Highly conducting gold nanoparticles-graphene nanohybrid films for ultrasensitive detection of carcinoembryonic antigen

A new label-free amperometric immunosensor was developed for detection of carcinoembryonic antigen (CEA) based on chitosan-ferrocene (CS-Fc) and nano-TiO₂ (CS-Fc + TiO₂) complex film and gold nanoparticles-graphene (Au-Gra) nanohybrid. CS-Fc + TiO₂ composite membrane was first modified on a bare glass carbon electrode. Then Au-Gra nanohybrid was formed on the CS-Fc + TiO₂ membrane by self-assembly strategy. Next, further immobilization of anti-CEA was constructed according to the strong interaction between Au-Gra and the amido groups of anti-CEA. Since Au-Gra nanohybrid films provided a congenial microenvironment for the immobilization of biomolecules, the surface coverage of antibody protein could be enhanced and the sensitivity of the immunosensor has been improved. The good electronic conductive characteristic might be attributed to the synergistic effect of graphene nanosheets and Au NPs. The modified process was characterized by scanning electron microscope (SEM) and cyclic voltammetry (CV). Under optimized conditions, the resulting biosensor displayed good amperometric response to CEA with linear range from 0.01 to 80 ng/mL and a detection limit of 3.4 pg/mL (signal/noise = 3). The results demonstrated that the immunosensor has advantages of high conduction, sensitivity, and long life time. This assay approach showed a great potential in clinical applications and detection of low level proteins.     

Probe-Integrated Label-Free Electrochemical Immunosensor Based on Binary Nanocarbon Composites for Detection of CA19-9

Convenient and sensitive detection of tumor biomarkers is crucial for the early diagnosis and treatment of cancer. Herein, we present a probe-integrated and label-free electrochemical immunosensor based on binary nanocarbon composites and surface-immobilized methylene blue (MB) redox probes for detection of carbohydrate antigen 199 (CA19-9), which is closely associated with gastric malignancies. Nanocarbon composites consisting of electrochemically reduced graphene oxides and carbon nanotubes (ErGO-CNT) are electrodeposited onto an indium tin oxide (ITO) electrode surface to form a 3D nanocomposite film, which could provide high surface area to immobilize abundant MB probes, facilitate the electron transfer of MB, and therefore, improve sensitivity. Polydopamine (PDA) served as a bifunctional linker is able to immobilize anti-CA19-9 antibodies and stabilize the inner probe, conferring the sensing interface with specific recognition capacity. Electrochemical detection of CA19-9 is achieved based on the decrease of the redox signal of MB after specific binding of CA19-9 with a wide linear range of 0.1 mU/mL to 100 U/mL and a limit of detection (LOD) of 0.54 nU/mL (S/N = 3). The constructed electrochemical immunosensor has good selectivity, repeatability, reproducibility, and stability. Furthermore, determination of CA19-9 in human serum samples is also realized.     

Antibody–Ferrocene Conjugates as a Platform for Electro-Chemical Detection of Low-Density Lipoprotein

Low-density lipoprotein (LDL) is a cardiac biomarker identified in the pathology of cardiovascular disease (CVD). Typically, the level of LDL is calculated using the Friedewald relationship based on measured values of total cholesterol, high-density lipoproteins (HDL), and triglycerides. Unfortunately, this approach leads to some errors in calculation. Therefore, direct methods that can be used for fast and accurate detection of LDL are needed. The purpose of this study was to develop an electrochemical platform for the detection of LDL based on an antibody–ferrocene conjugate. An anti-apolipoprotein B-100 antibody labeled with ferrocene was covalently immobilized on the layer of 4-aminothiophenol (4-ATP) on the surface of gold electrodes. Upon interaction between LDL and the antibody–ferrocene conjugate, a decrease in the ferrocene redox signal registered by square wave voltammetry was observed, which depends linearly on the concentration from 0.01 ng/mL to 1.0 ng/mL. The obtained limit of detection was equal to 0.53 ng/mL. Moreover, the satisfied selectivity toward human serum albumin (HSA), HDL, and malondialdehyde-modified low-density lipoprotein (MDA-LDL) was observed. In addition, the acceptable recovery rates of LDL in human serum samples indicate the possible application of immunosensors presented in clinical diagnostics.     

Biosensors Based on Ion-Sensitive Field-Effect Transistors for HLA and MICA Antibody Detection in Kidney Transplantation

This work demonstrates the ability of the Ion-Sensitive Field-Effect Transistor (ISFET)-based immunosensor to detect antibodies against the human leukocyte antigen (HLA) and the major histocompatibility complex class-I-related chain A (MICA). The sensing membrane of the ISFET devices was modified and functionalized using an APTES-GA strategy. Surface properties, including wettability, surface thickness, and surface topology, were assessed in each module of the modification process. The optimal concentrations of HLA and MICA proteins for the immobilization were 10 and 50 μg/mL. The dose-response curve showed a detection range of 1.98–40 µg/mL for anti-HLA and 5.17–40 µg/mL for anti-MICA. The analytical precision (%CV) was found to be 10.69% and 8.92% for anti-HLA and -MICA, respectively. Moreover, the electrical signal obtained from the irrelevant antibody was considerably different from that of the specific antibodies, indicating the specific binding of the relevant antibodies without noise interference. The sensitivity and specificity in the experimental setting were established for both antibodies (anti-HLA: sensitivity = 80.00%, specificity = 86.36%; anti-MICA: sensitivity = 86.67%, specificity = 88.89%). Our data reveal the potential of applying the ISFET-based immunosensor to the detection of relevant anti-HLA and -MICA antibodies, especially in the field of kidney transplantation.     

基于聚硫堇、DNA/纳米银复合物共修饰癌胚抗原免疫传感器的研究

将硫堇聚合到玻碳电极(GCE)表面形成带正电的多孔聚硫堇(PTH)复合膜, 通过静电吸附固定DNA/纳米银复合物, 利用复合物中纳米银大的比表面积和强的吸附能力将癌胚抗体(anti-CEA)固定到电极表面, 从而制得高灵敏的电流型癌胚抗原(CEA)免疫传感器. 通过循环伏安法考察了电极表面的电化学行为, 并对免疫传感器的性能进行了详细研究. 在最优的实验条件下, 用示差脉冲伏安法(DPV)对癌胚抗原进行检测, 其线性范围为1.0～10.0 ng•mL^－1和10.0～80.0 ng•mL^－1, 线性相关系数分别为0.9983和0.9970, 检测限为0.24 ng•mL^－1, 并将该免疫传感器用于血清样品中CEA的检测.     

脑钠素与充血性心力衰竭

制备了一种三维多孔镍钼硫化物纳米花修饰碳纳米管（CNT）的复合材料（NiMoS@CNT）. 通过扫描电子显微镜和X射线衍射表征所合成材料的形貌和结构. 利用循环伏安法和计时电流法对所制备材料的电化学催化性能进行研究. 基于NiMoS@CNT对过氧化氢（H₂O₂）优异的电催化性能,构建了一种检测脑利钠肽的夹心型电化学传感器. 在最优条件下,电流响应强度和脑利钠肽质量浓度的对数在0.20~20 ng/mL范围内呈线性关系. 结果表明免疫传感器具有高的灵敏度、选择性和稳定性,可用于实际样品的检测.     

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.     

Biomolecule‐Doped Organic/Inorganic Hybrid Nanocomposite Film for Label‐Free Electrochemical Immunoassay of α‐1‐Fetoprotein

A new electrochemical immunosensor for the detection of α‐1‐fetoprotien (AFP) was developed based on AFP antibody (anti‐AFP)‐functionalized organic/inorganic hybrid nanocomposite membrane. To fabricate such a hybrid composite membrane, 3,4,9,10‐perylenetetracarboxylic acid‐bound thionine molecules (PTCTH) were initially doped into titania colloids (TiO₂), and then gold nanoparticles and anti‐AFP were immobilized onto the composite film in turn. Comparison with the electrode fabricated only with thionine not 3,4,9,10‐perylenetetracarboxylic acid, the immunosensor with PTCTH exhibited high sensitivity and fast electron transfer. The presence of gold nanoparticles provided a good microenvironment for the immobilization of biomolecules, enhanced the surface coverage of protein, and improved the sensitivity of the immunosensor. The modified process was characterized by scanning electron microscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The surface topography of the membrane was investigated by scanning electron microscopy (SEM). Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 2.5 to 200.0 ng/mL towards AFP with a detection limit of 0.5 ng/mL (S/N=3). The stability, reproducibility and precision of the immunosensor were acceptable. Comparison with the conventional enzyme‐linked immunosorbent assay (ELISA), the present method did not require more labeled procedures and washing steps. Significantly, the detection methodology provides a promising approach for other proteins or biosecurities.     

Specific Detection of Salmonella typhi Using Renewable Amperometric Immunosensor

A renewable amperometric immunosensor was developed for the specific detection of Salmonella typhi (S. typhi) using flagellin specific antibodies. An immunocomposite comprising paraffin, graphite, and capturing antibodies against S. typhi was used to construct the electrode. The detection technique involved a sandwich ELISA system. The assay conditions were optimized for loading of capturing antibody, incubation time for S. typhi cells, rotation speed and minimum amount of substrate needed. 1‐naphthyl phosphate was used as the substrate with an amperometric detection of its enzymatic hydrolysis product 1‐naphthol at a potential of +400 mV vs Ag/AgCl reference electrode. The minimum detection limit for S. typhi was found to be 10⁵ cells/mL in 90 min, while ELISA detects 10⁶ cells/mL in five hours.     

Immunosensor for the determination of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> using a tyrosinase–mercaptopropionic acid modified electrode as an amperometric transducer

An amperometric immunosensor for the quantification of Staphylococcus aureus based on the coimmobilization of rabbit immunoglobulin G (RbIgG) and tyrosinase on a mercaptopropionic acid self-assembled monolayer modified gold electrode is reported. A competitive mode in which protein-A-bearing S. aureus cells and antiRbIgG labeled with alkaline phosphatase (AP) compete for the binding sites of immobilized RbIgG was used. Monitoring of the affinity reaction was carried out by the amperometric detection at -0.15 V of phenol generated in the enzyme reaction with AP, at the tyrosinase-modified electrode through the electrochemical reduction of the o-quinone formed. Optimization of the working variables, such as the immunosensor composition and incubation times, the applied potential, the working pH and the concentration of phenyl phosphate used as the AP substrate, was carried out. Under the optimized conditions, both the repeatability of the measurements and the reproducibility of the responses obtained with different immunosensors yielded relative standard deviation values for the steady-state current lower than 10%. The immunosensor showed a dynamic range from 4.4x10(5) to 1.8x10(7) S. aureus cells mL(-1), with a detection limit of 1.7x10(5) cells mL(-1). The limit of detection was remarkably improved by subjecting S. aureus cells to wall lysis by heat treatment. The value obtained was 2.3x10(3) cells mL(-1), which is adequate for the monitoring of S. aureus contamination levels in some foodstuffs. As an application, milk samples spiked with bacteria at the 4.8x10(3) cells mL(-1) level were analyzed.