Amperometric Immunosensor Research Articles

A novel immunosensor based on an alternate strategy of electrodeposition and self-assembly

A novel amperometric immunosensor for the determination of carcinoembryonic antigens (CEA) was developed. Firstly, ordered multilayer films of Prussian blue (PB) and multiwalled-carbon nanotube/polyethylenimine/Au (MWNT–PEI–Au) nanocomposite were fabricated onto the surface of a glassy carbon electrode via alternate electrodeposition and self-assembly. Then a layer of chitosan mixed with gold nanoparticles was cast onto the surface of the electrode. Subsequently, the electrode was coated with antibody (Ab1) and blocked with BSA. The morphology of the MWNT–PEI–Au nanocomposite was characterized by transmission electron microscopy (TEM). The fabrication process of the ordered multilayer structure and immunosensor were characterized by scanning electron microscopy (SEM) and electrochemical measurements, respectively. The proposed fabrication strategy effectively ensured the stability of the Prussian blue as electron mediator. Under optimal conditions, the fabricated immunosensor exhibited a good response to CEA, with a detection range from 0.5 to 160ng/mL and a detection limit of 0.08ng/mL at 3δ. The current fabricated immunosensor exhibited good sensitivity, selectivity, and long-term stability. Furthermore, current study demonstrated the promising application of the alternate strategy based on electrodeposition and self-assembly for the construction of biosensor.

Comparison of three immunosensor methods (surface plasmon resonance, screen-printed and classical amperometric immunosensors) for immunoglobulin G determination in human serum and animal or powdered milks

Within the framework of research carried out by our team aimed at developing new immunological methods to determine proteins such as immunoglobulins G in different biological matrixes, for instance, serum and milk, tests were performed on several immunosensors based on different transducer types, i.e. amperometric (classical or screen-printed) electrodes for hydrogen peroxide. Lastly the feasibility of constructing immunosensors based on surface plasmon resonance (SPR) was investigated. “Competitive” immunological procedures were used in the first two cases. Conversely, the surface plasmon resonance transduction technique allowed a “direct” measurement. Applications were performed on human serum, powdered milks for babies and particularly on several animal milks, in the case of buffalo milk seeking a routine control method to identify possible inflammatory affections in the animals.

A label-free amperometric immunosensor for alpha-fetoprotein determination based on highly ordered porous multi-walled carbon nanotubes/silica nanoparticles array platform

In this contribution, we develop a novel type of multi-walled carbon nanotubes/silica nanoparticles array (MWCNTs/SiO2) composite by the galvanostatic deposition technique. In this process, MWCNTs were mixed with ammonium fluorosilicate to form a doped precursory sol solution, the electrochemically generated hydroxyl ions at negative potentials promote the hydrolysis of ammonium fluorosilicate, and the simultaneously generated hydrogen bubbles assist the formation of three-dimensional porous silica matrix, further facilitating the construction of SiO2 NPs array with uniform distribution, as confirmed by scanning electron microscopy. The formation of SiO2 NPs array provides a higher surface area and biocompatible microenvironment for retaining the native activity of the immobilized biomolecules. Further incorporation of MWCNTs into SiO2 NPs array improves the electronic conductivity and enhances the electroactive surface area of the film, making the fabricated three-dimensional porous MWCNTs/SiO2 electrode an ideal platform for further immobilization of alpha-fetoprotein (AFP), as a model protein. This method achieves ultrasensitive detection of AFP antigen with ferricyanide as a probe. The obtained results provided a linear response range from 0.1 to 30.0ngmL−1 AFP antigen with a lower detection limit of 0.018ngmL−1. This work implies that the biocompatible and controllable three-dimensional porous SiO2 NPs array possessed potential applications for biosensing.

Ultrasensitive detection of kanamycin in animal derived foods by label-free electrochemical immunosensor.

A label-free amperometric immunosensor based on graphene sheet(GS)-nafion(Nf)/thionine(TH)/Pt nanoparticles modified electrode is proposed for the ultrasensitive detection of kanamycin. The anti-kanamycin antibody was immobilised onto the GS-Nf/TH/Pt modified glassy carbon electrode (GCE) through electrostatic adsorption. TH was selected as an electron transfer mediator, and the synergistic effects among GS, TH and Pt were investigated. The electroactivity of TH was greatly improved in the presence of GS and Pt due to their good electron-transfer ability. The proposed immunosensor shows good performance such as low detection limit (5.74 pg/mL), wide linear range (from 0.01 to 12.0 ng/mL), high stability, and good selectivity in the detection of kanamycin. The reliability of the developed immunosensor was proved by determination of kanamycin in animal derived foods with satisfactory results.

Development of a novel label-free amperometric immunosensor for the detection of okadaic acid

Okadaic acid (OA), a lipophilic phycotoxin is mainly produced by toxigenic dinoflagellates. The need to develop high performing methods for OA analysis able to improve the traditional ones is evident. In this work, a novel experimental methodology for label-free detection of OA was developed. Protein G magnetic beads (protein-G-MBs) modified gold electrode was used to immobilize anti-OA monoclonal antibody (anti-OA-MAb). Preliminary, colorimetric tests were performed in order to validate protein-G-MBs and anti-OA-MAb reaction. Electrochemical detection was carried out by differential pulse voltammetry in ferri/ferrocyanide solution. The limit of detection value obtained (0.5μgL−1) validated the developed electrochemical immunosensor as a promising tool for routine use. The matrix effect and the recovery rate were also assessed with real samples, showing a good percentage of recovery.

Probe functionalization with a Rhop-3 antibody: toward a Rhop-3 antigen immunosensor for detection of malaria

The antibody specific for the malaria protein, Rhop-3, and FL-Rhop-3, were immobilized on the surface of a gold electrode modified with cysteamine. Colloidal gold was used to enhance the detection signal for Rhop-3 antigens. The Rhop-3 antibody was also immobilized on gold electrodes preactivated with dithiobis(succinimidyl proprionate) (DSP). Immobilization was performed at room temperature and at 37 °C. Cyclic voltammetry (CV) was used to monitor the interaction between the immobilized antibody and its cognate antigen in solution, using ferricyanide, K3Fe(CN)6, as reporting electroactive probe. Tests indicate recognition of Rhop-3 protein by the immobilized antibody. Antigen recognition was enhanced by incubation at 37 °C compared with room-temperature incubation. Our results suggest that an immunosensor can be developed and optimized to aid detection of Rhop-3 antigens in samples from malaria patients. As far as we are aware, this is the first amperometric immunosensor targeting Rhop-3 antigen as a malaria biomarker.

Disposable Amperometric Immunosensor for Detecting Toxoplasma Gondii-Specific IgM Based on Graphene and CeO2-Au

Toxoplasma gondii-specific IgM (Tg-IgM) is a sensitive and reliable marker for early diagnosing toxoplasmosis infection in pregnant women. In this study, a disposable amperometric immunosensor for sensitive detecting Tg-IgM was constructed based on graphene (GP) and CeO2-Au nanoparticle. The GP was treated with chitosan (CS) to obtain a stable graphene-chitosan (GPCS) composite membrane. The CeO2-Au nanoparticle which was used for the preparation of the amperometric immunosensor was synthesized by the hydrothermal method. GPCS composite membrane was first assembled onto the screen-printed carbon electrode (SPCE) for adsorbing the CeO2-Au nanoparticle. The toxoplasma gondii antigen (Tg-Ag) was subsequently adsorbed by CeO2-Au nanoparticle to obtain the proposed immunosensor. Scanning electron microscope (SEM) and transmission electron microscope (TEM) were employed to characterize the construction process of the immunosensor. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) were used to study the electrochemical properties of the immunosensor. The results indicated that, CS employed in this study improved the dispersion and homogeneity of GP, as well as enhance the stability of the proposed immunosensor. The use of GPCS composite membrane and CeO2-Au nanoparticle had good conductivity and prominent biocompatibility, which excellently improved the sensitivity of the immunosensor. In the optimal conditions, the proposed immunosensor can be applied to quantify the concentration of Tg-IgM in a wide linear range from 7.5×10 AU•mL to 24 AU•mL with a correlation coefficient of -0.998 and a low detection limit of 4.4×10 AU•mL (S/N=3). The novel proposed immunosensor for detecting Tg-IgM in human serum specimens with satisfactory results had also been proved. In addition, the Tg-IgM contents determined by the immunosensor agreed well with the ELISA measurement. Furthermore, the proposed amperometric immunosensor exhibited some advantages, such as high selectivity, long-term stability, good repeatability, low sample consumable, and short analysis time, which were suitable for detecting toxoplasma infection in pregnant women.

Electrochemical immunosensor modified with self-assembled monolayer of 11-mercaptoundecanoic acid on gold electrodes for detection of benzo[a]pyrene in water

Well-oriented bio-conjugates on gold electrode surfaces will indirectly influence the molecular recognition of antigens to surface bound antibodies thus improving the detection performance of electrochemical immunosensors. This paper describes the modification of self-assembled monolayers (SAMs) on gold electrode surface with 11-mercaptoundecanoic acid (11-MUA). Activation of carboxylic acid terminal was performed by reaction of a mixture of water soluble carbodiimide and N-hydrosuccinimide (NHS) on the electrode surfaces. Characterisation of the SAM formation on the gold electrode was performed using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and contact angle measurements. An amperometric immunosensor was developed for the screening of polycyclic aromatic hydrocarbons (PAHs) in water. The system consists of gold as the working electrode, platinum as the counter electrode and a Ag/AgCl reference electrode. This three electrode system is integrated on a single chip. The measurement employs the enzyme-linked immunosorbent assay (ELISA) principle. Benzo[a]pyrene (BaP) was detected using an immunological reaction by measuring the alkaline phosphatase (AP) enzymatic reaction towards the substrate para-amino phenyl phosphate (pAPP). A competitive assay was performed within the electrode using AP as the labelled-enzyme. A lower limit of detection (5.6 ng ml(-1)) of BaP was achieved after the activation of the mixture of carbodiimide and succinimide with the alkanethiol SAM on the gold electrode in comparison to that obtained for the unmodified electrode (14.2 ng ml(-1)). The developed surface functionalised sensor demonstrated acceptable reproducibility and good stability, with a wide linear response to BaP (4-140 ng ml(-1)).

Reagentless amperometric immunosensor for α-1-fetoprotein based on assembly of nano-Au@SiO2 composite

A novel immunosensor for the direct amperometric determination of α-1-fetoprotein (AFP) was constructed by immobilization of PDDA/nano-Au@SiO2 onto a Prussian blue modified gold electrode surface, followed by adsorption of α-1-fetoprotein antibody onto nano-Au@SiO2 composite particles with large specific areas and good biocompatibility. Finally, BSA was employed to block the possible remaining active sites of the nano-Au@SiO2 layer to avoid non-specific adsorption. The modification procedure was electrochemically monitored by cyclic voltammetry. The performance and influencing factors of the resulting immunosensor were studied in detail. Under the optimized conditions, there is a linear relation between response current and the logarithm of the concentrations of AFP in the concentration range of 0.05–200 ng mL−1 with a detection limit of 0.02 ng mL−1 (S/N = 3).

A bienzymatic amperometric immunosensor exploiting supramolecular construction for ultrasensitive protein detection

Self-assembly of a ferrocene-appended polymer bearing an antigen fragment and lactate oxidase on a cyclodextrin-modified surface provides a highly sensitive, easy-to-operate and self-sufficient immunosensor.

Synergic effect of multi-walled carbon nanotubes and gold nanoparticles towards immunosensing of ricin with carbon nanotube–gold nanoparticles–chitosan modified screen printed electrode

An amperometric immunosensor for the specific detection of Ricinus communis is reported. Screen printed electrodes (SPEs) were modified with gold nanoparticles (GNPs) loaded multiwalled carbon nanotubes (MWCNTs)-chitosan (Ch) film. The ratio of MWCNT and GNP was optimised to get best electrochemically active electrode. Sandwich immunoassay format was used for the immunosensing of ricin. The revealing antibodies tagged with the enzyme alkaline phosphatase (ALP) converts the substrate 1-naphthyl phosphate into 1-naphthol that was determined with the amperometric technique. The amperometric current obtained was correlated with the concentration of ricin. The prepared GNP-MWCNT-Ch-SPE showed high stability due to the Ch film, short response time with good reproducibility and increased shelf life of the electrodes immobilised with antibodies. The electrochemical activity of the electrode improved because of optimization of composition of CNTs and gold nanoparticles. Under the optimal conditions, the modified electrode showed a wide linear response to the concentration of ricin in the range of 2.5-25 ng mL(-1) with a limit of detection of 2.1 ng mL(-1) and with a relative standard deviation of 5.1% and storage life of 32 days.

Amperometric immunosensor for the determination of IgA deficiency in human serum samples

An electrochemical immunosensor for the detection of human IgA deficiency in real human blood serum has been developed. The performance of the immunosensor presents a large but sensitive dynamic range that allows the determination of non-deficient IgA levels (>70μg/mL) as well as of severe IgA deficiencies (0.5–5.0μg/mL). The assay architecture involves the immobilisation of a coating antibody on an electrode surface using carboxylic-ended bipodal alkane-thiol self-assembled monolayers (SAMs). The long chain bipodal SAM presents intercalated poly(ethylenglycol) groups that confer the immunosensor the ability to retain its optimum performance in very complex matrices and serum with negligible non-specific adsorption phenomena. Amperometric optimisation of the assay resulted in limits of detection of 142ng/mL in just 30min total assay time. Real patients’ serum samples were analysed using the developed electrochemical immunosensor demonstrating an excellent correlation in terms of sensitivity and reproducibility compared with standard enzyme linked immunosorbent assays (ELISA).

Amperometric Immunosensor Based on a Protein A/Deposited Gold Nanocrystals Modified Electrode for Carbofuran Detection

In this paper, an amperometric immunosensor modified with protein A/deposited gold nanocrystals (DpAu) was developed for the ultrasensitive detection of carbofuran residues. First, DpAu were electrodeposited onto the Au electrode surface to absorb protein A (PA) and improve the electrode conductivity. Then PA was dropped onto the surface of DpAu film, used for binding antibody Fc fragments. Next, anti-carbofuran monoclonal antibody was immobilized on the PA modified electrode. Finally, bovine serum albumin (BSA) was employed to block the possible remaining active sites avoiding any nonspecific adsorption. The fabrication procedure of the immunosensor was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), respectively. With the excellent electroconductivity of DpAu and the PA’s oriented immobilization of antibodies, a highly efficient immuno-reaction and detection sensitivity could be achieved. The influences of the electrodeposition time of DpAu, pH of the detection solution and incubation time on the current response of the fabricated immunosensor were investigated. Under optimized conditions, the current response was proportional to the concentration of carbofuran which ranged from 1 to 100 ng/mL and 100 ng/mL to 100 μg/mL. The detection limit was 0.1924 ng/mL. The proposed carbofuran immnuosensor exhibited high specificity, reproducibility, stability and regeneration performance, which may open a new door for ultrasensitive detection of carbofuran residues in vegetables and fruits.

Electrochemical Immunosensor Coupled to Cyclone Air Sampler for Detection of Escherichia coli DH5α in Bioaerosols

AbstractThe portable detector ImmunoSMART was combined with the cyclone air sampler to detect the model bioagent E. coli DH5α in bioaerosols. The selected capture antibody was immobilized on gold electrodes, antibody‐peroxidase conjugate served as a tracer. In buffer, the amperometric immunosensor measured 103 to 108 CFU/mL within 30 min. Detection in air was realized in a closed aerosol chamber. Automated dissemination of the cells, sampling and measurement allowed remote testing of the cyclone/immunodetector. The level of 150 CFU/L in air was clearly indicated (20 min analysis including 5 min sampling) and independently confirmed using the slit sampler and agar cultivation. The results seem promising for further extended tests in both aerosol chamber and field trial conditions.

In vitro monitoring of i-NOS concentrations with an immunosensor: The inhibitory effect of endocrine disruptors on i-NOS release

The amperometric immunosensor has demonstrated the toxicity of endocrine disrupters (EDs) through monitoring the in vitro i-NOS concentration change, where the antibody of inducible nitric oxide synthase (i-NOS) was immobilized on the conducting polymer–gold nanoparticles composite. The performance of the sensor and the experimental parameters affecting the immunoreaction were optimized. Neuronal cells treated by EDs decreased in the in vitro i-NOS concentration. The effect of bisphenol A (BPA) on the i-NOS concentration released in the cells was investigated with different incubation times, and the interfering by nonspecific binding species present in a neuronal cell lysate was also examined. Of all the tested EDs, BPA showed the inhibitoriest effect and the minimum inhibitory concentration of BPA affecting the i-NOS concentration was 0.09 ± 0.005 μM. The result shows that monitoring of i-NOS in the neuronal cells treated by EDs will be a useful method to evaluate the toxic behavior of EDs.

Amperometric immunosensor for the detection of Escherichia coli O157:H7 in food specimens

A novel, label-free amperometric immunosensor has been developed for the rapid detection of heat-killed Escherichia coli O157:H7 ( E. coli O157:H7). This immunosensor was prepared as follows. First, the long-chain, amine-terminated alkanethiol 11-amino-1-undecanethiol hydrochloride (AUT) was self-assembled onto a gold electrode surface to form an ordered, oriented, compact, and stable monolayer possessing –NH 2 functional groups that could immobilize massive gold nanoparticles (GNPs). Next, chitosan-multiwalled carbon nanotubes–SiO 2/thionine (CHIT–MWNTs–SiO 2@THI) nanocomposites and GNPs multilayer films were prepared via layer-by-layer (LBL) assembly. The surface area enhancement from the LBL assembly of the multilayer films improves the stability of the immobilized CHIT–MWNTs–SiO 2@THI. More important, the sensitivity and stability of the immunosensor can be enhanced proportionally to the quantity of the THI mediator immobilized on the electrode surface. Finally, the E. coli O157:H7 antibody (anti- E. coli O157:H7) was covalently bound to the GNP monolayer and its bioactivity was measured by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy (TEM) was employed to characterize the morphology of the MWNTs, CHIT–MWNTs, and CHIT–MWNTs–SiO 2@THI. Under optimal conditions, the calibration curve for heat-killed E. coli O157:H7 has a working range of 4.12 × 10 2–4.12 × 10 5 colony-forming units (CFU)/ml, and the total assay time was less than 45 min.

A Sandwich-type Amperometric Immunosensor Based on One-dimensional Assembly of Magnetic DNA Nanoprobes

Abstract A new type of one-dimensional magnetic nanoprobe was fabricated based on a DNA chain fixed with numerous Fe 3 O 4 (core)/ZrO 2 (shell) nanomagnetic beads (ZMPs) modified with horseradish peroxidase (HRP)-labeled anti-α-fetoprotein secondary antibody (AFPAb 2 ). An electrochemical immunosensor for AFP was developed using the nanoprobe DNA/(ZMPs-HRP-AFPAb 2 ) n and AFP-antibody (AFPAb 1 )-immobilized glassy carbon electrode (GCE) modified with nanogold. A sandwich-type immunocomplex could be formed between the immobilized AFPAb 1 on the GCE, the AFP sample, and the probe DNA/(ZMPs-HRP-AFPAb 2 ) n ; moreover, the linked HRP on the probes could catalyze the electrochemical reaction of carbamide peroxide in the presence of hydroquinone. Under optimal conditions, the immunosensor response showed a linear relationship with the AFP concentration in the range 0.01–25 μg L −1 , with a detection limit of 4 ng L −1 . The immunosensor was used to determine the AFP level in serum samples, and the results were satisfactory. The proposed amperometric immunosensor was sensitive, provided a rapid detection, and was controllable by a magnetic field. This study provides a strategy for the one-dimensional assembly and development of highly sensitive immunosensors.

A strategy for signal amplification using an amperometric enzyme immunosensor based on HRP modified platinum nanoparticles

A novel sandwich-type electrochemical enzyme immunosensor for sensitive detection of α-1-fetoprotein (AFP) using a signal amplification strategy based on the enzyme and the catalytic activity of Pt nanoparticles (PtNPs) is investigated in this work. Sensitivity was greatly amplified by the detection mode which was based on dual signal amplification: (1) secondary antibody labeled HRP-functionalized PtNPs were introduced on the electrode surface through biotin–avidin interactions; (2) the biotin-labeled HRP was further attached to HRP-functionalized PtNPs to assemble a dense HRP film, which resulted in significant enhancement of the current response. The adjusted (but not random) orientation of anti-AFP on the nanohybrid film of gold nanoparticles/multi-walled carbon nanotubes (AuNP/MWNT) was accomplished using an avidin–biotin immobilization technique. The nanohybrid film AuNP/MWNT can improve the response of the immunosensors due to its higher exposed surface area, high catalytic activity which facilitates electron transfer and excellent biocompatibility. The performance of the amperometric immunosensor was evaluated and optimized for best performance. The detection limit for AFP was found to be 0.08 ng/mL at S/ N = 3 and the detection concentration were in the range between 0.25 and 100 ng/mL.

A Renewable Amperometric Immunosensor for hs-CRP Based on Functionalized Fe<sub>3</sub>O<sub>4</sub>@Au Magnetic Nanoparticles Attracted on Fe (III) Phthlocyanine/Chitosan-Membrane Modified Screen-Printed Carbon Electrode by a Magnet

A novel disposable screen-printed immunosensor for rapid determination of highly sensitive C reactiveprotein (hs-CRP) in human serum has been developed in the experiment. The sensor was constructed on one screen-printed carbon electrode (SPCE) with HRP labeled anti-hs-CRP antibody functionalized Fe3O4@Au magnetic nanoparticles (HRP labeled anti hs-CRP/ Fe3O4@Au) as the biorecognition probes attracted on the surface of Fe (III) phthalocyanine (FePc)/ chitosan membrane modified screen-printed carbon electrode (SPCE|FePc/Chit/chitosan) by external magnetic field. FePc was acted as electron immediate. The modified electrode shows an excellent electrocatalytic activity for hs-CRP in phosphate buffer solution (pH=7.0). After the immunosensor is incubated with hs-CRP antigen solution at 37°C for 20 min, the access of activity center of the HRP to electrode is partly inhibited, which leads to a linear decrease of the catalytic efficiency of the HRP to the reduction of immobilized FePc by H2O2 at –50 mV in hs-CRP’s concentration ranges from 1.2 to 200 ng/mL. The detection limit was 0.5ng/mL. The immunosensor was successfully utilized for determination of hs-CRP in real serum samples of heart disease patients, whose results were consistent with that by ELISA method. The accuracy and precision of the assay were 91.5-104.4% and 15.8-24.4%, respectively. The immunosensor was reusable once constructed and can be regenerated by adding new nanoprobes on the surface of basal electrode through magnet on its bottom. It can greatly reduce the detection cost which is valuable for the early diagnosis of tumors.

Amperometric Immunosensor for Chagas’ Disease Using Gold CD‐R Transducer

AbstractChagas’ disease has been spread mainly through blood transfusion or blood components, therefore the development of a device for neglected diseases with high specificity and sensitivity is important to public health. This work describes the development of an amperometric immunosensor for determination of Chagas’ disease through a gold‐based electrode obtained from a recordable compact disc modified with 4‐(methylmercapto)benzaldehyde for the immobilization of Tc85 protein of the Trypanosoma cruzi. The immunoassays were carried out using positive and negative sera from Chagas’disease patients and immunoglobulin conjugated with peroxidase enzyme. The immunosensor presented −0.949 µA as cut‐off value and was applied in sera samples.