An electrochemical immunosensor using ferrocenyl-tethered dendrimer

We report here an enzyme-amplified, sandwich-type immunosensor for detecting the biospecific interaction between an antibody and antigen using redox mediation. We employed biotin/anti-biotin IgG as a model immunosensing pair. Partially ferrocenyl-tethered dendrimer (Fc-D), whose ferrocene moiety acts as a redox mediator, was immobilized to the electrode surface by covalent binding between the dendrimer amines and the carboxylic acids of a self-assembled monolayer. The unreacted amines of the immobilized Fc-D were modified with biotin groups to allow the specific binding of goat anti-biotin IgG. Rabbit anti-goat IgG-conjugated alkaline phosphatase was bound to goat anti-biotin IgG to catalyze conversion of p-aminophenyl phosphate monohydrate to p-aminophenol. This product is oxidized to quinoimide by the reduction of ferrocenium back to ferrocene, producing an electrocatalytic anodic current. Cyclic voltammograms and surface plasmon resonance experiments showed that the binding of nonspecific proteins is not significant on the biotinylated Fc-D surface. We also examined the change in peak current according to the concentration of anti-biotin IgG and found that the detection range of this immunosensing scheme is between 0.1 and 30 microg mL(-1).     

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.     

Improved Detection Limit and Stability of Amperometric Carbon Nanotube-Based Immunosensors by Crosslinking Antibodies with Polylysine

Amperometric immunosensor configurations featuring covalently bound anti-biotin antibodies (Ab) embedded into a polylysine (PLL)-single walled carbon nanotube (SWCNT) composite layer were evaluated. Assemblies were made by first oxidizing pyrolytic graphite (PG) electrodes to form surface carboxylic acid groups, to which PLL, SWCNTs and anti-biotin were covalently linked. Incorporating SWCNT into PLL-antibody assemblies improved the amperometric detection limit for biotin (Ag) labeled with horseradish peroxidase to 10 fmol mL(-1). Anti-biotin embedded into the PLL matrix had improved thermal stability and retained its binding ability for biotin after exposure to temperatures of 42 degrees C for up to 3 hours, while the noncrosslinked antibody was inactivated at this temperature in several minutes.     

Development of an electrochemical immunosensor for the detection of HbA1c in serum

An electrochemical immuno-biosensor for detecting glycosylated haemoglobin (HbA1c) is reported based on glassy carbon (GC) electrodes with a mixed layer of an oligo(phenylethynylene) molecular wire (MW) and an oligo(ethylene glycol) (OEG). The mixed layer is formed from in situ-generated aryl diazonium cations. To the distal end of the MW, a redox probe 1,1'-di(aminomethyl)ferrocene (FDMA) was attached followed by the covalent attachment of an epitope N-glycosylated pentapeptide (GPP), an analogon to HbA1c, to which an anti-HbA1c monocolonal antibody IgG can selectively bind. HbA1c was detected by a competitive inhibition assay based on the competition for binding to anti-HbA1c IgG antibodies between the analyte in solution, HbA1c, and the surface bound epitope GPP. Exposure of the GPP modified sensing interface to the mixture of anti-HbA1c IgG antibody and HbA1c results in the attenuation of ferrocene electrochemistry due to free antibody binding to the interface. Higher concentrations of analyte led to higher Faradaic currents as less anti-HbA1c IgG is available to bind to the electrode surface. It was observed that there is a good linear relationship between the relative Faradaic current of FDMA and the concentration of HbA1c from 4.5% to 15.1% of total haemoglobin in serum without the need for washing or rinsing steps.     

An Electrochemical Impedance Immunosensor Based on Gold Nanoparticle‐Modified Electrodes for the Detection of HbA1c in Human Blood

A label‐free immunosensor for the detection of HbA1c was developed based on gold nanoparticle (AuNP)‐aryl diazonium salt modified glassy carbon (GC) electrode where transduction is achieved using electrochemical impedance spectroscopy (EIS). GC electrodes were first modified with 4‐aminophenyl (Ph‐NH₂) layers to which AuNPs were attached. Thereafter an oligo(ethylene glycol) (OEG‐COOH) species were covalently attached to the remaining free amine groups on the Ph‐NH₂ surface. The AuNP surfaces were further modified with Ph‐NH₂ followed by attachment of a glycosylated pentapeptide (GPP), an analogon to HbA1c. Exposure of this interface to anti‐HbA1c IgG resulted in a change in charge transfer resistance (R_ct) due to the anti‐HbA1c IgG selectively complexing to the surface bound GPP. To detect the amount of HbA1c, a competitive inhibition assay was employed where the surface bound GPP and HbA1c in solution compete for the anti‐HbA1c IgG antibodies. The higher the concentration of HbA1c, the less antibody binds to the sensing interface and the lower the change of R_ct. The response of the immunosensor is linear with the HbA1c% of total haemoglobin in the range of 0%–23.3%. This competitive inhibition assay can be used for the detection of HbA1c in human blood. The performance of the immunosensor for detection of HbA1c in human blood is comparable to the clinical laboratory method.     

Nanocomposites of gold nanoparticles and graphene oxide towards an stable label-free electrochemical immunosensor for detection of cardiac marker troponin-I

A stable label-free amperometric immunosensor is presented based on gold nanoparticles and graphene oxide nanocomposites for detection of cardiac troponin-I in the early diagnosis of myocardial infarction. For designing of the sensing platform, firstly the nanocomposites based on GO and AuNPs were prepared and anchored on electrode surfaces. The formed nanocomposites provided a platform with big surface area for loading anti-cTnI capture antibody, and worked as a bridge for fast electron transfer subsequently increased the sensitivity. Moreover, the linkages between AuNP, GO, and electrodes were based on covalent bonding by aryldiazonium salt coupling chemistry, which favors the stability of the sensing interface. Finally, the anti-cTnI detection antibody was immobilized on GO tailored with ferrocene molecules, functioning as the signal reporter for the detection of cTnI. The modification process was monitored using electrochemistry, SEM, XPS. The herein immunosensor demonstrates a good selectivity and high sensitivity against human-cTnI, and is capable of detecting cTnI at concentrations as low as 0.05 ng mL⁻¹, which is 100 times lower than that possible by conventional methods. It is potential to design the portable sensing platform based on AuNPs and GO nanocomposites for future point-of-care diagnostics.     

基于纳米金／壳聚糖掺杂碳纳米管修饰金电极非标记免疫传感器的研究

应用吸附法将羊抗人IgG抗体直接固定于纳米金(GNPs)/壳聚糖(Chit)掺杂碳纳米管(CNTs)修饰的金电极表面,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,用差分脉冲伏安法研究了测试底液的pH值对免疫传感器性能的影响.实验表明,在含不同浓度人IgG的...     

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 Label‐Free Amperometric Immunosensor Based on Redox‐Active Ferrocene‐Branched Chitosan/Multiwalled Carbon Nanotubes Conductive Composite and Gold Nanoparticles

A novel immunosensor has been developed by self‐assembling Au NPs onto a ferrocene‐branched chitosan/multiwalled carbon nanotubes (CS‐Fc/MWCNTs) modified electrode for the sensitive determination of hepatitis B surface antigen (HBsAg). The formation of CS‐Fc effectively avoids the leakage of Fc and retains its electrochemical activity. Incorporation of MWCNTs and Au NPs into CS‐Fc further increases the electrochemical active Fc in the CS films and provides interactive sites for the immobilization of HBsAb. The morphologies and electrochemistry of the formed biofilm were investigated by using scanning electron microscopy and electrochemical techniques. The immunosensor exhibits a specific response to HBsAg in the range of 1.0–420 ng mL^?1. Excellent analytical performance, fabrication reproducibility and operational stability of the proposed immunosensor indicated its promising application in clinical diagnostics.     

Biocatalytic precipitation induced by an affinity reaction on dendrimer-activated surfaces for the electrochemical signaling from immunosensors

We have developed a strategy of signal generation for immunosensors that transduces biospecific affinity recognition reactions into electrochemical signals. The cyclic voltammetric method, tracking the precipitation of insoluble products onto the sensing surface and the subsequent decrement in the electrode area, was chosen for signal registration. Precipitation of insolubilities was induced by the catalytic reaction of enzymes, which were labeled to the biospecifically attached protein or antibody molecules. As a model system for affinity recognition, we have investigated the functionalization of biotin groups to the sensing monolayer and their biospecific interactions with anti-biotin antibody molecules. The immunosensing interface was developed onto the dendrimer-activated self-assembled monolayers (SAMs), as the base template for the functionalization of the antigen moiety and signal generation. The advantages of using dendrimer-activated SAMs in comparison to the plain modified thiolate SAMs for the sensing surface were shown in terms of sensing performances, and the analytical characteristics of the resulting immunosensor were examined. Additionally, the sensing system was applied for biotin/(strept)avidin couples, extending the applicability of the developed strategy.     

The impact of antibody/epitope affinity strength on the sensitivity of electrochemical immunosensors for detecting small molecules

A displacement immunoassay involves having a labelled analogue of the analyte (the epitope) already bound to the antibody. The presence of the analyte causes a competition for antibodies, and some of the antibodies dissociates from the epitope so that it can bind with the analyte. Herein, the influence of the affinity of the surface-bound epitope for the antibody on the sensitivity and selectivity of a displacement immunosensor is explored both theoretically and experimentally. An electrochemical immunosensor described previously [1], where the dissociation of antibodies from an electrode surface causes an increase in current from surface-bound ferrocene species, is used for this purpose. As expected, the ease and effectiveness of the bound antibody being displaced is inversely related to the affinity of the antibody to the surface-bound epitope relative to the analyte in solution as expected. However, if the affinity constant is too low, selectivity and/or sensitivity are compromised. Experimental results are qualitatively compared with a simple mass-action model.

Biocompatible and label-free amperometric immunosensor for hepatitis B surface antigen using a sensing film composed of poly(allylamine)-branched ferrocene and gold nanoparticles

An amperometric immunosensor has been developed for sensitive determination of hepatitis B surface antigen as a model protein. A glassy carbon electrode was modified with an assembly of positively charged poly(allylamine)-branched ferrocene (PAA-Fc) and negatively charged gold nanoparticles (Au NPs). The formation of PAA-Fc effectively avoids the leakage of Fc, retains its electrochemical activity, and enhances the conductivity of the composite. The adsorption of Au NPs onto the PAA-Fc matrix provides sites for the immobilization of the antigen and a favorable micro-environment to maintain its activity. The morphologies and electrochemistry of the sensing film were investigated via scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. Factors influencing the performance of the immunosensor were studied in detail. The concentration of the antigen can be quantitated (by measuring the decrease of the amperometric response resulting from the specific binding between antigen and antibody) in the range between 0.1 and 150?ng?mL^?C1, with a detection limit of 40?pg?mL^?C1 (S/N = 3). The method is economical, efficient, and potentially attractive for clinical immunoassays.

磁性无机-生物复合粒子敏感膜新型电流型免疫传感器的研究

合成了磁性Fe3O4纳米粒子,利用3-氨基丙基三乙氧基硅烷(APS)进行硅烷化,形成表面带有氨基的磁性Fe3O4纳米复合粒子,再用戊二醛将羊抗人免疫球蛋白G抗体(anti-IgG)固定在该磁性粒子表面,通过磁力将其修饰于固体石蜡碳糊电极表面制作成免疫传感器。与标记HRP的二抗体anti-IgG结合,以对苯二酚作为电子媒介体,实现对人免疫球蛋白G(IgG)的定量检测。IgG测定线性范围为2.5~400μg/L,检出限为0.75μg/L。该免疫传感器制作简单,成本低,表面更新方便,可用于临床血清检测。     

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.     

Label-free amperometric immunobiosensor based on a gold colloid and Prussian blue nanocomposite film modified carbon ionic liquid electrode

A novel experimental methodology based on a Prussian blue (PB) and gold nanoparticles (AuNPs) modified carbon ionic liquid electrode (CILE) was developed for use in a label-free amperometric immunosensor for the sensitive detection of human immunoglobulin G (HIgG) as a model protein. The CILE was fabricated by using the ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate as binder. Controllable electrodeposition of PB on the surface of the CILE and coating with 3-aminopropyl triethylene silane (APS) formed a film with high electronic catalytic activity and large surface area for the assembly of AuNPs and further immobilization of HIgG antibody. The electrochemistry of the formed nanocomposite biofilm was investigated by electrochemical techniques including cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy. The HIgG concentration was measured through the decrease of amperometric responses in the corresponding specific binding of antigen and antibody. The decreased differential pulse voltammetric values were proportional to the HIgG concentration in two ranges, 0.05–1.25 ng mL⁻¹ and 1.25–40 ng mL⁻¹, with a detection limit of 0.001 ng mL⁻¹ (S/N = 3). This electrochemical immunoassay combined the specificity of the immunological reaction with the sensitivity of the AuNPs, ionic liquid, and PB amplified electrochemical detection and would therefore be valuable for clinical immunoassays.     

A Label-Free Electrochemical Immunosensor for Clostridium Difficile Toxin B Based on One-Step Immobilization of Thionine in a Silica Matrix

A label-free amperometric immunosensor was fabricated to test clostridium difficile toxin B. Multi-walled carbon nanotubes were modified on the surface of a glassy carbon electrode by electrodeposition. A sol-gel method was developed to encapsulate thionine in an electrochemically induced three-dimensional porous silica matrix by a one-step process. Gold nanoparticle layers were constructed by covalent bonds and electrostatic adsorption with thionine. The clostridium difficile toxin B antibody was immobilized on the gold nanoparticles to construct the immunosensor. Cyclic voltammetry and differential pulse voltammetry demonstrated that the formation of antibody-antigen complexes decreased the peak current of thionine. The morphologies of the nanocomposites were investigated by scanning electron microscopy and ultraviolet-visible spectrometry. The electrode was shown to be sensitive and specific to detect clostridium difficile toxin B from 1.0 to 80.0 ng/mL with a limit of detection of 0.3 ng/mL.     

Electrochemical immunosensor nanoarchitectonics with the Ag-rGO nanocomposites for the detection of receptor-binding domain of SARS-CoV-2 spike protein

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a grave threat to human life and health, it is essential to develop an efficient and sensitive detection method to identify infected individuals. This study described an electrode platform immunosensor to detect SARS-CoV-2-specific spike receptor-binding domain (RBD) protein based on a bare gold electrode modified with Ag-rGO nanocomposites and the biotin-streptavidin interaction system. The Ag-rGO nanocomposites was obtained by chemical synthesis and characterized by electrochemistry and scanning electron microscope (SEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to record the electrochemical signals in the electrode modification. The differential pulse voltammetry (DPV) results showed that the limit of detection (LOD) of the immunosensor was 7.2 fg mL⁻¹ and the linear dynamic detection range was 0.015 ~ 158.5 pg mL⁻¹. Furthermore, this sensitive immunosensor accurately detected RBD in artificial saliva with favorable stability, specificity, and reproducibility, indicating that it has the potential to be used as a practical method for the detection of SARS-CoV-2.

     

Label-free capacitive immunosensor based on quartz crystal Au electrode for rapid and sensitive detection of Escherichia coli O157:H7

A label-free capacitive immunosensor based on quartz crystal Au electrode was developed for rapid and sensitive detection of Escherichia coli O157:H7. The immunosensor was fabricated by immobilizing affinity-purified anti-E. coli O157:H7 antibodies onto self-assembled monolayers (SAMs) of 3-mercaptopropionic acid (MPA) on the surface of a quartz crystal Au electrode. Bacteria suspended in solution became attached to the immobilized antibodies when the immunosensor was tested in liquid samples. The change in capacitance caused by the bacteria was directly measured by an electrochemical detector. An equivalent circuit was introduced to simulate the capacitive immunosensor. The immunosensor was evaluated for E. coli O157:H7 detection in pure culture and inoculated food samples. The experimental results indicated that the capacitance change was linearly correlated with the cell concentration of E. coli O157:H7. The immunosensor was able to discriminate between cellular concentrations of 10²–10⁵ cfu mL⁻¹ and has applications in detecting pathogens in food samples. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were also employed to characterize the stepwise assembly of the immunosensor.     

Design of a voltammetric immunosensor for determination of 1-nitropyrene

Abstract  In this study a design of an immunosensor for 1-nitropyrene is illustrated. First the response of 1-nitropyrene reduced at a glassy carbon electrode between 0 and −0.8 V was investigated with respect to pH and solvent. Then polyclonal anti-1-nitropyrene antibodies isolated from a rabbit antiserum were covalently bound to the surface of the glassy carbon electrode. This modified electrode was immersed in standard solutions or sample extracts for 5–15 min. 1-Nitropyrene was bound by the antibodies, accumulated and then analyzed at pH 6.5 in another supporting electrolyte by differential pulse voltammetry. Afterwards the immunosensor could be regenerated for the next measurement by rinsing with acetonitrile–water (40:60, v/v). A linear response was found between 20 and 100 ng/cm³. The limit of detection was 10 ng/cm³ and the intraday reproducibility of three immunosensors assembled during two months was between 4.5 and 10%. This sensor was applied to the analysis of 1-nitropyrene in air particulate matter and smoked tea. Graphical abstract        

A Novel Magnetism‐assisted Electrochemical Immunosensor with Sub‐Picomolar Sensitivity

A novel electrochemical immunosensor based on a magnetic glassy carbon electrode (MGCE) was developed for the quantitative determination of human immunoglobulin G (IgG). The immunosensing interface was fabricated by initially depositing silver nanoparticles on the MGCE surface and then immobilizing anti‐human IgG antibodies via the magnetic force between MGCE and Fe₃O₄ nanoparticles. The antibodies were covalently bonded to the amine‐functionalized Fe₃O₄ nanoparticles. Under optimal conditions, the magnetism‐assisted immunosensor exhibited a wide linear range from 0.1 pg/mL to 1.0 µg/mL with the detection limit of 0.05 pg/mL. Furthermore, the immunosensor displayed the advantages of good reproducibility and satisfactory stability.