Competitive Electrochemical Immunosensor Research Articles

Construction of a competitive electrochemical immunosensor based on sacrifice of Prussian blue and its ultrasensitive detection of alpha-fetoprotein

Effective signal amplification is a prerequisite for ultrasensitive detection by electrochemical immunosensors. For quantitative and ultrasensitive detection of alpha-fetoprotein (AFP), we designed a competitive electrochemical immunosensor and transferred the immunoreactivity from the electrode surface to the cuvette. AFP antigen was captured using AFP primary antibody (Ab1) immobilized on magnetic nanobeads (MBs), and ZIF-8 nanomaterials attached to secondary antibody (Ab2) were used as probes. MBs helped retain the sandwich structure in the test tube through incubation and washing steps. Then, an appropriately fixed excess of sodium ethylenediaminetetraacetic acid (EDTA) solution was added to the cuvettes, resulting in etching of Zn ions from ZIF-8 and formation of Zn-EDTA complexes. After magnetic separation, a certain amount of supernatant is added dropwise to the Prussian blue (PB)-modified electrode (GCE), and Fe ions (from PB) complex with the remaining EDTA in the supernatant, thus reducing the signal response value of PB. The higher the AFP concentration, the lower the amount of free EDTA in the supernatant, the less the destruction of PB, and therefore the higher the current. Under optimal conditions, the immunosensor achieved ultra-sensitive detection of AFP in the range of 10−4 ng/mL-100 ng/mL with a limit of detection (LOD) as low as 0.032 pg/mL (S/N = 3). The excellent performance provides an important tool for the early screening and detection of AFP.

A nanozyme-based competitive electrochemical immunosensor for the determination of E-selectin.

Ananozyme-based competitive electrochemical immunosensor has been developedfor the quantitative determination of E-selectin, a common adhesion molecule expressed by activated endothelial cells. A glassy carbon electrode modified with poly(azure A) and E-selectin antibody (GCE/PAA/Ab) was prepared. Au-CuO nanocomposite-labeled E-selectin, CD62E-Au-CuO, was synthetized, and it could be captured on GCE/PAA/Ab owing to the immunoreaction. The immobilized nanocomposites on GCE/PAA/Ab/CD62E-Au-CuO acted as nanozymes and were involved in the electrocatalytic process that caused the high cathodic peak current. The assembly of GCE/PAA/Ab/CD62E-Au-CuO was inhibited by E-selectin due to the competitive immunoreaction, which resulted in adecrease ofthe current signal. The cathodic peak current difference at - 0.35V vs SCEwas proportional to the concentration of E-selectin in the range 0.500-500ngmL-1, and the limit of detection was estimated to be 226pgmL-1. The cell morphology observation, the cell viability test, and the electrochemical measurement indicate that the injury of human umbilical vein endothelial cells was aggravated, and the release of E-selectin from the injured cells was gradually accelerated when the NaCl content in the growth medium increased.

A Novel Label-Free Electrochemical Immunosensor Based on a Self-Assembled Monolayer-Modified Electrode for Polychlorinated Biphenyl (PCB) in Environmental Analysis

PCBs (polychlorinated biphenyls) are a very large group of organic compounds that have between two and ten chlorine atoms attached to the biphenyl. These compounds have an acute impact as environmental pollutants, causing cancer and other adverse health effects in humans. It is therefore imperative to develop techniques for the cost-effective detection of PCBs at very low concentrations in ecosystems. In this paper, a novel label-free, indirect, competitive electrochemical immunosensor was first developed with a PCB-BSA conjugate. It is shown herein to compete with free PCBs for binding to the anti-PCB polyclonal primary antibody (IgY). Then, we used a secondary antibody to enhance the sensitivity of the sensor for the detection of PCB in a sample. It has been successfully immobilized on an 11-mercaptoundecanoic acid (11-MUA)-modified gold electrode via a carbodiimide-coupling reaction using cross-linking 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) on the electrode surface. The immunosensor was investigated by cyclic voltammetry and differential pulse voltammetry in a standard solution of [Fe(CN)6]3−/4−. A linear range of 0.011–220 ng/mL−1 and a limit of detection (LOD) of 0.11 ng/mL−1 for PCBs detection were achieved by the developed immunosensor, showing advantages over conventional assays. The novel label-free electrochemical immunosensor discussed in this paper is a solution for simple, rapid, cost-effective sample screening in a portable, disposable format. The proposed immunosensor has good sensitivity, and it can prove to be an adequate real-time monitoring solution for PCBs in soil samples or other samples.

Competitive Electrochemical Immunosensor for Detection of Capsaicinoids in Edible Vegetable Oil Based on Computer Aided Molecular Modeling

The adulteration of edible vegetable oil is a serious threat to the health of consumers. Previous studies have shown that capsaicinoids could be used as a marker of adulteration to identify vegetable oils. The key sites of antibody and capsaicinoids were analyzed by computer aided molecular modeling (CAMM), which provided a basis for the selection of modified material and the implanting of antigen and antibody on the electrode in the subsequent construction of the electrochemical immunosensor. Simultaneously, it was determined that the different forces between capsaicinoids and antibody were the important reason for the differences in sensitivity of capsaicinoids. On this basis, combined with the good conductivity and excellent redox characteristic of Fe3O4NPs@CeO2, a competitive immunosensor was constructed. Under the optimal conditions, the proposed immunosensor provided a linear range of 10–3 to 104 ng ml−1 with a detection limit of 0.33 pg ml−1. This study may broaden the interaction between capsaicinoids and antibody, encourage the design of novel performance-enhanced antibody, and provide a new method for the detection of capsaicinoids.

Dual mode competitive electrochemical immunoassay for dibutyl phthalate detection based on PEI functionalized nitrogen doped graphene-CoSe2/gold nanowires and thionine-Au@Pt core-shell

In this work, a sensitive dual mode competitive electrochemical immunosensor based on differential pulse voltammetry (DPV) and square wave voltammetry (SWV) response modes for quantitative determination of dibutyl phthalate (DBP) was successfully established. According to diethylenetriamine (DETA) assisted hydrothermal strategy, a novel N-doped graphene/CoSe2 nanobelt composite was prepared. Afterwards, PEI functionalized NG-CoSe2 and gold nanowires (AuNWs) were further compounded as a preliminary signal amplification platform for immunosensor. Thionine (Thi)-loaded [email protected] and secondary antibody (Ab2) connected as a signal label. The immobilized coating antigen DBP-BSA and the DBP compete for limited antibody binding sites. The concentration of DBP can be determined by DPV detect the Thi signal loaded on the [email protected]2 label. It can also be determined by SWV mode to detect the current generated by the reaction system of H2O2 and o-phenylenediamine (o-PD) catalyzed by [email protected] with peroxidase like properties. Under optimum conditions, the linear ranges of the DPV and SWV modes were 1 pg mL−1∼1 μg mL−1. And LOD were 0.486 pg mL-1 (DPV) and 0.711 pg mL-1 (SWV), respectively. Besides, it exhibited outstanding selectivity, reproducibility, stability and can be used for sensitive detection of DBP in real samples.

Dopamine/mucin-1 functionalized electro-active carbon nanotubes as a probe for direct competitive electrochemical immunosensing of breast cancer biomarker

Abstract Mucin-1 (MUC-1) is associated with a broad range of human epithelia including gastric, lung and colorectal. In this work, a direct competitive electrochemical immunosensor based on gelatin modified transduction platform was designed. Dopamine (DA)/mucin-1 functionalized electro-active carbon nanotubes were employed as signal generating probes in the construction of electrochemical immunosensor for early stage diagnosis of breast cancer. The gelatin modified electrode served as a support to immobilize antibody (anti-MUC-1), while electrochemical response of functionalized electro-active carbon nano probes was used for quantitative measurement of MUC-1. Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) were carried out to characterize the transduction surface at different fabrication steps. The developed immunosensor permitted the detection of MUC-1 in the linear range of 0.05−940 U/mL, with a detection limit (LOD) of 0.01 U/mL. The immunosensor showed recovery values in the range of 96% for human serum sample analysis, demonstrating its practical applicability.

A competitive electrochemical immunosensor based on bimetallic nanoparticle decorated nanoflower-like MnO2 for enhanced peroxidase-like activity and sensitive detection of Tetrabromobisphenol A

In this work, a novel competitive electrochemical immunosensor was developed based on gold palladium bimetallic nanoparticles which were successfully synthesized and modified by amine (-NH2) functionalized-nanoflower-like manganese oxide (NH2-fMnO2) for the rapid and sensitive detection of TBBPA in environmental waters. The MWCNTs modified GCE was employed to load TBBPA-antigens (Ag) through the effects of adsorption for the design of a highly sensitive immunosensor, which provided a large surface area for the specific recognition between antigen-antibody reactions. Meanwhile, AuPd-NH2-fMnO2, the signal amplifier was employed as a carrier tag to label secondary antibody (Ab2), which showed excellent catalytic ability toward H2O2. The AuPd NPs boosted TBBPA oxidation, thereby generating improved signals. Hence, under optimal conditions, the competitive biosensor presented superior performances, as good sensitivity (LOD, 0.10 ng/mL; based on S/N = 3) and satisfactory accuracy (recoveries, 84–120 %; CV, 2.35–6.24 %), the proposed method was employed to analyze TBBPA in waters from various sources, indicating a great potential for the sensitive determination of trace TBBPA in aquatic environments.

Copper-Based Metal–Organic Frameworks Loaded with Silver Nanoparticles as Electrochemical Immunosensors for Diethylstilbestrol

An ultrasensitive competitive electrochemical immunosensor for the quantitative detection of diethylstilbestrol (DES) was constructed by linear-sweep voltammetry. In this work, copper-based metal–o...

Competitive electrochemical immunosensor for the detection of unfolded p53 protein in blood as biomarker for Alzheimer’s disease

Alzheimer's disease is one of the most common causes of dementia nowadays, and its prevalence increases over time. Because of this and the difficulty of its diagnosis, accurate methods for the analysis of specific biomarkers for an early diagnosis of this disease are much needed. Recently, the levels of unfolded isoform of the multifunctional protein p53 in plasma have been proved to increase selectively in Alzheimer's Disease patients in comparison with healthy subjects, thus entering the list of biomarkers that can be used for the diagnosis of this illness.We present here the development of an electrochemical immunosensor based on nanostructured screen-printed carbon electrodes for the quantification of unfolded p53 in plasma samples. The sensor shows a suitable linear range (from 2 to 50 nM) for its application in real blood samples and a very low limit of detection (0.05 nM). The concentration of unfolded p53 has been accurately detected in plasma of elderly people in healthy conditions, subjects with mild cognitive impairment (MCI) and Alzheimer's Disease (AD) subjects, obtaining results with no significant differences to those provided by an ELISA assay. These results support the possibility of measuring unfolded p53 levels with a cheap, simple and miniaturized device with a promising future for point-of-care applications in the early diagnosis of Alzheimer's dementia.

A highly sensitive competitive immunosensor based on branched polyethyleneimine functionalized reduced graphene oxide and gold nanoparticles modified electrode for detection of melamine

Branched polyethyleneimine functionalized reduced graphene oxide (BPEIGn) was prepared by a one-step reaction, catalyzed by NaOH, using branched polyethyleneimine (BPEI) and graphene oxide (GO) without reductant hydrazine hydrate or sodium borohydride. The branched polyethylenimine acted as both a grafting agent and a reducing agent of GO. An competitive electrochemical immunosensor based on the Au/sodium mercaptopropanesulfonate/BPEIGn/gold nanoparticles/melamine (Au/MPS/BPEIGn/AuNPs/Mel) modified electrode was constructed for the determination of melamine. The double amplification of BPEIGn and AuNPs increased the sensitivity of the sensor. The melamine was detected by differential pulse voltammetry (DPV) in buffer solution (pH 7.4) containing K3(Fe(CN)6]/K4[Fe(CN)6]. Under optimized conditions, the proposed melamine immunosensor showed a linear relationship in the concentration range of 1 × 10−6 to 1 μM, with a detection limit of 2.66 × 10−7 μM.

Competitive electrochemical immunosensor for maduramicin detection by multiple signal amplification strategy via hemin@Fe-MIL-88NH2/AuPt

Maduramicin (MD) is a type of monoglycoside polyether ionophore antibiotic that can effectively treat coccidiosis and facilitate animal growth. However, its extensive and excessive use brings potential risk to human health. Herein, an electrochemical immunosensor based on indirect competitive format was fabricated for analysis of MD residue in eggs by a multiple signal amplification system. Initially, Au nanoparticles were deposited onto glassy carbon electrode surface to load the coating antigen MD-BSA and to improve conductivity. Then the signal amplification platform was constructed by encapsulating hemin into Fe-MIL-88 NH2 metal-organic frameworks (hemin@MOFs), and then the obtained composites were decorated with AuPt nanoparticles. The synthesized hemin@MOFs/AuPt was not only used as a signal amplification mediator, but also utilized as a carrier for immobilization of horseradish peroxidase-conjugated affinipure goat anti-mouse antibody (Ab2-HRP) and horseradish peroxidase (HRP). The constructed hemin@MOFs/AuPt-Ab2-HRP bioconjugates could effectively amplify the current signal since hemin@MOFs, AuPt and HRP all exhibited high catalytic activity towards the hydrogen peroxide. Moreover, the established immunosensor showed high sensitivity and stability during the detection procedure. With the synergistic catalytic effect of hemin@MOFs, AuPt and HRP, a wide detection range of 0.1–50 ng mL−1 and a low detection limit of 0.045 ng mL−1 were achieved (S/N = 3), respectively. Ultimately, the developed method displayed excellent performance in practical applications, providing a promising probability to detect other veterinary drug residues to guarantee food safety.

A competitive immunosensor for ultrasensitive detection of sulphonamides from environmental waters using silver nanoparticles decorated single-walled carbon nanohorns as labels

A novel competitive electrochemical immunosensor based on Au nanodendrites (Au NDs)/silver nanoparticles (Ag NPs) @single-walled carbon nanohorns (SWCNHs) was established for sensitive determination of sulphonamides (SAs) in aquatic environments. The indirectly competitive binding system of the approach was composed of coating antigen that coated on Au NDs/glass carbon electrode (GCE), the target and primary antibody (Ab1). When Ab2@Ag NPs@SWCNHs was captured by coating antigen (Cag)- Ab1 complex, massive Ag+ will be released from electrode in the presence nitric acid (HNO3), consequently, the generated Ag+ will significantly amplify the electrochemical signal, which would be recorded by the linear sweep voltametry (LSV). Meanwhile, the used three-dimensional Au nanodendrites (Au NDs) could increase the conductivity of the electrode and the size of the active surface area to improve the antigen-loading. Under the optimal conditions, the immunosensor showed a good linear relationship for sulfamethazine (SMZ)ranged in 0.33–63.81 ng/mL with a detection limit of 0.12 ng/mL (LOD, based on 90% inhibition). In addition, the proposed approach exhibited satisfactory accuracy and precision (recoveries, 79.25–119.25%; CV, 2.14–9.58%), it can be applied for rapid analysis of the trace pollutants from environmental waters.

Dual mode competitive electrochemical immunoassay for B-type natriuretic peptide based on GS/SnO2/polyaniline-Au and ZnCo2O4/N-CNTs

A sensitive dual mode competitive electrochemical immunosensor for the detection of B-type natriuretic peptide (BNP) was successfully fabricated, which based on differential pulse voltammetry (DPV) and amperometric i-t curve response modes. Polyaniline (PAN) and tin dioxide (SnO2) were loaded on graphene sheets (GS), which could effectively promote the electron transfer process, thereby amplifying the current signal and increasing the sensitivity of the immunosensor. To promote biocompatibility, gold nanoparticles (Au) were incorporated on GS/SnO2/PAN (GS/SnO2/PAN-Au). GS/SnO2/PAN-Au complex was gotten to act as the platform which could provide a clearly DPV signals. N-doped carbon nanotubes (N-CNTs) embellished by ZnCo2O4 quantum dots (ZnCo2O4/N-CNTs) with excellent catalytic properties for the reduction of H2O2 was gotten to act as the label of the antibody-BNP (Ab), providing an obviously current signal through amperometric i-t curve method. A large quantity of BNP could be stable loaded in the modified electrode via GS/SnO2/PAN-Au with excellent electrical conductivity and good biocompatibility, which could compete with target-BNP to combine Ab that labelled by ZnCo2O4/N-CNTs. Under the optimum conditions, the immunosensor exhibited remarkable analytical performance of a linear range from 0.01 pg/mL to 1 ng/mL with a detection limit of 3.4 fg/mL for quantitative detection of BNP (S/N = 3). This method was able to become a universal strategy for other biological detection.

Electrochemical indirect competitive immunoassay for ultrasensitive detection of zearalenone based on a glassy carbon electrode modified with carboxylated multi-walled carbon nanotubes and chitosan

The authors describe an electrochemical immunoassay for ultrasensitive detection of the mycotoxin zearalenone (ZEA). A nanocomposite was prepared from carboxy-functionalized multi-walled carbon nanotubes and chitosan (cMWCNTs/Chit). The morphology and electrochemical performance of the materials was characterized by field-emission scanning electron microscopy, atomic force microscopy, differential pulse voltammetry, cyclic voltammetry, and electrochemical impedance spectroscopy. In this assay, ZEA–BSA conjugated covalently to activated cMWCNTs/Chit film, then the indirect competition between ZEA–BSA and free ZEA when immobilization of excess anti-ZEA. The secondary antibody is labeled with the enzyme alkaline phosphatase which can hydrolyze the substrate 1-naphthylphosphate to produce 1-naphthol which gives a stable and strong anodic electrochemical signal at a low working voltage of 0.3 V (vs. Ag/AgCl). The use of the modified GCE results in a strongly enhanced electrochemical current response. Compared with conventional methods, the established immunosensor exhibited a high level of sensitivity. Under optimal conditions, this immunoassay can quantify ZEA in the 10 pg·mL−1 to 1000 ng·mL−1 concentration range with a detection limit of 4.7 pg·mL−1 and the sensitivity is 0.51 μA·μM−1·cm−2. The method was applied to the determination of ZEA in cereal and feedstuff samples. Results showed satisfactory recovery and good consistency with high-performance liquid chromatography. Therefore, the indirect competitive electrochemical immunosensor provide a viable tool based on bioanalysis.

Indirect competitive electrochemical immunosensor for hepatitis A virus antigen detection

Indirect competitive electrochemical immunosensor for hepatitis A virus antigen (HAV) detection was developed by immobilizing HAV on the surface of carbon nanopowder paste electrode. The monitoring of the target HAV antigen was performed using a secondary antibody labeled with peroxidase. The analytical signal corresponds to an amperometric current resulting from the addition of hydrogen peroxide and hydroquinone as a redox mediator. A calibration curve was obtained by plotting the intensity of the amperometric signal versus the concentrations of HAV antigens. A low detection limit for HAV antigen was attained and estimated to be 26·10−5IU·mL−1. The sensitivity was also investigated and compared with indirect competitive ELISA. Moreover, the immunosensor provides exact results over a broad linear range from 2·10−4 to 5·10−3IU·mL−1, (R2=0.998). The average relative standard deviation (RSD) of this method was less than 3% for the assays performed on the same day and 7% for the measurements made on different days. The immunosensor exhibits a good stability and high biological selectivity for HAV antigen detection and was successfully applied for the determination of HAV in water samples.

Investigate electrochemical immunosensor of cortisol based on gold nanoparticles/magnetic functionalized reduced graphene oxide

A sensitively competitive electrochemical immunosensor for the detection of cortisol was successfully developed based on gold nanoparticles and magnetic functionalized reduced graphene oxide (AuNPs/MrGO). In order to construct the base of the immunosensor, the MrGO was initially fabricated by chemical cross-linking and used to modify the nafion pretreated glassy carbon electrode. Subsequently, the surface of electrode was modified by AuNPs via electrochemical deposition. A variety of cortisol (Cor) can be firmly loaded in the AuNPs/MrGO with large specific surface area and good bioactivity to construct the basic electrode (Cor/AuNPs/MrGO/Nafion@GCE), which was characterized by the cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), respectively. Due to the cortisol on the surface of basic electrode and samples can competitively combine with the cortisol antibody labelled by horseradish peroxidase (HRP-Strept-Biotin-Ab). Finally, the detection signal of electrochemical immunosensor (HRP-Strept-Biotin-Ab-Cor/AuNPs/MrGO/Nafion@GCE) in the test liquid had negative correlations with the concentration of cortisol in samples. The AuNPs/MrGO with excellent electrical conductivity being applied, the electrochemical response of the immunosensor was immensely amplified. The immunosensor displayed excellent analytical performance for the detection of cortisol range from 0.1 to 1000ng/mL with a detection limit of 0.05ng/mL at 3σ. Moreover, compared the developed immunoassay with commercially available enzyme linked immunosorbent assay, the proposed method showed good precision, acceptable stability and reproducibility, indicating the immunosensor could be used for the sensitive, efficient and real-time detection of cortisol in real samples. Therefore, the present strategy provides a novel and convenient method for clinical determination of cortisol.

Competitive electrochemical immunosensor for the detection of histamine based on horseradish peroxidase initiated deposition of insulating film

Competitive electrochemical immunosensor for the detection of histamine based on graphene modified electrode and horseradish peroxidase (HRP) initiated deposition of insulating film was developed. After the immobilization of anti-histamine antibodies onto graphene surface, free histamine and HRP tagged histamine molecules will compete to bind with the antibodies. The captured HRP could then catalyze the polymerization of 3,3′-dimethoxybenzidine (DB) in the presence of H2O2 to produce the deposition of an insulating polymer film, poly(3,3′-dimethoxybenzidine) (PDB) onto electrode surface. The deposited insulating PDB film resulted in the decrease of the electrochemical current of the electrode in Fe(CN)63−/4− solution, and the current change is proportional to the concentration of histamine detected. The proposed immunosensor displays wide linear range towards histamine detection (1pg/mL–1ng/mL) with a detection limit of 0.5pg/mL. The immunosensor was applied to the detection of histamine in serum samples with satisfactory results.

Competitive electrochemical immunosensor for amyloid-beta 1-42 detection based on gold nanostructurated Screen-Printed Carbon Electrodes

Alzheimer's disease is the most common form of dementia, characterized by the progressive accumulation of plaques with amyloid-beta peptide of 42 amino acids as one of the primary constituents. A disposable electrochemical immunosensor for the detection of amyloid-beta 1-42 is developed. Screen-Printed Carbon Electrodes nanostructured with gold nanoparticles generated “in situ” are used as the transducer surface. The immunosensing strategy consists in a competitive immunoassay: biotin-amyloid-beta 1-42 immobilized on the electrode surface and the analyte (amyloid-beta 1-42) compete for the anti-amyloid-beta 1-42 antibody. The electrochemical detection is carried out using an alkaline phosphatase labeled anti-rabbit IgG antibody. The analytical signal is based on the anodic stripping of enzymatically generated silver by cyclic voltammetry. The immunosensor achieved shows a low limit of detection (0.1ng/mL) and a wide linear range (0.5–500ng/mL).

A competitive electrochemical immunosensor for the detection of human interleukin-6 based on the electrically heated carbon electrode and silver nanoparticles functionalized labels

A facile one-step electrochemical reduction method was developed to prepare electrochemically reduced graphene oxide (ERGO) and gold-palladium bimetallic nanoparticles (AuPdNPs) as the platform of immunosensor. A novel competitive electrochemical immunosensor was then proposed by combining the ERGO-AuPdNPs platform with silver nanoparticles (AgNPs) functionalized polystyrene bionanolabel for the sensitive detection of human interleukin-6 (IL-6). An electrically heated carbon electrode (HCPE) was introduced in the detection procedure of the immunosensor, and further improved the sensitivity. The immunosensor exhibited a wide linear response to IL-6 ranging from 0.1 to 100000 pg mL(-1) with a detection limit of 0.059 pg mL(-1). The proposed method showed good precision, broad linear range, acceptable stability and high reproducibility, and could be used for the detection of IL-6 in real samples, which possessed promising application in clinical research.

An electrochemical immunosensor for digoxin using core–shell gold coated magnetic nanoparticles as labels

A simple, sensitive, and low-cost immunosensor was designed for the detection of digoxin through core-shell gold coated magnetic nanoparticles (Fe3O4-Au-NPs) as an electrochemical label. Having had such a large potential for a variety of applications, Fe3O4-Au-NPs have attracted a considerable attention and are actively investigated recently. Digoxin is a cardiac glycoside which, at high level, can indicate an increased risk of toxicity. This new competitive electrochemical immunosensor was developed based on antigen-antibody reaction employing antigen (Ag) labeled Fe3O4-Au-NPs and PVA modified screen-printed carbon electrode surface in order to detect the serum digoxin. The structures of Fe3O4-Au-NPs were studied by transmission electron microscopy, X-ray diffraction and Fourier transformed infrared spectroscopy. Cyclic voltammetry and differential pulse voltammetry (DPV) were employed to determine the physicochemical and electrochemical properties of immunosensor. DPV was employed for quantitative detection of digoxin in biological samples. The developed immunosensor was capable to detect digoxin in the range from 0.5 to 5ngmL(-1), with a detection limit as low as 0.05ngmL(-1). The proposed method represented acceptable reproducibility, stability, and reliability for the rapid detection of digoxin in serum samples.