Amperometric Immunosensor Research Articles

An Amperometric Immunosensor Based on an Ionic Liquid and Single-Walled Carbon Nanotube Composite Electrode for Detection of Tetrodotoxin in Pufferfish.

An amperometric immunosensor based on a composite electrode of single-walled carbon nanotubes and ionic liquid n-octylpyridinum afluorophosphate (SWCNT-ILE) was developed for the determination of tetrodotoxin (TTX). Compared with the glassy carbon electrode (GCE), the electrode combined advantages of carbon nanotubes and ionic liquid, which exhibited the excellent antifouling ability of p-nitrophenol (PNP) so that it remarkably improved the stability of the p-nitrophenyl phosphate-based sensor. Combining the enzyme-linked immune sorbent assay (ELISA) by alkaline phosphatase (AP) and magnetic particles immobilized with antigens, a real-time assay of tetrodotoxin was developed by amperometric immunosensors. Under the optimium condition, the developed sensor demonstrated a linear range of tetrodotoxin from 2 to 45 ng/mL with a low detection limit of 5 ng/mL. Furthermore, the amperometric immunosensor was applied to determine TTX in real samples and could be used as an effective and sensitive sensor for direct detection of tetrodotoxin within 20 min.

Disposable amperometric immunosensor for simple and sensitive determination of aflatoxin B1 in wheat

A disposable and label-free electrochemical immunosensor was developed for aflatoxin B1 (AFB1) determination in wheat based on immobilization of anti-AFB1 antibodies on the chitosan-gold nanoparticles (chitosan-AuNPs) modified gold microelectrode. The chitosan-AuNPs nanocomposite formed by one-step electrodeposition using chronoamperometry not only exhibited improved conductivity but also offered porous three-dimensional morphology, plentiful amine groups and good biocompatibility for antibody immobilization via covalent binding. To evaluate the effect of electrodeposition conditions on the surface structure and amperometric response of the chitosan-AuNPs modified microelectrode, both scanning electron microscope (SEM), cyclic voltammetry (CV) and energy dispersive spectroscopy (EDS) were studied under the different parameters of deposition potential and time. The morphology and electrochemical characterizations of the immunosensor were also investigated by atomic force microscopy (AFM) and CV. The developed immunosensor demonstrated good linearity between highly sensitive current response and AFB1 concentration in two ranges of 0.2–2ngmL−1 and 2–30ngmL−1, with a detection limit of 0.12ngmL−1 (S/N=3). For assay of AFB1 in wheat matrix, the linear range from 1.6 to 32ngmL−1 was obtained considering the extraction of the sample.

Amperometric IFN-γ immunosensors with commercially fabricated PCB sensing electrodes

Lab-on-a-Chip (LoC) technology has the potential to revolutionize medical Point-of-Care diagnostics. Currently, considerable research efforts are focused on innovative production technologies that will make commercial upscaling of lab-on-chip products financially viable. Printed circuit board (PCB) manufacturing techniques have several advantages in this field. In this paper we focus on transferring a complete IFN-γ enzyme-linked immune-sorbent assay (ELISA) onto a commercial PCB electrochemical biosensing platform, We adapted a commercially available ELISA to detect the enzyme product TMB/H2O2 using amperometry, successfully reproducing the colorimetry-obtained ELISA standard curve. The results demonstrate the potential for the integration of these components into an automated, disposable, electronic ELISA Lab-on-PCB diagnostic platform.

Electrochemical immunosensor for sensitive determination of transforming growth factor (TGF) - β1 in urine

The first amperometric immunosensor for the quantification of TGF-β1, a cytokine proposed as a biomarker for patients having or at risk for renal disease, is described in this work. The immunosensor design involves disposable devices using carboxylic acid-functionalized magnetic microparticles supported onto screen-printed carbon electrodes and covalent immobilization of the specific antibody for TGF-β1 using Mix&Go polymer. A sandwich-type immunoassay was performed using biotin-anti-TGF and conjugation with peroxidase-labeled streptavidin (poly-HRP-Strept) polymer. Amperometric measurements were carried out at −0.20V by adding hydrogen peroxide solution onto the electrode surface in the presence of hydroquinone as the redox mediator. The calibration plot allowed a range of linearity extending between 15 and 3000pg/mL TGF-β1 which is adequate for the determination of the cytokine in plasma and urine. The limit of detection, 10pg/mL, is notably improved with respect to those obtained with ELISA kits. The usefulness of the immunosensor for the determination of low TGF-β1 concentrations in real samples was evaluated by analyzing spiked urine at different pg/mL concentration levels.

Chlamydia trachomatis (CT) infection and Vγ9Vδ2 T cells in women with recurrent spontaneous abortions

This chapter presents a procedure for electrochemical synthesis of a biotinylated polypyrrole co-polymer film and electrochemical investigations for the determination of the most appropriate enzyme marker. Preparation of the electropolymerized biotinylated copolymer film include following steps: (1) electrolysis of a mixture of pyrrole-lactobioamide monomer (4mM) and pyrrole–biotin monomer (4mM) by controlled potential at 0.8V in acetonitrile electrolyte.(2) Platinum electrodes are to be used for GOX-B. (3) Glassy carbon electrodes are employed for HRP and PPO-B. Amperometric immunosensors is used for the detection of antitoxin. Immunosensor sensitivity was determined as the slope of the initial linear part of the calibration curve. From the results it clearly appears that the HRP system led to the best analytical performances—namely, the lowest detection limit recorded for a signal to noise ratio of 3 and the highest sensitivity. In addition, the comparison of the Imax values recorded for the theoretical formation of an anti-CT monolayer and hence for a HRP monolayer unambiguously corroborates the preceding results.

Amperometric sensing of HIF1α expressed in cancer cells and the effect of hypoxic mimicking agents

Hypoxia inducible factor 1 alpha (HIF1α) overexpression was detected in cancerous cells using an amperometric immunosensor with a nano-bioconjugate. The sensor probe was fabricated by covalently immobilizing the antibody (anti-HIF1α) onto a composite layer of functionalized conducting polymer [2,2:5,2-terthiophene-3-(p-benzoic acid)] (pTTBA) formed on a layer of gold nanoparticles (AuNPs). A nano-bioconjugate with hydrazine and a secondary antibody of HIF1α (sec-Ab2) attached on AuNPs reveals the immunoreaction at the sensor probe through the catalytic reduction of H2O2 by hydrazine at −0.35V vs. Ag/AgCl. Morphology and performance of the sensor probe were characterized using FE-SEM, XPS, EIS, and cyclic voltammetry. The calibration plot at optimized experimental conditions shows a dynamic range of 25–350pM/mL with a detection limit of 5.35±0.02pM/mL. The reliability of the sensor was evaluated using non-cancerous Vero and cancerous MCF-7 cell lysates, where the HIF1α expression was compared with three cancerous cell lines MCF-7, PC-3, and A549. Furthermore, the sensor probe confirms the stable expression of HIF1α in the A549 lung cancer cells when exposing them to hypoxic mimicking agents Co, Ni, and Mn ions. Of these, Co ions show the highest stabilization effect on HIF1α followed by Ni and Mn ions, respectively.

Portable detection of clenbuterol using a smartphone-based electrochemical biosensor with electric field-driven acceleration

We have developed a fast, highly sensitive and low-cost biosensing system for the detection of clenbuterol (CLB), using a homemade mobile electrochemical device with an electric field-driven acceleration strategy. This system consists of an embedded circuit in smartphone for signal processing and a screen-printed carbon electrode (SPCE) modified with multi-walled carbon nanotubes (MWNTs) and goat anti mouse-immunoglobulin G (IgG) sensing layer (MWNTs-I-layer). CLB monoclonal antibody was assembled through its binding to the surface-confined antibody. Such modified electrodes were used for rapid and sensitive amperometric immunosensing detection of CLB. Horseradish peroxidase-coupled CLB (CLB–HRP) competed with free CLB in the samples to bind the monoclonal antibody. By using this mobile system, we could detect CLB ranging from 0.3ng⋅mL−1 to 100ng⋅mL−1 with the detection limit of 0.076ng⋅mL−1. The whole competitive-type detection process was finished within 6min. We expect this device can meet the requirements for field detection of various food security-related species.

Competitive immunosensor based on gliadin immobilization on disposable carbon-nanogold screen-printed electrodes for rapid determination of celiotoxic prolamins

The first competitive disposable amperometric immunosensor based on gliadin-functionalized carbon/nanogold screen-printed electrodes was developed for rapid determination of celiotoxic prolamins. To date, no competitive spectrophotometric or electrochemical immunoassays have yet been successfully applied to gluten detection in processed food samples, which require the use of complex prolamin extraction solutions containing additives with denaturing, reducing and disaggregating functions. Thus, in this work, great effort was put into the optimization and performance evaluation of the immunosensor in terms of suitability as a screening tool for analysis of cereal-based food samples. For this purpose, aqueous ethanol or complex extraction mixtures, as the patented Cocktail Solution®, were proved effective in the extraction of gliadin. Good sensitivity was achieved after optimization of the immunocompetitive assay, giving limit of detection and limit of quantitation of 8 and 22ng/ml of gliadin, respectively, for ethanol extracts. The immunosensor was proved to be suitable also for samples extracted with Cocktail Solution® after a proper dilution. Analysis of real samples of different flours proved the suitability of the immunosensing device as a powerful tool for safety assessment of raw materials used for the formulation of dietary products for celiac disease patients. This immunosensor combines good analytical performance using a very simplified set-up protocol with suitability for rapid screening analysis performed using inexpensive and portable instrumentation. Graphical abstract Depiction of the development and working principle of the competitive immunosensor.

Electrochemical clenbuterol immunosensor based on a gold electrode modified with zinc sulfide quantum dots and polyaniline

A nanocomposite consisting of zinc sulfide quantum dots and polyaniline (ZnSQD@PANI) was placed on a gold electrode along with antibody against clenbuterol to give an amperometric immunosensor for clenbuterol. Compared to the use of pristine PANI, the electrode modified with the ZnSQD@PANI nanocomposite adsorbs clenbuterol antibody much better and therefore exhibits higher sensitivity to clenbuterol. The biosensor, when operated at a working potential of 0.21 V (vs. Ag/AgCl), displays a detection limit as low as 5.5 pg⋅mL−1 and works over the 0.01 to 10 ng⋅mL−1 concentration range. Related species such as salbutamol and ractopamine, urine components such as urea and uric acid, and the ions Ca(II), Na(I), and K(I) do not interfere.

Design and development of an amperometric immunosensor based on screen-printed electrodes

An amperometric immunosensor has been developed from laboratory fabricated screen-printed electrodes for the detection of bovine serum albumin antigen.

Fabrication of a label-free electrochemical immunosensor using a redox active ferrocenyl dendrimer

An amperometric immunosensor based on a redox active ferrocenyl end-grafted PAMAM dendrimer provides highly sensitive detection of immunoglobulin, down to 2 ng mL−1.

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.

Immunosensors for human cardiac troponins and CRP, in particular amperometric cTnI immunosensor

In this paper, the review of immunosensors for selected cardiovascular disease markers: human cardiac troponins and human C-reactive protein (CRP) is presented. In particular, (cTnI) amperometric immunosensor for cTnI measurements in the concentration range useful in medical diagnostics, based on the developed earlier human CRP amperometric immunosensor, is described. The human cTnI is recommended as one of specific myocardial damage biomarkers, and is considered as the “gold standard”, whereas the human CRP is used as the powerful, nonspecific, supplementary biomarker of cardiovascular disease. Carbon, graphite and platinum pastes, used for fabrication of our immunosensor working electrode (WE), were investigated. In the developed simple measuring procedure, based on a direct solid phase enzyme-linked immunosorbent assay (ELISA), for the first time ascorbic acid monophosphate was used for cTnI detection as a substrate in enzymatic reaction of alkaline phosphatase labelling antibodies. Disposable amperometric graphite immunosensors, made on polyester film by means of microdispensing robot, suitable for determination of cTnI in the concentration range 0–35μg/L with the sensitivity 0.67μA/(μg/L) and linear correlation coefficient 0.91 were obtained.

Network nanostructured polypyrrole hydrogel/Au composites as enhanced electrochemical biosensing platform.

In this work, a new network nanocomposite composed of polypyrrole hydrogel (PPy hydrogel) loaded gold nanoparticles (AuNPs) was prepared. The PPy hydrogel was directly synthesized by mixing the pyrrole monomer and phytic acid, and the mixed solution can be gelated to form hydrogel at once. The three-dimensional network nanostructured PPy hydrogel not only provided a greater effective surface area for increasing the quantity of immobilized biomolecules and facilitated the transport of electrons and ions, but also exhibited an improved conductivity. Meanwhile, the electrodeposited AuNPs on the PPy hydrogel can further increase the specific surface area to capture a large amount of antibodies as well as improve the capability of electron transfer. The network PPy hydrogel/Au nanocomposites were successfully employed for the fabrication of a sensitive label-free amperometric immunosensor. Carcinoembryonic antigen (CEA) was used as a model protein. The proposed immunosensor exhibited a wide linear detection range from 1 fg mL−1 to 200 ng mL−1, and an ultralow limit of detection of 0.16 fg mL−1 (S/N = 3), and it also possessed good selectivity. Moreover, the detection of CEA in ten human serums showed satisfactory accuracy compared with the data determined by ELISA, indicating that the immunosensor provided potential application for clinical diagnosis.

A new surface plasmon resonance immunosensor for triazine pesticide determination in bovine milk: a comparison with conventional amperometric and screen-printed immunodevices.

A detailed comparison was made of the analytical features of a new Surface Plasmon Resonance (SPR) immunodevice for triazine pesticide determination with those of two other amperometric (conventional and screen-printed) immunosensors and the advantages and disadvantages of the SPR method were thoroughly investigated. For conventional amperometric and screen-printed devices, “competitive” assays were used; conversely, the SPR transduction technique allowed a “direct” measurement format to be used. As far as the main analytical data are concerned, the SPR method does not seem to offer substantial advantages. Nevertheless the measurement time is much shorter and the measurement itself much easier to perform. Lastly several applications and recovery tests were carried out on bovine milk samples, before and after spiking, to check for triazine pesticides in the samples, obtaining satisfactory results.

A sensitive label-free amperometric immunosensor for alpha-fetoprotein based on gold nanorods with different aspect ratio.

A simple and accurate label–free amperometric immunosensor for α–fetoprotein (AFP) detection is developed based on gold nanorods (GNRs) with different aspect ratio and compared with gold particles (GNPs). The positively charged GNRs and GNPs due to the surface immobilized cetyltrimethyl ammonium bromide (CTAB) can adsorb the negatively charged AFP antibody (Ab) directly. The presence of the GNRs not only enhanced the immobilized amount of biomolecules, but also improved the electrochemical properties of the immunosensor. With the aid of GNRs, the electrochemical signal was greatly enhanced in comparison with GNPs. Under optimal conditions, the proposed immunosensor could detect AFP in a linear range from 0.1 to 200 ng/mL with a detection limit of 0.04 ng/mL (signal–to–noise ratio = 3), and it also possessed good reproducibility and storage stability. Moreover, the detection of AFP in five human serum samples also showed satisfactory accuracy. The proposed methodology was potentially attractive for clinical immunoassay.

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

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

Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

Tuberculosis (TB) has been a major public health problem, which can be better controlled by using accurate and rapid diagnosis in low-resource settings. A simple, portable, and sensitive detection method is required for point-of-care (POC) settings. This paper studies an amperometric biosensor using a microtip immunoassay for a rapid and low-cost detection of Mycobacterium tuberculosis (MTB) in sputum. MTB in sputum is specifically captured on the functionalized microtip surface and detected by electric current. According to the numerical study, the current signal on the microtip surface is linearly changed with increasing immersion depth. Using a reference microtip, the immersion depth is compensated for a sensing microtip. On the microtip surface, target bacteria are concentrated and organized by a coffee-ring effect, which amplifies the electric current. To enhance the signal-to-noise ratio, both the sample processing and rinsing steps are presented with the use of deionized water as a medium for the amperometric measurement. When applied to cultured MTB cells spiked into human sputum, the detection limit was 100 CFU mL−1, comparable to a more labor-intensive fluorescence detection method reported previously.

An amperometric immunosensor for detection of Streptococcus suis serotype 2 using a nickel–gold nanocomposite as a tracer matrix

For quick, sensitive detection of SS2, a simple sandwich-type amperometric immunosensor was developed. A complex between AuNCs and NiHCFNPs acted as an Ab2 substrate and signal probe, as well as an AA oxidation catalyst to amplify the response signal.

Polydopamine and graphene oxide synergistically modified Prussian blue electrochemical immunosensor for the detection of alpha-fetoprotein with enhanced stability and sensibility

In this paper, a simple and highly sensitive amperometric immunosensor for the determination of alpha-fetoprotein based on multi-functional gold nanoparticles–polydopamine-Prussian blue–graphene oxide nanocomposites (Au–PDA-PB–GO) was fabricated.