Label-free Electrochemical Impedimetric Immunosensor Research Articles

The development of an electrochemical immunosensor utilizing chicken IgY anti-spike antibody for the detection of SARS-CoV-2

This paper introduces a novel approach for detecting the SARS-CoV-2 recombinant spike protein combining a label free electrochemical impedimetric immunosensor with the use of purified chicken IgY antibodies. The sensor employs three electrodes and is functionalized with an anti-S IgY antibody, ELISA and immunoblot assays confirmed the positive response of chicken immunized with SARS-CoV2 S antigen. The developed immunosensor is effective in detecting SARS-CoV-2 in nasopharyngeal clinical samples from suspected cases. The key advantage of this biosensor is its remarkable sensitivity, and its capability of detecting very low concentrations of the target analyte, with a detection limit of 5.65 pg/mL. This attribute makes it highly suitable for practical point-of-care (POC) applications, particularly in low analyte count clinical scenarios, without requiring amplification. Furthermore, the biosensor has a wide dynamic range of detection, spanning from 11.56 to 740 ng/mL, which makes it applicable for sample analysis in a typical clinical setting.

Electrochemical immunosensor utilizing a multifunctional 3D nanocomposite coating with antifouling capability for urinary bladder cancer diagnosis

Currently available point-of-care tests for Bladder Cancer (BC) are qualitative and prone to high false positives, thus preventing them from replacing clinical cystoscopy as reliable stand-alone diagnostics. In this study, we have targeted this unmet need by developing a label-free electrochemical impedimetric immunosensor for detection of APO-A1, a major high-density lipoprotein that is overexpressed in the urine of early-stage bladder cancer patients. The immunosensor utilizes a novel multifunctional 3D nanocomposite coating consisting of a porous bovine serum albumin (BSA) matrix embedded with a network of highly conductive and biocompatible gold coated silver nanowires (Au@AgNWs). The main components of this nanocomposite coating and their specific functions include: (i) A 3D porous BSA matrix to enable oriented antibody conjugation and prevent non-specific protein adsorption while allowing diffusion of analyte with minimal hindrance as compared to traditional antifouling coatings (ii) Embedded Au@AgNWs that enhance biocompatibility and improve charge transfer to the underlying electrode. The nanocomposite coating demonstrated minimal decrease in electrochemical performance even after 1-month of incubation in 1% BSA, human serum and human urine. Furthermore, the low-cost and disposable screen-printed electrochemical immunosensor exhibited excellent feasibility for sensitive and specific APO-A1 detection in the clinically relevant sensing range of 100 pg/mL to 250 ng/mL with high reproducibility (n = 5, RSD=2.2%) and an impressive LOD of 22 pg/mL. These results highlight the potential of the proposed immunosensor to enable reliable early diagnosis of bladder cancer at the point-of-care and serve as a viable alternative to cystoscopy.

A label-free impedimetric immunosensor for zearalenone based on CS-CNT-Pd nanocomposite modified screen-printed disposable electrodes

A label-free electrochemical impedimetric immunosensor has been developed for the rapid and sensitive detection of zearalenone (ZEN) by immobilizing monoclonal anti-zearalenone antibodies (ZEN-MAb) onto the palladium nanoparticle embedded functionalized carbon nanotube-chitosan (SPCE/CS-CNT-Pd) modified electrode. This CS-CNT-Pd nanocomposite consists of finite Pd nanoparticles implanted onto the covalently functionalized CNTs by the chemical reduction method. The structure of the nanocomposite was studied by using X-ray diffraction (XRD), transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy (EDX). The major advantages of the coexistence of Pd nanoparticles with CNTs are greatly enhanced conductive, catalytical properties of transducer surface, and better immobilization of bio-recognition antibody. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to verify the stepwise assemblage of the developed immunosensor. The immobilization of antibodies and the binding of zearalenone to the modified electrode increased the electron-transfer resistance (Rct), which was directly measured with EIS using [Fe(CN)6]3/4- as a redox probe. The developed immunosensor was quite sensitive and simple compared with various conventional methods. The immunosensor performance was tested under the optimized conditions, towards the detection of ZEN, from 0.25 to 16 ng mL−1 with a detection limit of 0.25 ng mL−1. The new biocompatible CS-CNT-Pd based immunosensor was successfully applied to assay ZEN in corn samples, and the good recovery values were gained, this holds the considerable promise for the sensor performance in the food industries.

An ultrasensitive label-free electrochemical impedimetric immunosensor for vascular endothelial growth factor based on specific phage via negative pre-screening

As a vascular growth regulator, vascular endothelial growth factor (VEGF) exerts significant biological roles through specific binding to its receptors on the vascular endothelial cells. VEGF165 is generally referenced as a potential therapeutic target of many malignant tumors. In this study, a negative pre-screening strategy with structurally analogous members of VEGF121, VEGFC and VEGFD was first proposed for VEGF165 biopanning, aiming at significantly improving the specificity of the selected phage monoclones. Indirect ELISA experiment showed that the phage monoclone expressing peptide SPFLLRM demonstrates excellent affinity and specificity. Then a VEGF165 electrochemical impedimetric spectroscopy (EIS) immunosensor was constructed by above specific phage modified electrode. After optimizing the experimental conditions, the as-explored EIS immunosensor had a linear range of 0.5–1000 pg/mL with the limit of detection of 0.15 pg/mL VEGF165. In addition, the developed phage-based EIS immunosensor was applied to satisfactorily detect VEGF165 in human serum samples. Considering its ultra-sensitivity, good selectivity, batch reproducibility and stability, the screened selective phage-based EIS sensor is envisioned potential application in diagnosis and therapy.

A Label-Free Electrochemical Impedimetric Immunosensor with Biotinylated-Antibody for SARS-CoV-2 Nucleoprotein Detection in Saliva.

The timely detecting of SARS-CoV-2 coronavirus antigens for infection validation is an urgent request for COVID-19 pandemic control. This study constructed label-free electrochemical impedance spectroscopy (EIS)-based immunosensors based on gold nanostructured screen-printed carbon electrodes (AuNS/SPCEs) to detect the SARS-CoV-2 nucleocapsid protein (N-protein) in saliva. Using short-chain 3-mercaptopropionic acid (MPA) as a linker to covalently bond streptavidin (SA) and bovine serum albumin (BSA) for controlling the oriented immobilization of the biotinylated anti-N-protein antibody (BioAb) can offer a greater sensitivity, a lower limit of detection (LOD), and better reproducibility of immunosensors (defined as BioAb/SA-BSA/MPA/AuNS/SPCEs) than the antibody randomly immobilized immunosensors and the long-chain 11-mercaptoundecanoic acid (MUA)-modified immunosensors (BioAb/SA-BSA/MUA/AuNS/SPCEs). The BioAb/SA-BSA/MPA/AuNS/SPCE-based immunosensors presented good linearity from 0.01 ng/mL to 100 ng/mL and a low LOD of 6 pg/mL in a phosphate buffer solution (PBS) and PBS-diluted saliva. Moreover, the immunosensor exhibited little cross-activity with other viral antigens such as MERS-CoV N-protein, influenza A N-protein, influenza B N-protein, and SARS-CoV-2 spike protein, indicating the high specificity of the immunosensors. The disposable label-free EIS-based immunosensors have promising potential in facilitating the rapid and sensitive tests of saliva-based COVID-19 diagnostics.

Electrochemical Immunosensor for the Early Detection of Rheumatoid Arthritis Biomarker: Anti-Cyclic Citrullinated Peptide Antibody in Human Serum Based on Avidin-Biotin System.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that produces a progressive inflammatory response that leads to severe pain, swelling, and stiffness in the joints of hands and feet, followed by irreversible damage of the joints. The authors developed a miniaturized, label-free electrochemical impedimetric immunosensor for the sensitive and direct detection of arthritis Anti-CCP-ab biomarker. An interdigitated-chain-shaped microelectrode array (ICE) was fabricated by taking the advantage of microelectromechanical systems. The fabricated ICE was modified with a self-assembled monolayer (SAM) of Mercaptohexanoic acid (MHA) for immobilization of the synthetic peptide bio-receptor (B-CCP). The B-CCP was attached onto the surface of SAM modified ICE through a strong avidin-biotin bio-recognition system. The modified ICE surface with the SAM and bio-molecules (Avidin, B-CCP, Anti-CCP-ab and BSA) was morphologically and electrochemically characterized. The change in the sensor signal upon analyte binding on the electrode surface was probed through the electrochemical impedance spectroscopy (EIS) property of charge-transfer resistance (Rct) of the modified electrodes. EIS measurements were target specific and the sensor response was linearly increased with step wise increase in target analyte (Anti-CCP-ab) concentrations. The developed sensor showed a linear range for the addition of Anti-CCP-ab between 1 IU mL−1 → 800 IU mL−1 in phosphate buffered saline (PBS) and Human serum (HS), respectively. The sensor showed a limit of detection of 0.60 IU mL−1 and 0.82 IU mL−1 in the PBS and HS, respectively. The develop bio-electrode showed a good reproducibility (relative standard deviation (RSD), 1.52%), selectivity and stability (1.5% lost at the end of 20th day) with an acceptable recovery rate (98.0% → 101.18%) and % RSD’s for the detection of Anti-CCP-ab in spiked HS samples.

Quantitative Determination of Triiodothyronine by Electrochemical Impedance Spectroscopic Biosensor Using Gold Nanoparticle-Modified Electrode.

A label-free electrochemical impedimetric immunosensor for the detection of Triiodothyronine-a thyroid hormone that functions as the biomarker for monitoring for thyroid dysfunction was developed. The gold nanoparticle-modified electrode was employed to achieve the sensitive determination of Triiodothyronine at a low concentration level. The gold nanoparticle layer on the gold electrode was generated by chronoamperometry method and its resulting characteristics were investigated by scanning electron microscopy. Redox probe [Fe(CN)6]3-/4- and electrochemical impedance spec-troscopy was used for both evaluation of the immobilization of anti-Triiodothyronine antibody on the electrode surface and quantitative determination of target Triiodothyronine in different concentrations. The electrode with absorbed antibodies showed significant changes in charge transfer resistance upon binding the antigen, which resulted in an increase in normalized impedance change as the addition of antigen concentrations over a dynamic linear range of 0.01-100 ng/ml. These results indicated that the proposed immunosensor could be a potential alternative method for determination of Triiodothyronine in clinics with the advantage of low cost and less time-consuming.

Label-free electrochemical impedimetric immunosensor for sensitive detection of IgM rheumatoid factor in human serum

The authors report a label-free and direct detection of rheumatoid factor- Immunoglobulin M (IgM-RF) based on an impedimetric-interdigitated wave type microelectrode array (IDWμE). IDWμE was functionalized with a self-assembled monolayer (SAM) of thioctic acid for antigen immobilization. The SAM functionalized IDWμE were characterized by atomic force microscopy, Energy Dispersive X-Ray Spectroscopy, and X-ray photoelectron spectroscopy. The covalent immobilization of IgG-Fc onto the SAM modified electrode arrays was characterized morphologically via AFM and electrochemically via cyclic voltammetry and electrochemical impedance spectroscopy. Impedimetric measurements in the presence of redox probe (Potassium ferrocyanide and potassium ferricyanide) showed a significant change in both the impedance spectrum and charge transfer resistance upon IgM-RF binding. Impedance measurements were target specific and linear with an increase in IgM-RF concentrations between 1 and 200 IU mL−1 in redox probe and human serum, respectively. The sensor showed detection limits of 0.6 IU mL−1 in the presence of redox probe and 0.22 IU mL−1 in the human serum. The biosensor exhibited good reproducibility (relative standard deviation (RSD), 4.96%) and repeatability (RSD, 2.31%) with an acceptable selectivity towards IgM-RF detection. The sensor also showed a good stability for 3 weeks at 4 °C in 1X PBS. Therefore, the sensitive and stable immunosensor exhibiting IDWμE features can be integrated with a miniaturized potentiostat to develop a sensing system that detects IgM-RF for point-of-care applications.

Label-free impedimetric immunosensor based on one-step co-electrodeposited poly-(pyrrole-co-pyrrole-1-propionic acid) and reduced graphene oxide polymer modified layer for the determination of melamine

A label-free electrochemical impedimetric immunosensor was developed to monitor melamine, and it was successfully applied for the detection of melamine in spiked dairy samples. The poly (pyrrole-co-pyrrole-1-propionic acid) and reduced graphene oxide polymer layer (rGO-PPYPPA) were co-electrodeposited on the electrode using a one-pot method, thus obtaining a copolymer layer with favorable electrochemical properties. A competitive immunoassay was used for quantitative analysis of melamine. The rGO-PPYPPA copolymer improved the conductivity of the sensor and provided adequate anchor sites for antibody binding, thus enhancing immuno-binding. The specific immune-interactions at the electrode surface that restrict the electron transfer of the redox probe Fe(CN)63−/4− lead to changes in electrochemical impedance, and the changes in impedance correlated to specific concentrations of melamine. The immunosensor displays a wide detection range (10−11−10−2 mol L−1), low limit of detection (1.2 × 10−11 mol L−1) and acceptable recovery (84.6%–100.3%) in real samples. The results provide a simple and versatile impedimetric immunosensing platform, with great potential for real-world applications.

A highly sensitive signal-amplified gold nanoparticle-based electrochemical immunosensor for dibutyl phthalate detection

A simple and signal-amplified, label-free electrochemical impedimetric immunosensor for dibutyl phthalate (DBP), a type of phthalate ester, was developed using gold nanoparticles (AuNPs) induced signal amplification. The approach was based on an indirectly competitive binding system of coating antigen that coated on antigen/chitosan/MWCNTs@GONRs/GCE (modified electrodes), target (DBP) and anti-DBP antibody. Signal amplification was performed via the enlargement of AuNPs through NADH-promoted catalytic precipitation, which caused a marked increase in the electron-transfer resistance and electrostatic repulsion after an AuNP-labeled second antibody conjugated to anti-DBP. Under the optimized conditions, this method displayed: (i) low detection limits (7ng/mL, 10 times lower than the traditional ELISA method using the same antibody); (ii) satisfactory accuracy (recoveries, 86.0–120.4%) and agreement with corresponding ELISA method; (iii) high tolerance to some environmental interferents; and (iv) low cost and low sample consumption (6µL). Our results demonstrate the great potential and high efficiency of this immunosensor in practical applications for the cost-effective and sensitive monitoring of DBP in aquatic environments.

A novel impedimetric immunosensor for the detection of chicken interferon-gamma based on a polythionine and gold nanoparticle modified glassy carbon electrode

Currently, there is no effective detection method for chicken interferon-γ (ChIFN-γ), therefore it is of great significance to develop a new, accurate and sensitive method for the rapid detection of ChIFN-γ to solve the problems which are directly impeding the in-depth research of immune regulatory factors and poultry immune responses, and which are preventing the analysis and evaluation of the immunity of poultry in diseases or after vaccination. This paper is the first report of a novel label-free electrochemical impedimetric immunosensor for the sensitive detection of ChIFN-γ, based on a 3E3 monoclonal antibody against chicken interferon-γ (denoted as 3E3McAb) immobilized on a glassy carbon electrode modified with polythionine and gold nanoparticle self-assembled layers. The surface modification steps were characterized by scanning electron microscopy and electrochemical impedance spectroscopy (EIS). Detection was performed by measuring the relative change in impedance before and after the 3E3McAb–ChIFN-γ reaction by EIS. Under the optimized conditions, the relative change in impedance was proportional to the logarithmic value of ChIFN-γ concentrations in the range of 0.10 to 1.0 × 103 ng mL−1 (r = 0.9946) with a detection limit of 0.10 ng mL−1. Compared to the traditional method of enzyme-linked immunosorbent assay, the proposed method performed a low-cost, sensitive, quantitative detection of ChIFN-γ in real samples with satisfactory results, indicating that the developed immunoassay could be a promising alternative approach for the detection of ChIFN-γ.

Impedimetric immunoglobulin G immunosensor based on chemically modified graphenes

Immunosensors which display high sensitivity and selectivity are of utmost importance to the biomedical field. Graphene is a material which has immense potential for the fabrication of immunosensors. For the first time, we evaluate the immunosensing capabilities of various graphene surfaces in this work. We propose a simple and label-free electrochemical impedimetric immunosensor for immunoglobulin G (IgG) based on chemically modified graphene (CMG) surfaces such as graphite oxide, graphene oxide, thermally reduced graphene oxide and electrochemically reduced graphene oxide. Disposable electrochemical printed electrodes were first modified with CMG materials before anti-immunoglobulin G (anti-IgG), which is specific to IgG, was immobilized. The principle of detection lies in the changes in impedance spectra of the redox probe after the attachment of IgG to the immobilized anti-IgG. It was found that thermally reduced graphene oxide has the best performance when compared to the other CMG materials. In addition, the optimal concentration of anti-IgG to be deposited onto the modified electrode surface is 10 μg ml(-1) and the linear range of detection of the immunosensor is from 0.3 μg ml(-1) to 7 μg ml(-1). Finally, the fabricated immunosensor also displays selectivity for IgG.

Development of an impedimetric immunosensor for the determination of 3-amino-2-oxazolidone residue in food samples

The use of furazolidone in food animals has been banned in European Union (EU) because of its carcinogenicity and mutagenicity on human health, but its continued misuse is widespread. Therefore, there is an urgent need for a simple, reliable, and rapid method for the detection of its marker residue, 3-amino-2-oxazolidinone (AOZ), in food products. In this regard, a sensitive and reliable electrochemical method was presented to detect AOZ based on a novel label-free electrochemical impedimetric immunosensor to address this need. The immobilization of monoclonal antibody against AOZ (denoted as AOZ-McAb) on the gold electrode was carried out through a stable acyl amino ester intermediate generated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide (NHS), which could condense antibodies on the self-assembled monolayer (SAM). The detection of AOZ was performed by measuring the relative change in charge transfer resistance before and after AOZ and AOZ-McAb immunoreaction by electrochemical impedance spectroscopy (EIS). Under the optimized conditions, the relative change in charge transfer resistance was proportional to the logarithmic value of AOZ concentrations in the range of 20.0 to 1.0 × 10 4 ng mL −1 ( r = 0.9987). Moreover, the proposed immunosensor has a high selectivity to AOZ alone with no significant response to the metabolites of other nitrofuran antibiotics, such as 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), semicarbazide (SEM), and 1-aminohydantoin hydrochloride (AHD). This protocol has been applied to detect AOZ in food samples with satisfactory results.

Detecting 5-morpholino-3-amino-2-oxazolidone residue in food with label-free electrochemical impedimetric immunosensor

This paper reported a novel label-free electrochemical impedimetric immunosensor for sensitive detection of metabolite of furaltadone, 5-morpholino-3-amino-2-oxazolidone (denoted as AMOZ), based on monoclonal antibody against AMOZ (denoted as AMOZ–McAb) immobilized on gold electrode modified with 1,4-benzenedithiol and gold nanoparticle self-assembled layers. The sensitive steps of surface modification have been characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The immunoreaction between AMOZ and AMOZ–McAb directly triggered a signal via electrochemical impedance spectroscopy measurement. Under the optimized conditions, the relative change in impedance was proportional to the logarithmic value of AMOZ concentrations in the range of 1.0 to 1.0 × 106 ng/mL (r = 0.9991) with the detection limit of 1.0 ng/mL. The advantages of the immunosensor are exhibited in its high sensitivity, wide linear range and better stability. The proposed impedimetric immunosensor was successfully applied to the determination of AMOZ in animal derived food with excellent recoveries.

Label-free impedimetric immunosensor for sensitive detection of ochratoxin A

A novel label-free electrochemical impedimetric immunosensor for sensitive detection of ochratoxin A (OTA) was reported. A two-step reaction protocol was elaborated to modify the gold electrode. The electrode was first derivatized by electrochemical reduction of in situ generated 4-carboxyphenyl diazonium salt (4-CPDS) in acidic aqueous solution yielded stable 4-carboxyphenyl (4-CP) monolayer. The ochratoxin A antibody was then immobilized making use of the carbodiimide chemistry. The steps of the immunosensor elaboration and the immunochemical reaction between ochratoxin A and the surface-bound antibody were interrogated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The impedance change, due to the specific immuno-interaction at the immunosensor surface was utilized to detect ochratoxin A. The increase in electron-transfer resistance (ΔRet) values was linearly proportional to the concentration of OTA in the range of 1–20ngmL−1, with a detection limit of 0.5ngmL−1.