Electrochemical Magnetoimmunosensor Research Articles

Microfluidic electrochemical magnetoimmunosensor for ultrasensitive detection of interleukin-6 based on hybrid of AuNPs and graphene.

Cytokines are important factors in the early diagnosis of autoimmune diseases and require high sensitivity, high selectivity and quantitative detection. We proposed a miniaturized electrochemical magneto-immunosensor (EC-MIS) on portable interleukin-6 (IL-6) detection based on this requirement. Firstly, a micro-fabricated working electrode is electrochemically modified with a hybrid of reduced graphene oxide (rGO) and gold nanoparticles (AuNPs). Increased surface area and enhanced charge transfer rate improve the performance of this immunosensor on sensitivity. Secondly, magnetic beads attached with the capture antibody (cAb) are employed in sandwich immunoassay. This kind of immunoassay is immobilized on the working electrode surface by an external magnet to enrich the analyte IL-6. Thirdly, the last two features are combined and integrated on a microfluidic device in order to restrict the sample at certain areas and ease the operation of detection. With our prototypic EC-MIS operated in amperometric mode, we have achieved the detection of IL-6 with a linear range from 0.97 to 250pg/mL and a limit of detection (LOD) of 0.42pg/mL. Real serum samples were demonstrated and compared with benchtop equipment's results.

An array-based electrochemical magneto-immunosensor for early neonatal sepsis diagnostic: Fast and accurate determination of C-reactive protein in whole blood and plasma samples

An array-based electrochemical magneto-immunosensor (a-EMI) constituted by eight screen-printed carbon electrodes is proposed for the determination of C-reactive protein (CRP). Remarkably, the analytical usefulness of the approach was demonstrated not only by analyzing whole blood samples, but plasma ones from newborns with suspicion of sepsis, which sample volume is hardly available.The a-EIM enabled the accurate determination of CRP in these unique samples (Er < 3%) with excellent intra (CV<4%, n=8) and inter-assays (CV<9%, n=8) precision, using only 5 µL of clinical sample, covering the sepsis diagnostic range and employing only 15 min for total assay. The electrode array enabled to work with up to 8 tailored dilutions simultaneously accordingly to the CRP clinical diagnostic range. It constitutes by itself a valuable advantage for speed and reliability improvements in the sample analysis and further sepsis diagnosis.These results non-only highly met the clinical needs for sepsis newborns diagnosis, but they opened new perspectives in low volumes-based diagnostics.

Paper microfluidics on screen-printed electrodes for simple electrochemical magneto-immunosensor performance

Using magnetic beads (MB) allows producing relatively fast and simple electrochemical magneto-immunosensors. However, this type of sensor implies carrying out serial MB incubation and washing steps in tubes using external magnets, a procedure that requires user training and is tedious and time consuming. In this work, a paper device has been developed that allows performing MB washing and electrochemical detection on-chip with less intervention of the user.In order to achieve this goal, we optimized a single-step magneto-immunoassay for matrix metalloproteinase 9 (MMP-9) detection. Electrochemical detection was then carried out using a customized multiplexed holder that incorporated 8 screen-printed carbon electrodes (SPCE) and a movable multiplexed fluidic module. This paper device provided along the assay sequential MB confinement under static conditions, reagent adsorption and MB washing under flow conditions, and electrochemical detection under static conditions. As we show, the sensor produced in this way achieved detection of MMP-9 in about 10 min, with lineal response spanning 0.03–2 ng mL−1, detection limit of 0.01 ng mL−1 and quantification limit of 0.09 ng mL−1. Furthermore, MMP-9 quantification in 18 plasma samples from patients correlated linearly with the estimates of a reference ELISA that took >5 h. These results demonstrate that paper microfluidics can be exploited to simplify magneto-immunosensor manipulation, providing fast, simple and sensitive assay formats.

An immunomagnetic separation and bifunctional Au nanoparticle probe-based multiamplification electrochemical strategy

A novel electrochemical magnetoimmunosensor for the rapid and sensitive detection of carcinoembryonic antigen (CEA) was fabricated based on a combination of high-efficiency immunomagnetic separation, bifunctional Au-nanoparticle (bi-AuNP) probes, and enzyme catalytic amplification. The reaction carrier magnetic beads (MBs) effectively reduced the toxicity of the complex sample to the working electrode, and the signal carrier bi-AuNP probes loaded a large amount of signal molecules, both of which enhanced the signal-to-noise ratio and further improved the detection sensitivity. A detection limit as low as 0.11 pg/mL was achieved for CEA detection based on the immunomagnetic separation and bi-AuNP probe-based multiamplification strategy, and the strategy was further successfully applied in human serum samples. The transducer was regenerated via a simple washing procedure, which enabled the detection of all samples on a single electrode with high reproducibility. The proposed strategy, which has the merits of high sensitivity, selectivity, and reproducibility exhibits great potential for detection in complex samples.

Magnetic Nanoparticles Enhance Pore Blockage-Based Electrochemical Detection of a Wound Biomarker.

A novel pore blockage-based electrochemical immunosensor based on the combination of 100 nm-magnetic nanoparticles (MNPs), as signal enhancers, and 200 nm-pore diameter nanoporous anodic alumina (NAA) membranes, as sensing platform, is reported. A peptide conjugate mimicking flightless I (Flii), a wound healing biomarker, was chosen as target analyte. The sensing platform consists of an anti-Flii antibody (Ab1)-modified NAA membrane attached onto a gold electrode. Anti-KLH antibody (Ab2)-modified MNPs (MNP-Ab2) were used to selectively capture the Flii peptide conjugate in solution. Sensing was based on pore blockage of the Ab1-modified NAA membrane caused upon specific binding of the MNP-Ab2-analyte complex. The degree of pore blockage, and thus the concentration of the Flii peptide conjugate in the sample, was measured as a reduction in the oxidation current of a redox species ([Fe(CN)6]4−) added in solution. We demonstrated that pore blockage is drastically enhanced by applying an external magnetic field at the membrane backside to facilitate access of the MNP-Ab2-analyte complex into the pores, and thus ensure its availability to bind to the Ab1-modified NAA membrane. Combining the pore blockage-based electrochemical magnetoimmunosensor with an externally applied magnetic field, a limit of detection (LOD) of 0.5 ng/ml of Flii peptide conjugate was achieved, while sensing in the absence of magnetic field could only attain a LOD of 1.2 μg/ml. The developed sensing strategy is envisaged as a powerful solution for the ultra-sensitive detection of an analyte of interest present in a complex matrix.

Diagnostic accuracy and usefulness of intraepithelial lymphocytes immunophenotyping in intestinal mucosa for the diagnosis of celiac disease

An electrochemical magneto immunosensor for the detection of anti-transglutaminase antibodies (ATG2) in celiac disease was developed. The immunological reaction is performed on magnetic beads (MBs) as a solid support in which the transglutaminase enzyme (TG2) is covalently immobilized (TG2-MB) and then ATG2 were revealed by an antibody labeled with peroxidase. The electrochemical response of the enzymatic reaction with o-phenilendiamine and H2O2 as substrates by square wave voltammetry was correlated with the ATG2. Graphite–epoxi composite cylindrical electrodes and screen printed electrodes were used as transducers in the immunosensor. A total number of 29 sera from clinically confirmed cases of celiac disease and 19 negative control sera were tested by the electrochemical magneto immunosensor. The data were submitted to the receiver-operating characteristic plot (ROC) analysis which indicated that 16.95 units was the most effective cut-off value (COV) to discriminate correctly between celiac and non-celiac patients. Using this point for prediction, sensitivity was found to be 100%, while specificity was 84%.

Detection of plasma MMP-9 within minutes. Unveiling some of the clues to develop fast and simple electrochemical magneto-immunosensors

Magnetic beads (MB) have been extensively used to produce sensitive and efficient electrochemical magneto-immunosensors. However, MB effective handling requires training, and MB washing after each incubation step is time consuming and contributes to raise result variability. Consequently, most of the electrochemical magneto-immunosensors reported to date, which entailed relatively long and complex multi-step procedures, would be difficult to carry out at point-of-care (POC) settings or by laypersons. For this reason, here we targeted the development of a simplified detection path, which is fast and simple enough to be operated at a POC setting, sufficiently efficient to provide analyte quantitation comparable to classical diagnostic methods, and dependent on minimal technical requirements to facilitate method global exploitation. As a proof-of-concept, we optimized an extremely simple, fast and efficient electrochemical magneto-immunosensor for detection of matrix metalloproteinase 9 (MMP-9). To accomplish this, we optimized MB immunomodification, produced an immunomodified Poly-HRP signal amplifier, developed a single-step magneto-immunoassay, and optimized electrochemical detection using a multiplexed magnetic holder and a ready-to-use commercial substrate solution. The sensor was finally calibrated by detecting MMP-9 in clinical samples. This electrochemical magneto-immunosensor detected MMP-9 in just 12–15 min, displaying linear response between 0.03 and 2 ng mL−1 of MMP-9, limits of detection (LOD) and quantification (LOQ) of 13 pg mL−1 and 70 pg mL−1, respectively, %CV< 6%, and accurate quantification of MMP-9 in patient plasma samples. These results were comparable to those afforded by a 5-h reference ELISA that used the same antibodies, confirming the applicability of our simplified method.

Electrochemical magneto immunosensor based on endogenous β-galactosidase enzyme to determine enterotoxicogenic Escherichia coli F4 (K88) in swine feces using square wave voltammetry

Diseases caused by enterotoxicogenic Escherichia coli F4 (K88) (ETEC F4) are a problem in swine production establishments. Due to the high rate of mortality and morbidity of E. coli infections, a rapid and accurate diagnosis is important in order to choose an appropriate treatment to reduce the economic impact. Therefore, an electrochemical magneto-immunosensor (EMI) was developed to detect and quantify ETEC F4 in swine feces samples through a direct non-competitive immunoassay. ETEC F4 was selectively captured by immunomagnetic separation. The detection principle was based on the activity of β-galactosidase endogenous enzyme (β-gal), which hydrolyses the p-aminophenyl-β-D-galactopyranoside (p-APG) producing p-aminophenol (p-AP), which was oxidized on a carbon screen printed electrode (CSPE) using square wave voltammetry (SWV). All parameters related to construction and electrochemical responses were optimized. The total analysis time to quantify ETEC F4 using the EMI was less than 2h and the limit of detection (LOD) was 33CFUmL−1. The perceptual relative error (%Er) was 20%. The magneto-immunosensor was validated versus conventional method of culture and plate count, obtaining a very good agreement. The EMI is simple, fast and economical to detect and quantify ETEC F4 in swine feces samples, being thus a valuable tool in swine production.

Developing enhanced magnetoimmunosensors based on low-cost screen-printed electrode devices

Abstract Electrochemical magnetoimmunosensors combine a number of issues that guarantee extremely high performance and also compatibility with the study of complex sample matrices. First, analyte immunocapture exploits the high affinity and specificity of antibodies. Second, magnetic particles (MP) provide faster and more efficient immunocapture than binding on two-dimensional structures, separation from nontarget sample components, and concentration of the target analyte. Finally, electrochemical detection supplies sensitivity and fast signal generation using robust and potentially miniaturized measurement equipment and transducers. On the contrary, MP handling is slightly more complex for end-users and more difficult to integrate in point-of-care devices than the manipulation of a classical biosensor. Attempts have been made to automate immunomagnetic binding, and the first robotized systems and platforms for the fluorescent and spectrophotometric detection of magnetoimmunoassays have already reached the market. Among the different types of electrodes available, screen-printed electrodes (SPE) stand out because of their low production cost and yet acceptable performance and interdevice reproducibility, which make them an excellent choice for analytical applications. In addition, each SPE entails a whole electrochemical cell stamped on a planar physical substrate, which makes it possible detection in small volumes and is especially favorable for the magnetic confinement of MP and the integration of microfluidic structures. In this article, we discuss the advantages obtained by using SPE and MP for the production of electrochemical magnetoimmunosensors and the clues for the successful development of such devices. We then revise some of the most outstanding works published in the literature.

Estrogen receptor α determination in serum, cell lysates and breast cancer cells using an amperometric magnetoimmunosensing platform

An electrochemical magnetoimmunosensor for the determination of estrogen receptor α (ERα) protein in complex samples (serum and cell lysates) able to discriminate between ERα positive and negative breast cancer cells is reported. Specifically functionalized magnetic microbeads with sandwich immunocomplexes and amperometric detection at disposable screen-printed carbon electrodes (SPCEs) resulted in highly selective and sensitive ERα detection with a detection limit of 19pgmL−1. This magnetoimmunosensing platform was successfully applied to the quantitation of ERα in spiked human serum and cell lysates samples without any matrix effect with an advantageous performance in terms of simplicity and assay times over commercial ELISA assays. The biosensor capability for assessing ERα in intact breast cancer cells makes it competitive with conventional strategies providing rapidly quantitative and reliable results on this relevant biomarker currently used in the clinical practice for diagnosis, follow-up and monitoring of metastatic breast cancer.

Electrochemical Magnetoimmunosensor for Progesterone Receptor Determination. Application to the Simultaneous Detection of Estrogen and Progesterone Breast‐cancer Related Receptors in Raw Cell Lysates.

AbstractAn electrochemical magnetoimmunosensor for the determination of progesterone receptor (PR) making use of functionalized magnetic microbeads (MBs), a sandwich‐type immunoassay and amperometric detection at disposable screen‐printed carbon electrodes (SPCEs) was described for the first time in this work. This selective and sensitive magnetoimmunosensor, with a detection limit of 22 pg mL−1, was successfully applied to the determination of PR in raw cell lysates with just a dilution as sample treatment and exhibiting no matrix effect. The advantageous performance in terms of simplicity and assay time over commercial ELISAs makes it competitive with conventional strategies providing rapidly quantitative and reliable results on this biomarker currently used in the clinical practice for diagnosis, follow‐up and monitoring of metastatic breast cancer. In addition, a dual magnetoimmunosensor using screen‐printed dual carbon electrode (SPdCE) has been implemented for the simultaneous determination of PR and estrogen receptor α (ERα) in cell lysates. This dual receptor determination allows a more accurate diagnosis enabling a more efficient treatment. The usefulness of this dual magnetoimmunosensing platform was demonstrated by discriminating raw lysates of two types of cancer cells (MCF‐7 and MDA‐MB‐436) with significantly different PR/ERα expression levels.

Disposable electrochemical magneto immunosensor for simultaneous simplified calibration and determination of Ochratoxin A in coffee samples

In food producing and developing countries having a reliable and inexpensive tool for the analysis of agricultural commodities is essential. This article describes a reliable disposable electrochemical magnetoimmunosensor for monitoring of coffee samples for Ochratoxin A contamination. The electrochemical detection performed on carbon screen-printed electrodes (CSPEs) used magnetic beads as antibody immobilization support and enzyme horseradish peroxidase as tracer. A remarkable limit of detection of 0.32μgL−1, well below of the legislative requirements, demonstrated the suitability of the proposed immunosensor as a screening tool for the analysis of Ochratoxin A in coffee samples.A simultaneous simplified Ochratoxin A calibration and coffee sample analysis strategy allowed the accurate determination of the mycotoxin with recoveries of 90% reducing the long times used in conventional calibration. This strategy enhanced the analytical merits of immunosensor approach towards truly disposable tools for screening of Ochratoxin A in coffee samples and opens novel avenues for food-safety monitoring.

Disposable and reliable electrochemical magnetoimmunosensor for Fumonisins simplified determination in maize-based foodstuffs

An electrochemical magnetoimmunosensor involving magnetic beads and disposable carbon screen-printed electrode (CSPE) for Fumonosins (FB1, FB2 and FB3) has been developed and evaluated through a certified reference material (CRM) and beer samples. Once the immunochemical reactions took place on the magnetic beads solution, they were confined on the surface of CSPE, where electrochemical detection is achieved through the addition of suitable substrate and mediator for enzymatic tracer (Horseradish peroxidase – HRP). A remarkable detection limit of 0.33μgL−1, outstanding repeatability and reproducibility (RSDintraday of 5.6% and 2.9%; RSDinterday of 6.9% and 6.0%; both for 0 and 5μgL−1 FB1 respectively), and excellent accuracy with recovery rate of 85–96% showed the suggested approach to be a very suitable screening tool for the analysis of Fumonisin B1 and B2 in food samples.A simultaneous simplified calibration and analysis protocol allows a fast and reliable determination of Fumonisin in beer samples with recovery rate of 87–105%. This strategy enhanced the analytical merits of immunosensor approach towards truly disposable tools for food-safety monitoring.

Carbon nanotube wiring for signal amplification of electrochemical magneto immunosensors: application to myeloperoxidase detection

In this work, chronoamperometric myelo-peroxidase (MPO) detection was accomplished using immunofunctionalized magnetic microparticles (MPs), disposable carbon screen-printed electrodes (C-SPEs), and a ready-to-use commercially available tetramethylbenzidine (TMB)-based enzymatic substrate. In order to reach the limit of detection (LOD) needed to study real blood serum samples, assay performance was additionally improved by exploiting CNT wiring, which amplified the signal and decreased the LOD. The optimized assay can be performed in 30min and yields LODs of 6 and 55ngmL(-1) in PBS and undiluted human serum, respectively, making it useful for the identification of patients at risk of cardiovascular disease. These results demonstrate that electrode nanostructuring can be accomplished "post-assay," which favors the development of enhanced magneto immunosensors based on the exploitation of cheap and simple SPE devices.

A portable electrochemical magnetoimmunosensor for detection of sulfonamide antimicrobials in honey

A new electrochemical magnetoimmunosensor (EMIS) has been developed for the screening of residues of sulfonamide antimicrobials in honey samples. The immunosensor is able to detect up to ten different sulfonamide congeners at levels below the action points established in some European countries (25μgkg(-1)) after a hydrolysis step in which the sulfonamides are released from the corresponding conjugates formed in samples of this type. In spite of the complexity of the sample after the hydrolysis procedure, the EMIS could perform quantitative measurements, directly in these samples, without any additional sample cleanup or extraction step. For example, sulfapyridine, used as a reference, can be detected in hydrolyzed honey with a limit of detection (IC90) of 0.1 ± 0.03μgkg(-1). Considering that the use of antibiotics for bee treatment is prohibited in the European Union, the immunosensor presented here could be an excellent screening tool. Moreover, several samples can be processed in parallel, which facilitates the analysis, reducing the necessity to use more costly confirmatory methods for just screening. As a proof of concept, a set of blind honey samples (spiked and incurred) were analyzed and the results were compared with those obtained by high-performance liquid chromatography-tandem mass spectrometry, demonstrating the potential of the EMIS as a screening tool.

An electrochemical magneto immunosensor (EMIS) for the determination of paraquat residues in potato samples

An electrochemical magneto immunosensor for the detection of low concentrations of paraquat (PQ) in food samples has been developed and its performance evaluated in a complex sample such as potato extracts. The immunosensor presented uses immunoreagents specifically developed for the recognition of paraquat, a magnetic graphite-epoxy composite (m-GEC) electrode and biofunctionalized magnetic micro-particles (PQ1-BSAMP) that allow reduction of the potential interferences caused by the matrix components. The amperometric signal is provided by an enzymatic probe prepared by covalently linking an enzyme to the specific antibodies (Ab198-cc-HRP). The use of hydroquinone, as mediator, allows recording of the signal at a low potential, which also contributes to reducing the background noise potentially caused by the sample matrix. The immunocomplexes formed on top of the modified MP are easily captured by the m-GEC, which acts simultaneously as transducer. PQ can be detected at concentrations as low as 0.18 ± 0.09 μg L(-1). Combined with an efficient extraction procedure, PQ residues can be directly detected and accurately quantified in potato extracts without additional clean-up or purification steps, with a limit of detection (90% of the maximum signal) of 2.18 ± 2.08 μg kg(-1), far below the maximum residue level (20 μg kg(-1)) established by the EC. The immunosensor presented here is suitable for on-site analysis. Combined with the use of magnetic racks, multiple samples can be run simultaneously in a reasonable time.

Electrochemical Magnetoimmunosensing Approach for the Sensitive Detection of H9N2 Avian Influenza Virus Particles

A novel electrochemical magnetoimmunosensor for fast and ultrasensitive detection of H9N2 avian influenza virus particles (H9N2 AIV) was designed based on the combination of high-efficiency immunomagnetic separation, enzyme catalytic amplification, and the biotin-streptavidin system. The reusable, homemade magneto Au electrode (M-AuE) was designed and used for the direct sensing. Immunocomplex-coated magnetic beads (IMBs) were easily accumulated on the surface of the M-AuE to obtain the catalytically reduced electrochemical signal of H2 O2 after the immunoreaction. The transducer was regenerated through a simple washing procedure, which made it possible to detect all the samples on a single electrode with higher reproducibility. The magnetic-bead-based electrochemical immunosensor showed better analytical performance than the planar-electrode-based immunosensor with the same sandwich construction. Amounts as low as 10 pg mL(-1) H9N2 AIV could be detected even in samples of chicken dung. This electrochemical magnetoimmunosensor not only provides a simple platform for the detection of the virus with high sensitivity, selectivity, and reproducibility but also shows great potential in the early diagnosis of diseases.

Electrochemical magneto immunosensor for the detection of anti-TG2 antibody in celiac disease

An electrochemical magneto immunosensor for the detection of anti-transglutaminase antibodies (ATG2) in celiac disease was developed. The immunological reaction is performed on magnetic beads (MBs) as a solid support in which the transglutaminase enzyme (TG2) is covalently immobilized (TG2-MB) and then ATG2 were revealed by an antibody labeled with peroxidase. The electrochemical response of the enzymatic reaction with o-phenilendiamine and H2O2 as substrates by square wave voltammetry was correlated with the ATG2. Graphite–epoxi composite cylindrical electrodes and screen printed electrodes were used as transducers in the immunosensor. A total number of 29 sera from clinically confirmed cases of celiac disease and 19 negative control sera were tested by the electrochemical magneto immunosensor. The data were submitted to the receiver-operating characteristic plot (ROC) analysis which indicated that 16.95 units was the most effective cut-off value (COV) to discriminate correctly between celiac and non-celiac patients. Using this point for prediction, sensitivity was found to be 100%, while specificity was 84%.

Label‐Free Amperometric Magnetoimmunosensors for Direct Determination of Lactoperoxidase in Milk

AbstractNovel label free electrochemical magnetoimmunosensors for the determination of lactoperoxidase (LPO) were developed based on the use of streptavidin‐magnetic beads (Strep‐MBs) and graphite‐Teflon composite or screen‐printed carbon electrodes (SPCEs) as electrochemical transducers. Biotinylated antiLPO antibodies were immobilized onto the Strep‐MBs and a direct‐type immunoassay by detecting the enzymatic activity of the captured target enzyme using amperometric detection at −0.20 V and the H2O2/hydroquinone (HQ) system was employed. The SPCEs‐based magnetoimmunosensors allowed a detection limit (LOD) of 0.12 μg mL−1 and a good selectivity against nontarget proteins. Moreover, their analytical usefulness was successfully demonstrated by analyzing different types of milk samples.

Ultrasensitive determination of human growth hormone (hGH) with a disposable electrochemical magneto-immunosensor

In this paper, an electrochemical magneto-immunosensor for the detection of human growth hormone (hGH) is described for the first time. The immunosensor involves the use of tosyl-activated magnetic microparticles (TsMBs) to covalently immobilize a monoclonal mAbhHG antibody. A sandwich-type immunoassay with a secondary pAbhGH antibody and anti-IgG labelled with alkaline phosphatase (anti-IgG-AP) was employed. TsMBs–mAbhGH–hGH–pAbhGH–anti-IgG-AP conjugates were deposited onto the surface of a screen-printed gold electrode using a small neodymium magnet, and electrochemical detection was performed by square-wave voltammetry upon the addition of 4-aminophenyl phosphate as the AP substrate. All the variables involved in the preparation of immunoconjugates and in the immunoassay protocol were optimized. A calibration curve for hGH was constructed with a linear range between 0.01 and 100 ng/mL (r = 0.998) and a limit of detection of 0.005 ng/mL. This value is nearly three orders of magnitude lower than that obtained using surface plasmon resonance (Treviño et al., Talanta 78:1011-1016, 2009). Furthermore, good repeatability, with RSD = 3% (n = 10) at the 1-ng/mL hGH level, was obtained. Cross-reactivity studies with other hormones demonstrated good selectivity. The magneto-immunosensor was applied to the analysis of human serum spiked with hGH at the 4- and 0.1-ng/mL levels. Mean recoveries of 96 ± 6% and 99 ± 2%, respectively, were obtained.