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%。     

A novel antibody–antigen based impedimetric immunosensor for low level detection of HER2 in serum samples of breast cancer patients via modification of a gold nanoparticles decorated multiwall carbon nanotube-ionic liquid electrode

A highly sensitive impedimetric immunosensor based on a gold nanoparticles/multiwall carbon nanotube-ionic liquid electrode (AuNPs/MW-CILE) was developed for the determination of human epidermal growth factor receptor 2 (HER2). Gold nanoparticles were used to enhance the extent of immobilization and to retain the immunoactivity of the antibody Herceptin on the electrode. Cyclic voltammetry and electrochemical impedance spectroscopy were employed for characterization of various layers coated onto the AuNPs/MW-CILE. The impedance measurements at different steps were based on the charge transfer kinetics of the [Fe(CN)₆]^3−/4− redox pair. The immobilization of antibody and the corresponding antigen–antibody interaction at the electrode surface altered the interfacial electron transfer. The interactions of antibody with various concentrations of antigen were also monitored via the change of impedance response. The results showed that the charge transfer resistance increases linearly with increasing concentrations of HER2 antigen. The linear range and limit of detection were found as 10–110 ng mL⁻¹ and 7.4 ng mL⁻¹, respectively. The sensitivity and specificity of the immunosensor were validated. The results showed that the prepared immunosensor is a useful tool for screening of trace amounts of HER2 in serum samples of breast cancer patients.     

Sensitive and selective magnetoimmunosensing platform for determination of the food allergen Ara h 1

A highly sensitive disposable amperometric immunosensor based on the use of magnetic beads (MBs) is described for determination of Ara h 1, the major peanut allergen, in only 2 h. The approach uses a sandwich configuration involving selective capture and biotinylated detector antibodies and carboxylic acid-modified MBs (HOOC-MBs). The MBs bearing the immunoconjugates are captured by a magnet placed under the surface of a disposable screen-printed carbon electrode (SPCE) and the affinity reactions are monitored amperometrically at −0.20 V (vs a Ag pseudo-reference electrode) in the presence of hydroquinone (HQ) as electron transfer mediator and upon addition of H₂O₂ as the enzyme substrate. The developed immunosensor exhibits a wide range of linearity between 20.8 and 1000.0 ng mL⁻¹ Ara h 1, a detection limit of 6.3 ng mL⁻¹, a great selectivity, a good reproducibility with a RSD of 6.3% for six different immunosensors and a useful lifetime of 25 days. The usefulness of the immunosensor was demonstrated by determining Ara h 1 in different matrices (food extracts and saliva). The results correlated properly with those provided by a commercial ELISA method offering a reliable and promising analytical screening tool in the development of user-friendly devices for on-site determination of Ara h 1.     

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.     

EIS microfluidic chips for flow immunoassay and ultrasensitive cholera toxin detection

A flow-injection impedimetric immunosensor for the sensitive, direct and label-free detection of cholera toxin is reported. A limit of detection smaller than 10 pM was achieved, a value thousands of times lower than the lethal dose. The developed chips fulfil the requirement of low cost and quick reply of the assay and are expected to enable field screening, prompt diagnosis and medical intervention without the need of specialized personnel and expensive equipment, a perspective of special relevance for use in developing countries. Since the chip layout includes two sensing areas each one with a 2 × 2 sensor array, our biochips can allow statistical or (alternatively) multiplex analysis of biorecognition events between antibodies immobilized on each working electrode and different antigens flowing into the chamber.     

Electrochemical Detection of Human Interleukin-15 using a Graphene Oxide-Modified Screen-Printed Carbon Electrode

Biofunctionalizing a simple and disposable graphene oxide-modified screen-printed carbon electrode with anti-interleukin-15 antibodies has been successfully demonstrated for the first time for the label-free electrochemical detection of interleukin-15, a biomarker of early HIV infection. To improve the electrochemical reactivity and introduce carboxylic groups on the surface of screen-printed carbon electrode, high-quality graphene oxide was used for the modification of screen-printed carbon electrode. With simple modification of the screen-printed carbon electrode, the device exhibited satisfactory sensitivity, selectivity, stability, reproducibility, and regenerability. The immunosensor provided a detection limit of 3.51?ng?mL^?1 and a sensitivity of 0.5655?µA cm^?2?mL?ng^?1. The simply constructed immunosensor thus rendered promising device for immunoreactions on the surface of the electrode.     

A disposable immunosensor based on gold colloid modified chitosan nanoparticles-entrapped carbon paste electrode

A new strategy is described to construct disposable electrochemical immunosensors for the assay of human immunoglobulin. It is based on a carbon paste electrode constructed from chitosan nanoparticles modified with colloidal gold. The stepwise assembly process of the immunosensor was characterized by means of cyclic voltammetry and electrochemical impedance spectroscopy. Assay conditions that were optimized included the amount of chitosan nanoparticles in the preparation of carbon paste electrode, antibody concentration, and the incubation time of the antibody immobilization. Using hexacyanoferrate as a mediator, the current change increased with the concentration of human immunoglobulin G. A linear relationship in the concentration range 0.3 to 120 ng mL^?1 was achieved, with a detection limit of 0.1 ng mL^?1 (S/N?=?3). The method combines the specificity of the immunological reaction with the sensitivity of the gold colloid amplified electrochemical detection, and it has potential application in clinical immunoassay.     

An electrochemical immunosensor for the tumor marker α-fetoprotein using a glassy carbon electrode modified with a poly(5-formylindole), single-wall carbon nanotubes,and coated with gold nanoparticles and antibody

We report on a label-free electrochemical immunosensor for α-fetoprotein (α-FP). It is based on the use of a glassy carbon electrode that was first modified with conducting poly(5-formylindole) and single-walled carbon nanotubes (P5FIn/SWNTs), and then coated with gold nanoparticles and the respective antibody. The presence of aldehyde groups warrants direct immobilization of the antibody and results in a convenient method for fabricating of the immunosensor. Gold nanoparticles (GNPs) were deposited on the P5FIn/SWNTs composite material, and the modified electrode was applied to the detection of α-FP. The analytical signal is obtained by measuring the change of amperometric response at a typical working voltage of 100 mV before and after the immunoreaction. The detection limit is 200 fg mL^?1. The immunosensor is simple, sensitive, specific and reproducible. It has the potential for reliable point-of-care diagnosis of tumor or other diseases. Figure

A simple electrochemical immunosensor based on conducting poly(5-formylindole) and single-walled carbon nanotubes composite was fabricated to detect alpha-fetoprotein. The detection limit is 200 fg mL^?1. This immunosensor is simple, sensitive, specific and reproducible.     

Ultrasensitive electrochemical immunosensor for zeranol detection based on signal amplification strategy of nanoporous gold films and nano-montmorillonite as labels

Nano-montmorillonites belong to aluminosilicate clay minerals with innocuity, high specific surface area, ion exchange, and favorable adsorption property. Due to the excellent properties, montmorillonites can be used as labels for the electrochemical immunosensors. In this study, nano-montmorillonites were converted to sodium montmorillonites (Na-Mont) and further utilized for the immobilization of thionine (TH), horseradish peroxidase (HRP) and the secondary anti-zeranol antibody (Ab₂). The modified particles, Na-Mont-TH-HRP-Ab₂ were used as labels for immunosensors to detect zeranol. This protocol was used to prepare the immunosensor with the primary antibody (Ab₁) immobilized onto the nanoporous gold films (NPG) modified glassy carbon electrode (GCE) surface. Within zeranol concentration range (0.01–12 ng mL⁻¹), a linear calibration plot (Y = 0.4326 + 8.713 X, r = 0.9996) was obtained with a detection limit of 3 pg mL⁻¹ under optimal conditions. The proposed immunosensor showed good reproducibility, selectivity, and stability. This new type of immunosensors with montmorillonites and NPG as labels may provide potential applications for the detection of zeranol.     

Monitoring microbial populations of sulfate-reducing bacteria using an impedimetric immunosensor based on agglutination assay

An impedimetric immunosensor was fabricated for rapid and non-labeled detection of sulfate-reducing bacteria, Desulforibrio caledoiensis (SRB) by immobilizing lectin-Concanavalin A using an agglutination assay. The immobilization of lectin was conducted using amine coupling on the surface of a gold (Au) electrode assembled with 11-Mercaptoundecanoic acid. Electrochemical impedance spectroscopy (EIS) was used to verify the stepwise assembly of the sensor system. The work conditions of the impedimetric immunosensor, such as pH of the buffer solutions and the incubation time of lectin, were optimized. Faradic impedance spectra for charge transfer for the redox probe Fe(CN)₆^3−/4−were measured to determine SRB concentrations. The diameter of the Nyquist diagram that is equal to the charge-transfer resistance (R_ct) increased with increasing SRB concentration. A linear relationship between R_ct and SRB concentration was obtained in SRB concentration range of 1.8 to 1.8 × 10⁷ cfu/ml. The variation of the SRB population during the growth process was also monitored using the impedimetric immunosensor. This approach has great potential for simple, low-cost, and time-saving monitoring of microbial populations.     

Development of an enrofloxacin immunosensor based on label-free electrochemical impedance spectroscopy

Enrofloxacin is the most widespread antibiotic in the fluoroquinolone family. As such, the development of a rapid and sensitive method for the determination of trace amounts of enrofloxacin is an important issue in the health field. The interaction of the enrofloxacin antigen to a specific antibody (Ab) immobilized on an 11-mercapto-undecanoic acid-coated gold electrode was quantified by electrochemical impedance spectroscopy. Two equivalent circuits were separately used to interpret the obtained impedance spectra. These circuits included one resistor in series with one parallel circuit comprised of a resistor and a capacitor (1R//C), and one resistor in series with two parallel RC circuits (2R//C). The results indicate that the antigen-antibody reaction analyzed using the 1R//C circuit provided a more sensitive resistance increment against the enrofloxacin concentration than that of the 2R//C circuit. However, the 2R//C circuit provided a better fitting for impedance spectra, and therefore supplies more detailed results of the enrofloxacin-antibody interaction, causing the increase of electron transfer resistance selectively to the modified layer, and not the electrical double layer. The antibody-modified electrode allowed for analysis of the dynamic linear range of 1-1000 ng/ml enrofloxacin with a detection limit of 1 ng/ml. The reagentless and label-free impedimetric immunosensors provide a simple and sensitive detection method for the specific determination of enrofloxacin.     

Characterization of the gold-catalyzed deposition of silver on graphite screen-printed electrodes and their application to the development of impedimetric immunosensors

This paper describes the characterization of the gold-catalyzed deposition of silver on graphite screen-printed electrodes (SPEs) using electrochemical impedance spectroscopy (EIS) and the application of this approach to the development of impedimetric immunosensors. After applying −0.1 V for 45 s, the amount of electrodeposited silver quantitatively changes the magnitude of two elements of the electrical equivalent circuit: the interface capacitance, Ci, and the charge-transfer resistance, R_CT. Better correlations have been found when considering the R_CT since this parameter is almost exclusively dependent on the amount of deposited silver under these experimental conditions. This approach has been successfully applied to the development of an impedimetric immunosensor for aflatoxin M₁. The R_CT magnitude shows good correlation with the amount of gold immobilized on the electrode surface after a competitive assay and thus, with the toxin concentration. This approach has been found sensitive in a wide range of concentrations, from 15 to 1000 free-AFM₁ ppt with a limit of detection of 12 ppt.     

Screen-printed electrodes as impedimetric immunosensors for the detection of anti-transglutaminase antibodies in human sera

The concentration of anti-transglutaminase antibodies in human sera is an important analytical marker for the diagnosis of the autoimmune disorder celiac disease. In this work, an immunosensor for the electrochemical detection of anti-transglutaminase antibodies in human sera was developed. The immunosensor is based on the immobilization of transglutaminase onto screen-printed gold electrodes which were covered with a polyelectrolyte layer of poly (sodium-4-styrensulfonic acid). The antigen-antibody interaction was evaluated using an amplification step: incubation with peroxidase (POD)-labeled immunoglobulins and subsequent biocatalytic oxidation of 3-amino-9-ethylcarbazole (AEC). Changes in the interfacial properties of the sensor electrode were determined by electrochemical impedance spectroscopy (EIS). Impedance spectra could be fitted to a Randles equivalent circuit containing a constant phase element (CPE). Furthermore, it was shown that impedance measurements could be simplified by performing EIS at only two selected frequencies, without loss of reliability. Incubation of these disposable immunosensor chips with various anti-transglutaminase antibody concentrations resulted in changes in their charge transfer resistance (R_ct). Thereby, a calibration graph could be established. Finally, immunosensors were used for characterizing different human sera with respect to their anti-transglutaminase autoantibody concentration of the IgG and IgA type.     

A sensitive and label-free impedimetric biosensor based on an adjunct probe

A highly sensitive and label-free impedimetric biosensor is achieved based on an adjunct probe attached nearby the capture probe. In this work, the adjunct probe was co-assembled on the surface of gold electrode with the capture probe hybridized with the reporter probe, and then 6-mercapto-1-hexanol was employed to block the nonspecific binding sites. When target DNA was added, the adjunct probe functioned as a fixer to immobilize the element of reporter probe displaced by the target DNA sequences and made the reporter probe approach the electrode surface, leading to effective inhibition of charge transfer. The increase in charge transfer resistance is related to the quantity of the target DNA in a wide range. The linear range for target DNA with specific sequences was from 0.1 nM to 0.5 μM with a good linearity (R = 0.9988) and a low detection limit of 6.3 pM. This impedimetric biosensor has the advantages of simplicity, sensitivity, good selectivity, and large dynamic range.     

A Simple and Low-Cost Electrochemical Immunosensor for Ultrasensitive Determination of Calreticulin Biomarker in Human Serum

An early on time detection of breast cancer significantly affects the treatment process and outcome. Herein, a new label-free impedimetric biosensor is developed to determine the lowest change in the level of calreticulin (CALR), which is a new biomarker of breast carcinoma. The proposed immunosensor is fabricated by using reduced graphene oxide/amino substituted polypyrrole polymer (rGO-PPyNH₂) nanocomposite modified disposable electrode. The anti-CALR antibodies are first attached on the rGO-PPyNH₂ nanocomposite coated electrode through glutaraldehyde crosslinking; the CALR antigens are then immobilized with the addition of CALR antigens to form an immunocomplex on the sensing surface. This immunocomplex induces considerably larger interfacial electron transport resistance (R_ct). The variation in the R_ct has a linear relationship with CALR level in the detection range of 0.025 to 75 pg mL⁻¹, with a detection limit of 10.4 fg mL⁻¹. The suggested biosensor shows high selectivity to CALR, good storage stability (at least 5 weeks) and suitable reproducibility results as shown in quality control chart. The designed immunosensor is utilized to analyze CALR levels in human sera with satisfying results. This immunosensor provides a novel way for the clinical determination of CALR and other cancer biological markers.     

A novel lable-free electrochemical immunosensor for carcinoembryonic antigen based on gold nanoparticles-thionine-reduced graphene oxide nanocomposite film modified glassy carbon electrode

In this paper, gold nanoparticle-thionine-reduced graphene oxide (GNP-THi-GR) nanocomposites were prepared to design a label-free immunosensor for the sensitive detection of carcinoembryonic antigen (CEA). The nanocomposites with good biocompatibility, excellent redox electrochemical activity and large surface area were coated onto the glassy carbon electrode (GCE) surface and then CEA antibody (anti-CEA) was immobilized on the electrode to construct the immunosensor. The morphologies and electrochemistry of the formed nanocomposites were investigated by using scanning electron microscopy (SEM), ultraviolet-visible (UV-vis) spectrometry, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). CV and differential pulse voltammetry (DPV) studies demonstrated that the formation of antibody-antigen complexes decreased the peak current of THi in the GNP-THi-GR nanocomposites. The decreased currents were proportional to the CEA concentration in the range of 10-500 pg/mL with a detection limit of 4 pg/mL. The proposed method was simple, fast and inexpensive for the determination of CEA at very low levels.     

A Reagentless Amperometric Immunosensor for Alpha‐Fetoprotein Based on Gold Nanoparticles/TiO2 Colloids/Prussian Blue Modified Platinum Electrode

A novel reagentless amperometric immunosensor for the determination of alpha‐fetoprotein (AFP) was prepared by immobilizing TiO₂ colloids on Prussian blue (PB) modified platinum electrode, which yielded a positively charged interface with strong adsorption to deposit gold nanoparticles for immobilization of alpha‐fetoprotein antibody (anti‐AFP). The factors influencing the performance of the proposed immunosensors were studied in detail. Under the optimized conditions, cyclic voltammograms determination of AFP showed a specific response in two concentration ranges from 3.0 to 30.0 ng/mL and from 30.0 to 300.0 ng/mL with a detection limit of 1.0 ng/mL at a signal‐to‐noise ratio of 3. The proposed immunosensor exhibited high selectivity, good reproducibility, long‐term stability (>2 months) and good repeatability.     

Double‐Layer Nanogold and Double‐Strand DNA‐Modified Electrode for Electrochemical Immunoassay of Cancer Antigen 15‐3

Various sensor‐based immunoassay methods have been extensively developed for the detection of cancer antigen 15‐3 (CA 15‐3), but most often exhibit low detection signals and low detection sensitivity, and are unsuitable for routine use. The aim of this work is to develop a simple and sensitive electrochemical immunoassay for CA 15‐3 in human serum by using nanogold and DNA‐modified immunosensors. Prussian blue (PB), as a good mediator, was initially electrodeposited on a gold electrode surface, then double‐layer nanogold particles and double‐strand DNA (dsDNA) with the sandwich‐type architecture were constructed on the PB‐modified surface in turn, and then anti‐CA 15‐3 antibodies were adsorbed onto the surface of nanogold particles. The double‐layer nanogold particles provided a good microenvironment for the immobilization of biomolecules. The presence of dsDNA enhanced the surface coverage of protein, and improved the sensitivity of the immunosensor. The performance and factors influencing the performance of the immunosensor were evaluated. Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 1.0 to 240 ng/mL with a relatively low detection limit of 0.6 ng/mL (S/N=3) towards CA 15‐3. The stability, reproducibility and precision of the as‐prepared immunosensor were acceptable. 57 serum specimens were assayed by the developed immunosensor and standard enzyme‐linked immunosorbent assay (ELISA), respectively, and the results obtained were almost consistent. More importantly, the proposed methodology could be further developed for the immobilization of other proteins and biocompounds.     

电位无标型糖化血红蛋白免疫微传感器

研制了基于标准CMOS工艺和微加工技术的电位无标型免疫微传感器,可实现糖化血红蛋白浓度与血红蛋白浓度的简便检测。该微传感器包括含有信号读出电路的场效应型微传感集成芯片和一次性测试试条。微传感集成芯片由本实验室设计并经新加坡Chartered半导体公司流片制备。一次性测试试条采用微加工技术制备于柔性塑料片上,包括敏感电极阵列和三维微结构测量池。基于自组装单层膜并引入纳米金颗粒的方法,在测试试条电极表面固定抗体。采用循环伏安法和交流阻抗法对电极表面的修饰过程进行了测试和分析。该传感器对糖化血红蛋白和血红蛋白检测的线性范围分别为4～24mg/L和60～180mg/L。     

A new antibody immobilization strategy based on electrodeposition of nanometer-sized hydroxyapatite for label-free capacitive immunosensor

A new antibody immobilization strategy was proposed for the fabrication of a label-free capacitive immunosensor based on electrodeposition of nanometer-sized bioactive hydroxyapatite (HAP). By a procedure of constant current cathodal electrodeposition, a nano-HAP film with bioactivity was formed on a self-assembled β-mercaptoethanol monolayer-modified gold electrode. A suitable amount of chitosan was added into the electrodeposition solution with the aim of obtaining a strong and homogeneous HAP-coating film. After blocking with long-chain alkylthiol and then embedding antibody by coupling with divinylsulphone, the electrode was possessed of a higher initial capacitance value, which was suitable for capacitive transduction. The sensitive layer was characterized by Fourier transform infrared spectrum, scanning electron microscopy and electrochemical method. Human transferrin immunoassay was selected as the testing system. The linear response range of the sensor for transferrin was between 1 and 100 ng/mL with a detection limit of 0.15 ng/mL. After simply rinsing with subacidity solution, the regenerated sensor achieved up to 10 assay cycles without significant loss of sensitivity.