) -Nafion复...     

Electrochemical immunosensor for salbutamol detection based on CS-Fe3O4-PAMAM-GNPs nanocomposites and HRP-MWCNTs-Ab bioconjugates for signal amplification

An ultrasensitive electrochemical immunosensor based on chitosan-iron oxide-poly(amino-amine) dendrimers-gold nanoparticles (CS-Fe₃O₄-PAMAM-GNPs) nanocomposites and horseradish peroxidase-multiwall carbon nanotubes-antibody (HRP-MWCNTs-Ab) bioconjugates was developed for the detection of salbutamol (SAL). CS-Fe₃O₄-PAMAM-GNPs nanocomposites as immobilization matrix were used to enhance the electroactivity and stability of the electrode. HRP-MWCNTs-Ab bioconjugates as label were used to improve catalytic activity for hydrogen reduction of the electrode. Under the optimized conditions, a calibration plot for SAL was obtained with a linear range between 0.11 ng/mL and 1061 ng/mL (r = 0.9984). The detection limit was 0.06 ng/mL. The immunosensor was examined in real samples for the analysis of SAL.     

Highly sensitive label-free immunosensor for ochratoxin A based on functionalized magnetic nanoparticles and EIS/SPR detection

A label-free immunosensor for the detection of ochratoxin A (OTA) based on use of magnetic nanoparticles (MNPs) was developed. A gold electrode was modified using bovine serum albumin conjugate with a glutaraldehyde-thiolamine linker, creating a layer that prevents non-specific binding of OTA on gold. The OTA antibodies were attached to MNPs using the carbodiimide chemistry and afterwards were immobilized on the modified gold electrode using a strong magnetic field. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) were used to characterize each step in immunosensor development. The impedance variation due to the specific antibody-OTA interaction was correlated with the OTA concentration in the samples. The increase in electron-transfer resistance values was proportional to the concentration of OTA on a linear range between 0.01 and 5 ng/mL, with a detection limit of 0.01 ng/mL. SPR measurements showed a larger response range (1-50 ng/mL) with a detection limit of 0.94 ng/mL. Analytical results were in accordance with standard ELISA test kit.     

基于金属—有机骨架材料的C反应蛋白传感器研制

在常温下合成了Au纳米颗粒负载的超纳米结构金属有机骨架化合物(Au NPs/SNHKUST—1),并以其为标记材料标记C反应蛋白抗体.同时合成了中空状石墨烯纳米材料和Au纳米颗粒作为固定基质,制备了夹心型的C反应蛋白免疫传感器.通过检测标记物对H2 O2还原反应的催化电流,实现了对C反应蛋白的定量测定.传感器在C反应蛋白浓度为0.2~200 ng/mL的范围内线性良好的,线性相关系数R2=0.9947,检测下限为0.05 ng/mL,为C反应蛋白测定提供了一种新的简单快速的检测方法.     

检测B型葡萄球菌肠毒素的Love波免疫传感器研究

报道了一种可用于检测B型葡萄球菌肠毒素(SEB)的Love模式声表面波压电免疫传感器。传感器本身由压电石英基片、叉指换能器和S iO2声波导层构成。通过APTES和戊二醛层交联蛋白A,实现抗体分子的定向固定,在Love波传感器的表面构建出抗体分子探针的传感界面。建立了基于网络分析仪的传感器测试系统,对抗体分子固定化过程以及10-6g/mL SEB的响应过程进行了实时监测,相位的偏移随时间表现出明显而规律性的改变,与戊二醛共价交联法进行了对比,并用原子力显微镜(AFM)对蛋白A法固定抗体以及捕获了SEB后的表面进行了成像表征。     

Sensitive electrochemical immunosensor based on enlarged and surface charged gold nanoparticles mediated electron transfer

A simple and sensitive electrochemical immunosensor for the detection of protein biomarker interleukin-6 (IL-6) based on gold nanoparticles (AuNPs) as label was reported. The signal of the immunosensor was originated from enlarged and positively charged AuNPs mediated electron transfer between insulating self-assemble monolayer (SAM) modified electrode and K₃Fe(CN)₆ solution. The gold electrode was first modified with SAM to block the electron transfer between the electrode and K₃Fe(CN)₆ solution. After the preparation of the immunosensor, the AuNPs attached onto electrode was enlarged and positively charged when treated in solution containing HAuCl₄, ascorbic acid and cetyltrimethylammonium bromide (CTAB). The enlarged and positively charged AuNPs then mediated electron transfer between the electrode and K₃Fe(CN)₆ solution, creating redox current that is proportional to the concentration of IL-6 detected. The reported immunosensor has high sensitivity and wide linear range. This novel signal amplification strategy can be applied to the detection of different kinds of protein biomarkers.     

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.     

Shrink-induced ultrasensitive mercury sensor with graphene and gold nanoparticles self-assembly

Here we report an ultrasensitive trace mercury(II) micro sensor based on heat-shrinkable polymer (polyolefins, PO). The layer-by-layer self-assembly (LBL SA) method was employed to modify mixed gold nanoparticle (Au NPs) and graphene solution on a micro gold electrode with PO substrate. The unique wrinkle structure of the electrode surface and superior properties of modification film enhanced the performance of LBL SA graphene–Au NPs shrink sensor greatly in determination of Hg(II) using anodic stripping voltammetry (ASV): compared with a shrink gold electrode without surface modification, the sensitivity was improved for about 3.7 times from 0.197 to 0.721 μA/ppb; compared with a same-sized sensor without surface modification nor shrink, the sensitivity was improved for over 50 times. This sensor’s detection limit of Hg(II) was achieved as 0.931 ppb with a sensitivity of 0.721 μA/ppb. This simple but highly sensitive sensor can be widely used in applications of on-line environmental monitoring of Hg(II).

     

Investigation of anti-stiction coating for ohmic contact MEMS switches with thiophenol and 2-naphthalenethiol self-assembled monolayer

Anti-stiction coating with a conductive self-assembled monolayer (SAM) formed by π-conjugated bonds was investigated for ohmic contact microelectromechanical system (MEMS) switches with low-load contacts. SAMs of thiophenol (C₆H₅SH, TP) or 2-naphthalenethiol (C₁₀H₇SH, 2NT) were coated on Au samples with different surface roughness to investigate the effects of the surface asperities on the adhesion force and contact resistance. The adhesion force was measured using a silicon tipless cantilever in the relative humidity range of 10-85% and the contact resistance was measured in the contact force range of 0-70 μN using a conductive tipless cantilever coated with Au for the SAM coated samples and compared with those for a Au sample surface. The adhesion force measurements indicate that the TP and 2NT coatings can prevent a liquid meniscus from forming on device surfaces due to their hydrophobic character caused by the protruding aromatic group. In addition, it was confirmed that these coatings could reduce van der Waals forces more than Au coating. Contact resistance measurements revealed that an electric current begins to flow with smaller contact force for TP and 2NT coated samples than for Au coated samples. The measured contact resistances of the SAM and Au coated samples were comparable. Based on these results, SAMs of TP and 2NT have excellent potential as anti-stiction coating for MEMS switch contacts.     

Direct-binding quartz crystal microbalance immunosensor to detect carp metallothionein

Metallothionein (MT) is a valuable biomarker against xenobiotic heavy metal contamination in organs and blood of fish. In this study, MT was induced in carp blood by cadmium injection and was purified to homogeneity by DEAE-cellulose ion exchange column chromatography to be used dually as a standard and immunogen. A sensitive detection on carp MT was then followed using a batch-type direct-binding quartz crystal microbalance immunosensor system installed with a well cell. Among antibody immobilization methods tested, two methods that were carried out via a heterobifunctional thiolation cross-linker, and via the cross-linker and protein G showed their respective advantages in simplicity and sensitivity. When analyzed using the immunosensor for carp MT in the concentration range of 50-4000 ng/mL, the limit of detection around 250 ng/mL was obtained. Based on its reasonable sensitivity and real-time presentation for the sensor signal, the immunosensor of this study was presumed as a screening tool to monitor possible heavy metal contamination in a fish farm and imported fish.     

多孔阳极氧化铝的化学修饰及其应用于过氧化氢的测定

多孔阳极氧化铝经化学修饰后吸附细胞色素C,制备了过氧化氢生物传感器电极。多孔阳极氧化铝通过电化学和化学腐蚀阻挡层后,用两步无电沉积方法制备了纳米金修饰的多孔阳极氧化铝电极,再在含有L-半胱氨酸的细胞色素C的溶液中通过吸附制备细胞色素C电极。用循环伏安法和计时电流法测试细胞色素C电极的电化学性能及催化对过氧化氢的还原。结果表明,包覆的细胞色素C电极显示较好的稳定性,在扫描速度为80 mV/s时于-50 mV、-190 mV附近出现一对稳定的氧化还原峰。该电极对过氧化氢具有良好的电催化还原性能,在1.5×10-5 mol/L～4.8×10-4 mol/L浓度范围内,电流与浓度呈良好的线性关系。多孔阳极氧化铝经化学修饰后,可应用于生物传感器。     

日本血吸虫抗体电化学免疫传感器研究

该文研制了一种高灵敏的基于辣根过氧化物酶(HRP)放大的电化学免疫传感器用于日本血吸虫抗体的检测.实验采用夹心法的模式,在电极表面固定了日本血吸虫抗原(SjAg),用以捕获血清中的日本血吸虫抗体(SjAb),然后再与辣根过氧化物酶(HRP)标记的羊抗兔IgG二抗形成免疫复合物.用计时安培法检测HRP酶催化H2O2还原对苯二酚底物产牛的电流,其大小与日本血吸虫抗体(SjAb)浓度在一定范围内成正比.对免疫反应的实验条件如电极表面固定抗原的浓度,HRP酶标二抗的浓度进行了优化.此外,还考察了其它蛋白质对测定结果的影响.结果表明,该方法操作简单,灵敏度高,线性范围宽,检测下限低.     

Conductimetric immunosensor for atrazine detection based on antibodies labelled with gold nanoparticles

A novel conductimetric immunosensor for atrazine detection has been designed and developed. This immunosensor is mainly based on antibodies labelled with gold nanoparticles. Additionally, the immunosensor consists of an array of two coplanar non-passivated interdigitated metallic μ-electrodes (IDμE) and immunoreagents specifically developed to detect this pesticide. The chemical recognition layer was covalent immobilized on the interdigital space. Immunochemical detection of the concentration of atrazine is achieved by a competitive reaction that occurs before the inclusion of the labelled antibodies. It is shown that the gold nanoparticles provide an amplification of the conductive signal and hence makes possible to detect atrazine by means of simple DC measurements.The conductimetric immunosensor and its biofunctionalization steps have been characterized by chemical affinity methods and impedance spectroscopy.This work describes the immunosensor structure, fabrication, physico-chemical and analytical characterization, and the immunosensor response using conductivity measurements. The immunosensor developed detects atrazine with limits of detection in the order of 0.1–1 μg L⁻¹, far below the maximum residue level (MRL) (100 μg L⁻¹) established by European Union (EU) for residues of this herbicide in the wine.Although in this paper the competitive reaction occurs in buffer, an initial study of the wine matrix effect is also described.     

基于薄膜体声波谐振器的免疫球蛋白传感器制备

研制了一种基于薄膜体声波谐振器（ FBAR）的生物免疫球蛋白传感器,该FBAR采用AlN作为压电层,3对Ti/W金属层作为布拉格声学反射层,工作频率为2.047 GHz,回波损耗为－32 dB。利用自组装膜法修饰顶部金电极敏感区域。测试了免疫球蛋白G抗体和抗原的特异性结合前后传感器的指标变化。结果得到传感器的Q值和灵敏度分别达到846,3.38 kHz·cm2/ng,远高于广泛使用的石英晶体微天平（ QCM）,具有广阔的应用前景。     

利用喷墨打印技术制备的类神经气体传感器

利用喷墨打印技术沉积生物高分子溶液的方法,打印出金的梳状微电极的阵列图形,并采用NaX型沸石分子筛作为敏感膜,研制了探测神经类毒气沙林的相似物DMMP气体的阻抗型传感器。电极图形使用简单的绘图软件autoCAD画出,通过简单改进过的办公用喷墨打印机在金衬底上打印一层自组装膜的阻挡层,经过湿法刻蚀后得到了梳状微电极阵列。将制得的传感器对1ppm(即1×10-6)DMMP气体进行检测,测得在0.01Hz处,其电阻的相对变化值为10.7%。与传统MEMS工艺相比较,喷墨打印方法制备传感器具有工艺步骤简化,成本低,可在柔性等不同材料上制作等优点,有着广泛的应用前景。