高灵敏甲胎蛋白夹心免疫传感器的制备

构建了新型甲胎蛋白(AFP)夹心免疫传感器.采用金纳米粒子-氧化石墨烯-普鲁士蓝纳米立方体(AuNP-GO-PBNCs)纳米复合材料标记甲胎蛋白(AFP)二抗,将制备的金-聚多巴胺-四氧化三铁(Au-PDA-Fe3O4)磁性纳米复合物固定在自制的磁性电极表面,通过吸附作用固定AFP一抗,用牛血清白蛋白(BSA)封闭电极上的非特异性吸附位点.在37℃下与AFP抗原溶液孵育50 min,最后将电极放入AuNP-GO-PBNCs纳米复合材料标记的二抗溶液中孵育,基于此建立了采用普鲁士蓝(PB)标记的的夹心免疫传感器检测AFP的方法.在最佳实验条件下,PB催化H2O2氧化的响应电流与AFP的浓度表现出两段线性关系,线性范围分别为0.005~1.000 ng/mL和1~20 ng/mL, 检出限(LOD, S/N=3)为1.0 pg/mL.本方法具有灵敏度高、选择性好的特点.     

基于碳纳米管固定抗原的电化学免疫传感器法测定IgG抗体

以1-萘酚磷酸酯为底物,将IgG抗原吸附固定在碳纳米管修饰玻碳电极的表面,利用待测IgG抗体和碱性磷酸酯酶标记IgG抗体共同竞争免疫传感器表面的IgG抗原,研制了一种新型的电化学免疫传感器用于IgG抗体的测定。碱性磷酸酯酶可以催化底物1-萘酚磷酸酯水解生成1-萘酚,在电极表面氧化产生电信号。在电位为+0.30 V(vs.SCE)时,响应电流与IgG抗体浓度在5.0×10-9～5.0×10-7g/mL范围内呈良好的线性关系,检出限为2.0×10-9g/mL。     

基于普鲁士蓝-碳纳米管-纳米金复合材料的电化学免疫传感器的构建

构建一个基于普鲁士蓝–碳纳米管–纳米金复合物(PB–CNTs–CNPs)增效的新型免疫传感器检测大肠杆菌。普鲁士蓝–碳纳米管–纳米金复合物能够增强电子的传递效率和电极的稳定性。当大肠杆菌抗存在时,辣根过氧化氢酶(HRP)标记的大肠杆菌抗体也通过特异性作用结合到PB–CNTs–CNPs修饰的金电极表面,形成一个夹心型结构。通过大肠杆菌抗体上标记的HRP酶催化底夜中双氧水的还原对大肠杆菌进行定量。该传感器具有很好的特异性、重现性和稳定性。在最优条件下,大肠杆菌浓度在10~1×107 cfu/mL的范围内与该传感器的电流响应I存在I=33.68 lg C_(E.coli)+7.19的线性关系,检出限为9.2 cfu/mL(S/N=3)。将该传感器应用于实际样品中大肠杆菌的检测,样品加标回收率为91.3%~103.0%,与平板计数法的实验结果相比较,结果具有高度一致性。     

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%。     

基于金微粒修饰的聚苯胺乙脑疫苗传感器的研究

采用聚合物掺杂法将金微粒固定在聚苯胺膜中,再通过静电吸附固定抗体,制成非标记电流型乙脑疫苗传感器。根据抗原抗体特异性结合形成的免疫复合物使电极敏感膜有效扩散面积减小,电流减少的特性,提出以嵌入金微粒的聚苯胺膜作为电子媒介体,用0.1mol/L磷酸盐缓冲液(pH7.4)为测试底液检测乙脑疫苗效价的新方法。用该传感器检测乙脑疫苗效价,线性范围6.1×10-8～4.1×10-6lgpfu/mL,相关系数r=0.994,检测限为3.0×10-8lgpfu/mL(S/N=3)。该传感器可用于生物制品的测定。     

石墨烯-壳聚糖-纳米金修饰的免疫传感器应用于1-芘丁酸的检测研究

本文采用石墨烯(GS)-壳聚糖(CS)-纳米金(Nano-Au)复合材料修饰玻碳电极,构建性能良好的生物识别界面,制备一种无标记的电流型免疫传感器,并应用于1-芘丁酸(PBA)的高灵敏检测。研究结果表明,GS、CS和Nano-Au的协同作用,极大地提高了anti-PAHs抗体在电极表面的覆盖量,从而提高了免疫传感器的灵敏度和检测性能。采用示差脉冲伏安法(DPV)对PBA进行检测,PBA在0.001~10ng/mL和10~200ng/mL浓度范围内与峰电流值呈良好的线性关系,检出限为0.001ng/mL。该传感器应用于实际水样中痕量PBA的检测,加标回收率为90%~105%。     

基于纳米四氧化三铁-二茂铁构建甲胎蛋白免疫传感器的研究

用对交联剂1-乙基-3-(3-二甲基氨苪基)-碳化二亚胺/N-羟基琥珀酰亚胺将壳聚糖中的氨基与二茂铁甲酸中的羧基交联,并将制备好的纳米四氧化三铁加入其中反应形成富含氨基和羧基的磁性纳米复合物。该纳米复合物修饰到金电极表面,通过氨基及静电作用,吸附纳米金溶胶及甲胎蛋白抗体(anti-AFP)含量,从而成功制得高灵敏电流型甲胎蛋白免疫传感器。通过循环伏安法考察了电极表面的电化学特性。研究了孵育时间、温度、pH对免疫传感器的影响。在最优实验条件下,甲胎蛋白(AFP)质量浓度在0.01~10 ng/mL和10~120 ng/mL范围内,该免疫传感器的还原峰电流值与AFP的质量浓度有良好的线性关系,检出限为3 pg/mL。     

Nano-ZnO微叉指电极生物传感器检测大肠杆菌

设计了一种基于纳米ZnO材料检测大肠杆菌(E.coli O157:H7)的微叉指阻抗生物传感器,利用电化学方法在氧化铟锡(ITO)叉指电极表面沉积上纳米ZnO,然后将链霉亲和素固定在纳米ZnO表面,利用生物素亲和素的高亲和性原理将大肠杆菌抗体绑定在传感器表面,完成传感器的构建。实验表明,传感器检测E.coli O157:H7线性范围为40~4×10^6cfu/mL,检出限为40 cfu/mL,传感器的特异性、重现性、实用性较好。     

非标记型河虾免疫传感器的制备及应用

四乙氧基硅在HCl催化下水解形成硅溶胶。将硅溶胶与河虾抗体混合均匀后,涂于玻碳电极表面制备得非标记型河虾抗体免疫传感器。采用Fe(CN)63-/4-的磷酸盐缓冲溶液(pH 6.5)为测试底液,研究此传感器在免疫反应中的循环伏安和交流阻抗特性。结果显示免疫反应后传感器的循环伏安图上未出现新的氧化-还原峰,表明该免疫反应属于非氧化还原过程。采用交流阻抗监测电极表面,发现传感器阻抗值随虾过敏原浓度的增加而增大。对交流阻抗图进行分析,结果表明电极表面的免疫反应是一个受电子转移控制的过程。当虾过敏原浓度在0~10 ng/mL之间,电极表面电子转移阻抗的增加值与虾过敏原浓度呈线性关系,检出限为0.1 ng/mL(S/N=3)。该方法已成功应用于河虾制品中过敏原的测定。     

基于立方体纳米氧化亚铜修饰的安培型葡萄糖生物传感器的制备及性能研究

将合成的立方体纳米氧化亚铜用于修饰玻碳电极,在其上固定葡萄糖氧化酶,构建了高灵敏的安培型葡萄糖生物传感器.采用X射线衍射(X RD)、扫描电镜(SEM)对合成的立方体纳米氧化亚铜及其修饰电极进行了表征.结果表明,合成的纳米氧化亚铜为均匀的立方体形状.采用循环伏安法(CV)、交流阻抗谱(EIS)、差分脉冲伏安法(DPV)及计时电流法(CA)考察了修饰电极的电化学行为.在含0.1 mmol/L葡萄糖的磷酸盐缓冲溶液(pH 7.4)中研究了立方体纳米氧化亚铜修饰电极的循环伏安(CV)响应,实验结果表明,此修饰电极对葡萄糖显示出良好的电催化性能.DPV响应电流与葡萄糖的浓度在5.0×10-6 ～4.0× 10-3mol/L范围内呈良好的线性关系,线性相关系数R2=0.9983,检出限为6.8×10-7 mol/L(S/N=3).CA实验结果表明,尿酸、抗坏血酸、D-果糖对传感器不产生干扰.本传感器具有较好的重现性和稳定性,可用于实际样品中葡萄糖的检测.     

One‐step Synthesis of AuAg Alloy Nanodots and its Electrochemical Studies towards Nitrobenzene Reduction and Sensing

Efficient, stable, and low‐cost electrocatalysts for the degradation and sensing of environment pollutants are essential components of clean environment monitoring. Here we report, one‐step synthesis and characterization of 1–3 nm diameter sized bi‐metallic AuAg nanodots (NDs) embedded in amine functionalized silicate sol‐gel matrix (SSG) and its electrochemical studies toward nitrobenzene. The SSG was used as a reducing agent as well as stabilizer for the prepared mono‐ and bi‐metallic nanoparticles (NPs). From the HRTEM, STEM‐EDS and XPS analyses, the bi‐metallic AuAg NDs were identified as an alloy and not the mixtures of Au and Ag NPs. Characteristic surface plasmon resonance (SPR) band between the Au and Ag NPs SPR absorption region was noticed for the prepared AuAg NDs. The AuAg alloy NDs with different concentrations of Au and Ag (Au₂₅Ag₇₅, Au₅₀Ag₅₀ and Au₇₅Ag₂₅ NDs) modified electrodes exhibited synergistic electrocatalytic effect than did the Au and Ag NPs towards nitrobenzene reduction and detection. Together with ultra‐small size and exceptional colloidal stability features within these SSG‐AuAg NDs pave convenient way for nanotechnology‐based catalysts development and sensor applications.     

A competitive microcystin-LR immunosensor based on Au NPs@metal-organic framework (MIL-101)

A porous metal organic frameworks (MOFs) material (MIL-101) based on trivalent chromium skeleton were synthesized by hydrothermal synthesis method, and loaded with Au nanoparticles (Au NPs) to prepare Au NPs@MIL-101 composite materials which were used as a marker to label anti microcystin-LR (Anti-MC-LR). The composite materials have strong catalytic properties to the oxidation of ascorbic acid. Anti-MC-LR was immobilized on glassy carbon electrode surface using electrodeposition graphene oxide (GO) as a fixed matrix to construct a competitive microcystin-LR immunosensor.     

An ultrasensitive electrochemical immunosensor for CA72-4 based on a signal amplification strategy of MoS2 nanoflower-supported Au nanoparticles

Accurate detection of cancer antigen 72-4 (CA72-4), a tumor-associated glycoprotein, is of great significance for gastric cancer diagnosis and immunotherapy monitoring. Modification of noble metal nanoparticles on transition metal dichalcogenides can significantly enhance functions, such as electron transport. Molybdenum disulfide gold nanoparticles nanocomposites (MoS₂-Au NPs) were prepared in this study and a series of characterization studies were carried out. In addition, a label-free, highly sensitive electrochemical immunosensor molybdenum disulfide -Au nanoparticles/Glassy carbon electrode (MoS₂-Au NPs/GCE) was also prepared and used for the detection of CA72-4. The electrochemical performance of the immunosensor was characterized by electrochemical techniques, such as cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). The results indicated that better MoS₂-Au NPs nanomaterials have been synthesized, and the prepared electrochemical immunosensor, MoS₂-Au NPs/GCE, showed excellent electrochemical performance. The sensor exhibited high detection sensitivity under optimal conditions, including an incubation time of 30 min, an incubation temperature of 25 °C, and a pH of 7.0. The electrochemical immunosensor also had a low detection limit of 2.0 × 10^?5 U/mL (S/N = 3) in a concentration range of 0.001–200 U/mL, with good selectivity, stability, and repeatability. In conclusion, this study provided a theoretical basis for the highly sensitive detection of tumor markers in clinical biological samples.     

Ultrasensitive electrochemical immunosensor of carcinoembryonic antigen based on gold-label silver-stain signal amplification

A novel gold-label silver-stain electrochemical immunosensor based on polythionine-gold nanoparticles (PTh-Au NPs) modified glassy carbon electrode (GCE) as a platform and secondary antibody labeled Au NPs (Ab₂-Au NPs) as immumoprobe for carcinoembryonic antigen (CEA) detection. The sandwich-type biosensor adopted anodic stripping voltammetry to detect silver stripping signal when the Ab₂-Au NPs of the formed immunocomplexes were stained with silver.     

AuAg bimetallic nanoparticles film fabricated based on H2O2-mediated silver reduction and its application

A method to fabricate AuAg bimetallic nanoparticles film by H₂O₂-mediated reduction of silver was reported. Gold nanoparticles (Au NPs) were first adsorbed onto the surface of a self-assembled 2-aminoethanethiol monolayer-modified gold film or 3-aminopropyltriethoxysilane (APTES) monolayer-modified quartz slide. Upon further treatment of this modified film with the solution containing silver nitrate (AgNO₃) and H₂O₂, silver was deposited on the surface of Au NPs. The size of the AuAg bimetallic particles could be readily tuned by manipulating the concentration of H₂O₂. Surface plasmon resonance (SPR) was used to investigate the process, the deposition of silver on Au NPs modified gold film resulted in an obvious decrease of depth in the SPR reflectance intensity and minimum angle curves (SPR R-θ curves), which may be utilized for the quantitative SPR detection of the analyte, H₂O₂. Combination of the biocatalytic reaction that could yield H₂O₂ by using the enzyme, glucose oxidase, with the deposition of silver may enable the design of a glucose biosensor by SPR technique. Furthermore, we evaluated the AuAg bimetallic nanoparticles film for their ability to be an effective substrate for surface-enhanced Raman scattering (SERS).     

Dual Amplified Electrochemical Immunosensor for Hepatitis B Virus Surface Antigen Detection Using Hemin/G‐Quadruplex Immobilized onto Fe3O4‐AuNPs or (Hemin‐Amino‐rGO‐Au) Nanohybrid

A sensitive electrochemical immunosensor for Hepatitis B virus surface antigen (HBsAg) detection was fabricated based on hemin/G‐quadruplex interlaced onto Fe₃O₄‐AuNPs or hemin ‐amino‐reduced graphene oxide nanocomposite (H‐amino‐rGO‐Au). G‐quadruplex DNAzyme, which is composed of hemin and guanine‐rich nucleic acid, is an effective signal amplified tool for its outstanding peroxidase activity and Fe₃O₄‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites with quasi‐enzyme activity provide appropriate support for the immobilization of hemin/G‐quadruplex. The target protein was sandwiched between the primary antibody immobilized on the GO and secondary antibody immobilized on the Fe₃O₄‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites and glutaraldehyde was used as linking agent for the immobilization of primary antibody on the surface of GO. Both Fe₃O₄‐AuNPs and H‐amino‐rGO‐Au nanocomposite and also hemin/G‐quadruplex can cooperate the electrocatalytic reduction of H₂O₂ in the presence of methylene blue as mediator. The proposed immunosensor has a wide linear dynamic range of 0.1 pg/ml to 300 pg/ml with a detection limit of 60 fg/ml when Fe₃O₄‐AuNPs was used for immobilization of hemin/G‐quadruplex, while the dynamic range and DL were 0. 1–1000 pg/mL and 10 fg/mL, respectively in the presence of H‐amino‐rGO‐ Au nanocomposite as platform for immobilizing of hemin/G‐quadruplex. The proposed immunosensor was also used for analysis of HBsAg in spiked human serum samples with satisfactory results.     

Electrochemical and Electrochemiluminescence Determination of Cancer Cells Based on Aptamers and Magnetic Beads

The electrochemical and electrochemiluminescence (ECL) detection of cell lines of Burkitt’s lymphoma (Ramos) by using magnetic beads as the separation tool and high‐affinity DNA aptamers for signal recognition is reported. Au nanoparticles (NPs) bifunctionalized with aptamers and CdS NPs were used for electrochemical signal amplification. The anodic stripping voltammetry technology employed for the analysis of cadmium ions dissolved from CdS NPs on the aggregates provided a means to quantify the amount of the target cells. This electrochemical method could respond down to 67 cancer cells per mL with a linear calibration range from 1.0×10² to 1.0×10⁵ cells mL^?1, which shows very high sensitivity. In addition, the assay was able to differentiate between target and control cells based on the aptamer used in the assay, indicating the wide applicability of the assay for diseased cell detection. ECL detection was also performed by functionalizing the signal DNA, which was complementary to the aptamer of the Ramos cells, with tris(2,2‐bipyridyl) ruthenium. The ECL intensity of the signal DNA, replaced by the target cells from the ECL probes, directly reflected the quantity of the amount of the cells. With the use of the developed ECL probe, a limit of detection as low as 89 Ramos cells per mL could be achieved. The proposed methods based on electrochemical and ECL should have wide applications in the diagnosis of cancers due to their high sensitivity, simplicity, and low cost.     

基于金纳米颗粒负载的金属有机骨架材料的癌胚抗原传感器的研制

以负载Au的金属有机骨架材料(AuNPs/Cu-TPA)标记CEA抗体(Ab2)为信号探针,通过电还原的方法将氧化石墨烯还原到电极上,研制了一种捕获CEA抗体(Ab1)的电化学免疫传感器,并将其应用于癌胚抗原(CEA)检测.所合成的MOFs材料中含有大量Cu2+,且电化学信号比较稳定,因此可以通过检测MOFs材料中Cu2+的信号实现对CEA的检测.此信号探针不需要预处理和酸处理,易负载贵金属从而固定抗体,大大简化了检测步骤并缩短了检测时间.此传感器对CEA的检测灵敏度好,操作简便.在最优实验条件下,此传感器的线性范围为0.1～ 80 ng/mL,检出限为0.03 ng/mL,线性相关系数为0.9887,可用于真实样品中CEA的测定.     

Amplified inhibition of the electrochemical signal of ferrocene by enzyme-functionalized graphene oxide nanoprobe for ultrasensitive immunoassay

A nanoprobe-induced signal inhibition mechanism was designed for ultrasensitive electrochemical immunoassay at a chitosan-ferrocene (CS-Fc) based immunosensor. The nanoprobe was prepared by covalently loading signal antibody and high-content horseradish peroxidase (HRP) on the graphene oxide (GO) nanocarrier. The immunosensor was prepared through the stepwise assembly of gold nanoparticles (Au NPs) and capture antibody at a CS-Fc modified electrode. After sandwich immunoreaction, the GO-HRP nanoprobes were quantitatively captured onto the immunosensor surface and thus induced the production of a layer of insoluble film through the enzymatically catalytic reaction of the HRP labels. Both the dielectric immunocomplex formed on the immunosensor surface and the enzymatic precipitate with low electroconductivity led to the electrochemical signal decease of the Fc indicator, which was greatly amplified by the multi-enzyme signal amplification of the nanoprobe. Based on this amplified signal inhibition mechanism, a new ultrasensitive electrochemical immunoassay method was developed. Using carcinoembryonic antigen as a model analyte, this method showed a wide linear range over 5 orders of magnitude with a detection limit down to 0.54 pg/mL. Besides, the immunosensor showed good specificity, acceptable reproducibility and stability as well as satisfactory reliability for the serum sample analysis.     

Fabrication of a Sensitive Electrochemical Biosensor for Detection of DNA Hybridization Based on Gold Nanoparticles/CuO Nanospindles Modified Glassy Carbon Electrode

In this work, a sensitive electrochemical DNA biosensor for the detection of sequence‐specific target DNA was reported. Firstly, CuO nanospindles (CuO NS) were immobilized on the surface of a glassy carbon electrode (GCE). Subsequently, gold nanoparticles (Au NPs) were introduced to the surface of CuO NS by the electrochemical deposition mode. Probe DNA with SH (HS‐DNA) at the 5′‐phosphate end was covalently immobilized on the surface of the Au NPs through Au? S bond. Scanning electron microscopy (SEM) was used to elucidate the morphology of the assembled film, and electrochemical impedance spectroscopy technique (EIS) was used to investigate the DNA sensor assembly process. Hybridization detection of DNA was performed with differential pulse voltammetry (DPV) and the methylene blue (MB) was hybridization indicator. Under the optimal conditions, the decline of reduction peak current of MB (ΔI) was linear with the logarithm of the concentration of complementary DNA from 1.0×10^?13 to 1.0×10^?6 mol·L^?1 with a detection limit of 3.5×10^?14 mol·L^?1 (S/N=3). In addition, this DNA biosensor has good selectivity, and even can distinguish single‐mismatched target DNA.