Electrochemical Sandwich Immunoassay Research Articles

Rapid diagnosis of bloodstream infections using a culture-free phenotypic platform

BackgroundBloodstream infections (BSIs) are a life-threatening acute medical condition and current diagnostics for BSIs suffer from long turnaround time (TAT). Here we show the validation of a rapid detection-analysis platform (RDAP) for the diagnosis of BSIs performed on clinical blood samplesMethodsThe validation was performed on a cohort of 59 clinical blood samples, including positive culture samples, which indicated confirmed bloodstream infections, and negative culture samples. The bacteria in the positive culture samples included Gram-positive and Gram-negative pathogenic species. RDAP is based on an electrochemical sandwich immunoassay with voltage-controlled signal amplification, which provides an ultra-low limit of detection (4 CFU/mL), allowing the platform to detect and identify bacteria without requiring culture and perform phenotypic antibiotic susceptibility testing (AST) with only 1–2 h of antibiotic exposure. The preliminary diagnostic performance of RDAP was compared with that of standard commercial diagnostic technologies.ResultsUsing a typical clinical microbiology laboratory diagnostic workflow that involved sample culture, agar plating, bacteria identification using matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry, and AST using MicroScan as a clinical diagnostic reference, RDAP showed diagnostic accuracy of 93.3% and 95.4% for detection-identification and AST, respectively. However, RDAP provided results at least 15 h faster.ConclusionsThis study shows the preliminary feasibility of using RDAP to rapidly diagnose BSIs, including AST. Limitations and potential mitigation strategies for clinical translation of the present RDAP prototype are discussed. The results of this clinical feasibility study indicate an approach to provide near real-time diagnostic information for clinicians to significantly enhance the treatment outcome of BSIs.

Bio-functionalized carbon dots for signaling immuno-reaction of carcinoembryonic antigen in an electrochemical biosensor for cancer biomarker detection

Early diagnosis of cancer demands sensitive and accurate detection of cancer biomarkers in blood. Carbon dots (CDs) bio-functionalization with antibodies, peptides or aptamers have played significant role in cancer diagnosis and targeted cancer therapy. Herein, a biosensor for detection of cancer biomarker carcinoembryonic antigen (CEA) in blood serum has been designed using CDs bio-functionalized with HRP-conjugated CEA antibody (CUCDs@CEAAb2) as detection probe. CDs were synthesized by upscaling of cow urine, a nitrogen rich biomass waste, by hydrothermal method. Detection probe based on CDs resulted in 3.5 times higher sensitivity as compared to conventional electrochemical sandwich immunoassay. To further improve the sensor performance, hyper-branched polyethylenimine grafted poly amino aniline (PEI-g-PAANI) was used as the sensing interface, which enabled immobilization of higher amount of capture antibody. Detection of CEA in human blood serum coupled with wide linear range (0.5–50 ng/ml), good specificity, stability, reproducibility and low detection limit (10 pg/ml) signified the excellence of CUCDs based CEA immunosensor. CUCDs exhibited excitation wavelength dependent fluorescence property and showed strong blue emission under UV irradiation. MTT assay indicated that the material is not toxic towards human dental pulp stem cells (hDPSCs) and MG63 osteosarcoma cells (cell viability > 90%). The present study demonstrates a methodology for valorization of animal waste to a cost-effective carbon based functional nanomaterial for clinical detection of cancer biomarkers.

Multiple and simultaneous detection for cytokines based on the nanohole array by electrochemical sandwich immunoassay

Monitoring of cytokine storm markers is critical for determining the disease progression and treatment efficacy. In this study, we developed a novel fourplex electrochemical (EC) biosensor using nanohole array (NHA) structures containing several nanoholes on a single electrode for detecting cytokines with a low limit. A framework was developed for fabricating and optimizing multiplexed EC sandwich immunoassays using NHA electrodes because an NHA can enhance the EC detection of molecular binding by controlling the binding sites of captured biomolecules. For detecting multiple cytokines, the captured antibodies were immobilized on the surface-modified NHA and sandwich immunoassays with amperometric signal amplifications using poly-streptavidin horseradish peroxidase conjugates and 3,3′,5,5′-tetramethylbenzidine. Under the optimal conditions, the EC NHA immunosensors exhibited high sensitivity and specificity with a limit of detection of 1 pg/mL and a detection range of 1–300 pg/mL. The results revealed that the developed NHA sensor exhibits reliable EC characteristics and could be used to analyze multiple cytokines using small sample volume and subsequently developed for other applications.

Simultaneous determination of BoNT/A and /E using an electrochemical sandwich immunoassay based on the nanomagnetic immunosensing platform

Developing new ultrasensitive assays for the detection of the presence, and determination of the serotype of the most poisonous material known i.e. botulinum neurotoxin (BoNT) is vital to human health and the wellbeing of the surrounding environment. Here, an electrochemical sandwich immunoassay with high sensitivity is adopted to achieve simultaneous determination of BoNT serotypes A and E based on polystyrene@polydopamine/Cd2+ and Ag nanoparticles acting as monoclonal antibody labels. Two well-separated peaks with strong electrochemical signals are generated by the labels, allowing for the simultaneous detection of two analytes existing on the electrode. To obtain well-oriented polyclonal antibodies immobilization, boronic acid is directly attached to the magnetic core/metal-organic framework (MOF) shell nanoagent surfaces without the requirement of a long and flexible spacer. Accordingly, it is possible to directly detect the metal ion labels through square wave voltammetry without the metal pre-concentration step. This results in distinct and well-defined voltammetric peaks, pertaining to each sandwich-type immunocomplexes. The limits of detection of BoNT/A and BoNT/E analyses were found to be 0.04 and 0.16 pg mL−1 with the linear dynamic ranges of 0.1–1000 and 0.5–1000 pg mL−1, respectively. Based on the obtained results, this immunosensor has the wide linear ranges, while also exhibiting low limits of detection along with good stability and reproducibility.

Detection of Amyloid-β(1-42) Aggregation With a Nanostructured Electrochemical Sandwich Immunoassay Biosensor.

Amyloid-β(1–42) [Aβ(1–42)] oligomer accumulations are associated with physiologic alterations in the brains of individuals with Alzheimer’s disease. In this study, we demonstrate that a nanostructured gold electrode with deposited gold nanoparticles, induced via electrochemical impedance spectroscopy (EIS), may be used as an Aβ(1–42) conformation biosensor for the detection of Alzheimer’s disease. Monoclonal antibodies (12F4) were immobilized on self-assembled monolayers of the electrochemical sandwich immunoassay biosensor to capture Aβ(1–42) monomers and oligomers. Western blot and fluorescence microscopy analyses were performed to confirm the presence of Aβ(1–42) monomers and oligomers. EIS analysis with an equivalent circuit model was used to determine the concentrations of different Aβ(1–42) conformations in this study. We identified conformations of Aβ(1–42) monomers and Aβ(1–42) oligomers using probe antibodies (12F4) by employing EIS. indicates the sum resistance of impedance measured during Aβ(1–42) immobilization. refers to the concentration of probe antibody (12F4) binding with Aβ(1–42). The concentration of Aβ(1–42) oligomer was defined as the percentage of Aβ(1–42) aggregation . The experimental results show that the biosensor has high selectivity to differentiate Aβ(1–40) and Aβ(1–42) monomers and Aβ(1–42) oligomers and that it can detect Aβ(1–42) oligomer accurately. The linear detection range for Aβ(1–42) oligomers was between 10 pg/ml and 100 ng/ml. The limit of detection was estimated to be 113 fg/ml.

Evaluation of Analytical Performances of Magnetic Force-Assisted Electrochemical Sandwich Immunoassay for the Quantification of Carcinoembryonic Antigen.

Carcinoembryonic antigen (CEA) is a biomarker indicated in different cancers, targeted for quantitative analysis via immunoassay. Here we introduce a new technique called magnetic force-assisted electrochemical sandwich immunoassay (MESIA) for determination of CEA level in a drop of human serum using a fully automated point-of-care testing (POCT) device. The analytical performances of the assay are assessed based on precision, accuracy, limit of blank (LoB), limit of detection (LoD) and limit of quantitation (LoQ), linearity, Hook effect, interference, cross-reactivity, and method comparison following the guidelines of the Clinical Laboratory Standards Institute (CLSI). The LoD is 0.50 ng/ml. A linear relationship is shown in the range of 0.5–200 ng/ml. A high dose effect is not seen up to approximately 500,000 ng/ml. The recovery range is from 94.7 to 108.9%. The %CV of run-to-run and within-lab variations are less than 2.04 and 4.41% across the CEA concentrations, respectively, whereas reproducibility is 4.45–6.24%. Method comparison shows that the assay correlates well with the reference device (R 2 = 0.9884). The assay demonstrates acceptable precision, accuracy, LoB, LoD and LoQ, hook effect, linearity, interference, cross-reactivity, and high correlation with its reference device. Thus, the system is suitable for the quantification of CEA in clinical practices with a POCT manner.

Detection of Cytochrome C Using a Selective and Sensitive Methylene Blue-Based Electrochemical Aptasensor

The present research aimed to detect the cytochrome C using methylene blue (MB)-anchored ultrasensitive electrochemical aptamer conjugated with Au nanoparticles (NPs), as a new electrochemical sandwich immunoassay. Aptamer-Au NPs could act as sandwich amplification element and MB accumulation reagent. According to the results, the interaction of MB and guanine occurred strongly on aptamer probe. After switching the aptamer structure by the cytochrome C, the MB- labeled aptamer probe was forced to detach from the sensing interface. Moreover, the cytochrome C concentrations (0.05-10.0 µM) with 5.0 nM detection limit reduced linearly the peak current of MB. In conclusion, an acceptable selectivity was observed for the constructed aptasensor in detecting the cytochrome C.

Electrochemical sandwich immunoassay for quantification of therapeutic drugs based on the use of magnetic nanoparticles and silica nanoparticles

Adalimumab (Ada) is widely used to treat autoimmune diseases, and a correct dose of Ada can maintain therapeutic effectiveness and relieve symptoms. This study develops a rapid quantifying method of Ada using magnetic nanoparticle (MNP)-based immunoreaction and disposable screen-printed carbon electrodes (SPCEs). Anti-Ada Fab fragment and mouse anti-human antibody is respectively used as primary antibody (Ab1) and secondary antibody (Ab2). Glucose oxidase (GOD) and the Ab2 were co-immobilized on silica nanoparticles (SiNPs) with the concentration ratio of 3:1 to promise good immune affinity and the more amount of immobilized GOD. After sequentially immunoreacting Ada with Ab1@MNPs and GOD-Ab2@SiNPs, the separated GOD-Ab2@SiNPs/Ada/Ab1@MNPs complex was dripped on the glucose/ferricyanide-coated SPCEs for GOD catalysis, and then the anodic current of reduced ferrocyanide was used for the quantification of Ada. The MNP-based immunoassays have good linearity from 0.1 μg/mL to 2 μg/mL and a limit of detection of 15.5 ng/mL. Moreover, the immunoassays can practically measure the Ada concentrations in 10-fold diluted patient serum samples with good recovery. The operation time of MNP-based immunoassays takes only about 0.5 h. The disposable MNP-based immunoassays have great promise to develop a rapid quantification platform for therapeutic drug monitoring.

MESIA: Magnetic force-assisted electrochemical sandwich immunoassays for quantification of prostate-specific antigen in human serum

We propose a new immunoassay technique, called magnetic-force assisted electrochemical sandwich immunoassay (MESIA), where serum biomarkers can be determined by magnetic actuation and electrochemical detection of gold-coated iron oxide nanoparticles as probes for immunocomplex formation. In MESIA, neither washing buffer nor fluidic parts are necessary, because the formation of immunocomplexes and the removal of unbound probes are controlled by magnetic forces. Electrochemical pretreatment and measurement of the gold-coated magnetic probes allows highly sensitive, precise, and robust system for quantification of target analytes. Using MESIA, the concentration of prostate-specific antigen (PSA) in 10 μl of human serum is determined within 5 min. The limit of detection is 0.085 ng/mL, and the average coefficient of variance is 8.85% for five different PSA concentrations ranging from 0 to 25 ng/mL. This method shows good precision and reproducibility (<10%) and high correlation with cobas e 801 (r = 0.997) for clinical patient samples. We believe this technique to be useful in the development of a point-of-care testing platform for diagnosis and prognosis of various diseases, such as cancer, based on quantification of biomarkers in a drop of blood.

Electrochemical sandwich immunoassay for insulin detection based on the use of gold nanoparticle-modifiedMoS2 nanosheets and thehybridization chain reaction.

A sandwich-type of electrochemical immunoassay is described for the determination of insulin. It is based on the use of a glassy carbon electrode that was modified with MoS2 nanosheets decorated with gold nanoparticles (AuNPs) to immobilize a large amount of first antibody (Ab1). Following exposure to insulin, secondary antibody (Ab2) that was cross-linked to a DNA initiator strand (T0) to form an Ab2@T0 conjugate was added to undergo a sandwich immunoreaction. Subsequently, the long dsDNA concatemer was formed by a hybridization chain reaction between Ab2@T0 and auxiliary probes (H1, H2). Finally, the electrochemical probe ruthenium(II) hexaammine was intercalated into the dsHCR products via electrostatic interaction between the anionic DNA phosphate backbones and the cationic probe. The electrochemical response, best measured at a potential of around-0.21 V (vs Ag/AgCl) has a dynamic range that extends from 0.1pmolL-1 to 1nmolL-1 insulin, and the detection limit is as low as 50 fmol L-1. The assay was acceptably specific, reproducible and stable. In our perception, it represents a viable new tool for determination of this important clinical parameter. Graphical abstract Schematic of a sandwich-type of electrochemical immunoassay for the determination of insulin based on the use ofMoS2 nanosheets modified with gold nanoparticles (AuNP@MoS2) and hybridization chain reaction (HCR).

Electrochemical sandwich immunoassay for Escherichia coli O157:H7 based on the use of magnetic nanoparticles and graphene functionalized with electrocatalytically active Au@Pt core/shell nanoparticles.

A highly sensitive electrochemical sandwich immunoassay is described fordetermination of Escherichia coli O157:H7 (E. coli O157:H7). Silica coated magnetite nanoparticles (Fe3O4) were modified with primary antibody to capture E. coli O157:H7. Gold-platinum core/shell nanoparticles (Au@Pt NPs) with different Pt shell thicknesses were prepared via changing the molar ratio of H2PtCl6 to HAuCl4 in the precursor solution. The optimized Au@Pt NPs exhibit enhanced activity in the electrocatalytic reduction of hydrogen peroxide (H2O2). The Au@Pt NPs were modified with graphene that was functionalized with Neutral Red, and then used as an electrochemical label for secondary antibodies and horseradish peroxidase (HRP). The sandwich immunocomplexes were magnetically absorbed on a 4-channel screen printed carbon electrode. Due to the catalysis of the Au@Pt NPs and HRP, the signal is strongly amplified in the presence of H2O2 when using thionine as the electron mediator. Under optimal conditions, the immunoassay has a linear response in the 4.0 × 102 to 4.0 × 108CFU·mL-1 concentration range, with a limit of detection of 91CFU·mL-1 (at an S/N ratio of 3). Graphical abstract Preparation of Au@Pt core/shell nanoparticles with different Pt shell thickness (A), rGO-NR (B), rGO-NR-Au@Pt-Ab2-HRP (C) and the preparation and the detection process of the immunoassay (D). rGO: reduced graphene oxide, GO: graphene oxide, NR: Neutral Red, HRP: horseradish peroxidase, AuNPs: gold nanoparticles, Fe3O4@SiO2: Silica coated magnetite nanoparticles, 4-SPCE: 4-channel screen printed carbon electrode.

Comparative evaluation of the performance of electrochemical immunosensors using magnetic microparticles and nanoparticles. Application to the determination of tyrosine kinase receptor AXL

Electrochemical sandwich immunoassay strategies involving the use of carboxyl-functionalized magnetic microbeads (cMBs) and magnetic nanoparticles (cMNPs) have been evaluated and compared. The proteolytically cleaved soluble tyrosine kinase receptor sAXL was used as the target analyte. Antibodies against AXL were covalently immobilized on cMBs or cMNPs. Immunobinding of AXL was detected by means of a secondary biotinylated antibody and a streptavidin-horseradish peroxidase conjugate. The electrochemical transduction was accomplished by capturing the cMBs or cMNPs bearing the immunoconjugates onto screen-printed carbon electrodes (SPCEs) by using a small magnet. The amperometric response was measured at −0.20 V (vs the silver pseudo-reference electrode of the SPCE) upon the addition of H2O2 in the presence of hydroquinone as the redox mediator. The calibration plots for AXL extended up to 7.5 ng mL−1 when cMBs were used for the preparation of the immunosensor and up to 40 ng mL−1 in the case of using cMNPs. The respective slope values were 158 (cMBs) and 43 nA mL ng−1 (cMNPs), while the achieved LODs were 74 (cMBs) and 75 pg mL−1 (cMNPs). Although the immunosensors prepared with cMBs provided a shorter range of linearity, they exhibited a 3.7-times larger sensitivity than those constructed with cMNPs. The successful application of the new strategies was demonstrated for the determination of the endogenous content of sAXL in real human serum samples (a cut-off value of 71 ng mL−1 have been established for patients with risk of heart failure). The immunosensors constructed using cMBs or cMNPs can be advanta geously compared, in terms of sensitivity and fabrication time, with the only immunosensor for AXL previously reported. In addition, these new immunosensors took approximately half time than ELISA to perform the assay.

Electrochemical sandwich immunoassay for the prostate specific antigen using a polyclonal antibody conjugated to thionine and horseradish peroxidase

The authors present a voltammetric immunoassay for the quantification of the prostate specific antigen (PSA), a well-established tumor marker. Biotinylated anti- PSA antibodies were immobilized on the surface of a nano-TiO2-modified carbon paste electrode using specific interaction between streptavidin and biotin. Streptavidin itself was covalently grafted to the electrode surface via 4-aminobenzoic acid film. Signal transduction was then performed using polyclonal antibodies conjugated with horseradish peroxidase (HRP) and thionine. An amplified catalytic reduction current was observed in the presence of H2O2 because more than one polyclonal antibody linked to each antigen. Each step of the preparation of the immunosensor was monitored via electrochemical impedance spectroscopy. The electrode, if operated at a typical potential of 0.3 V vs. Ag/AgCl and using hexacyanoferrate as an electrochemical probe, exhibits linear responses in the 0.10 to 5.0 ng·mL−1 and 5.0 to 100 ng·mL−1 PSA concentration ranges, with a detection limit of 200 pg·mL−1. The accuracy of the biosensor was confirmed by analyzing a certified human serum sample, and this indicated that the immunosensor is well suited for the quantification of PSA.

Electrochemical sandwich immunoassay for human epididymis-specific protein 4 using a screen-printed electrode modified with graphene sheets and gold nanoparticles, and applying a modular magnetic detector device produced by 3D laser sintering

The authors describe an electrochemical sandwich immunoassay for the human epididymis-specific protein 4 (HE4). A commercially available electromagnetic detector device and a screen-printed electrode (SPE) modified with graphene sheets and gold nanoparticles were used to fabricate the detector. First, a nanocomposite suspension consisting of graphene sheets and gold nanoparticles was deposited onto the SPE. This results in an enlarged electrochemically active area and improves electron transfer. Next, biotinylated monoclonal antibody against HE4 (anti-HE4) was bound to streptavidin-modified magnetic beads via biotin-avidin binding. Nonspecific binding sites were blocked with bovine serum albumin. Stepwise changes in the microscopic features of the surfaces and electrochemical properties upon the formation of each layer were studied by scanning electron microscopy, transmission electron microscopy and cyclic voltammetry. After magnetic separation and washing, the biotinylated anti-HE4 beads were deposited on the SPE, which then was inserted into the electromagnetic modular detector. Following the immunoreaction with HE4, a sandwich-type immunoassay was performed on the SPE using horseradish peroxidase (HRP)-labeled HE4 as a tracer label. Electrochemical detection was carried out after addition of H2O2 as a substrate for HRP. Under optimal conditions, differential pulse voltammetry can detect HE4 in the 0 to 400 pM concentration range with a detection limit of 0.5 pM (at an S/N ratio of 3). The system was applied to the determination of HE4 with good accuracy and selectivity.

Electrochemical sandwich immunoassay for the peptide hormone prolactin using an electrode modified with graphene, single walled carbon nanotubes and antibody-coated gold nanoparticles

We describe a new kind of electrochemical immunoassay for the peptide hormone prolactin. A glassy carbon electrode (GCE) was modified with a hybrid material consisting of graphene, single walled carbon nanotubes and gold nanoparticles (AuNPs) in a chitosan (CS) matrix. The graphene and the single wall carbon nanotubes were first placed on the GCE, and the AuNPs were then electrodeposited on the surface by cyclic voltammetry. This structure results in a comparably large surface for immobilization of the capturing antibody (Ab1). The modified electrode was used in a standard sandwich-type of immunoassay. The secondary antibody (Ab2) consisted of AuNPs with immobilized Ab2 and modified with biotinylated DNA as signal tags. Finally, alkaline phosphatase was bound to the biotinylated DNA-AuNPs-Ab2 conjugate via streptavidin chemistry. The enzyme catalyzes the hydrolysis of the α-naphthyl phosphate to form α-naphthol which is highly electroactive at an operating voltage as low as 180 mV (vs. Ag/AgCl). The resulting immunoassay exhibits high sensitivity, wide linear range (50 to 3200 pg∙mL‾1), low detection limit (47 pg∙mL‾1), acceptable selectivity and reproducibility. The assay provides a pragmatic platform for signal amplification and has a great potential for the sensitive determination of antigens other than prolactine.

Dually Electroactive Disulfide‐Confined Aryldiazonium Salt Used as a Linker Molecule for Preparing Immunosensor Platforms

AbstractA new electroactive disulfide‐confined aryl diazonium (DSAD) salt was synthesized and used as a linker for biomolecules immobilization to prepare two kinds of immunoassay platforms. DSAD was electrodeposited on ITO electrode surfaces by cyclic voltammetry. Disulfide group of DSAD attached on the surfaces were electrochemically oxidized into thiosulfinate or thiosulfonate groups. For the first work, a detection of rabbit antigen was performed on ITO microelectrodes array by spatially‐selective approach. In the second work, DSAD was deposited on electrochemically reduced graphene oxide‐modified ITO surfaces, which were used as a platform for electrochemical sandwich immunoassay for detecting mouse antigen.

Ultrasensitive electrochemical immunoassay of proteins based on in situ duple amplification of gold nanoparticle biolabel signals

An electrochemical sandwich immunoassay method that can be sensitive to a few protein molecules (human immunoglobulin G or human prostate-specific antigen) is reported, based on HAuCl4-NH2OH redox reaction to enlarge the size of second antibody labeled gold nanoparticles and in situ microliter-droplet anodic stripping voltammetry analysis with enhanced cathodic preconcentration of gold.

Ultrasensitive electrochemical immunosensors for clinical immunoassay using gold nanoparticle coated multi-walled carbon nanotubes as labels and horseradish peroxidase as an enhancer

A novel electrochemical immunosensor for the ultrasensitive detection of α-fetoprotein (AFP) is described with the use of a gold nanoparticle/(3-mercaptopropyl) trimethoxysilane modified electrode as a desirable platform and gold nanoparticle functionalized multi-walled carbon nanotube (MWCNT) nanocomposite labeled secondary antibodies (Ab2) as a signal tag. At first, gold nanoparticles (AuNPs) were attached on the MWCNTs surface using poly(diallyldimethylammoniumchloride) (PDDA) as a linkage reagent to obtain AuNP–MWCNT nanocomposites. Subsequently, horseradish peroxidase (HRP) and thionine (Thi) labeled secondary antibodies (Thi-Ab2) were used to bind AuNP–MWCNT nanocomposites with a high load amount and good biological activity. Moreover, the immunosensor can provide amplified signals because of the increased surface area and biocompatibility of AuNP–MWCNT nanocomposites. On the other hand, the AuNP functionalized (3-mercaptopropyl) trimethoxysilanes (MPTS) were used for the biosensor platform to increase the surface area as well as improving the electronic transmission rate to capture a large amount of primary antibodies (Ab1). Thus, amplified signals can be obtained by an electrochemical sandwich immunoassay protocol in the presence of H2O2. The proposed sensing strategy provides a wide linear dynamic range from 0.01 to 50 ng mL−1 with a low detection limit of 3 pg mL−1 (S/N = 3). Significantly, the developed immunoassay method showed a good stability, selectivity and reproducibility.

Electrochemical Sandwich Immunoassay for the Ultrasensitive Detection of Human MUC1 Cancer Biomarker

A new electrochemical sandwich immunoassay for the ultrasensitive detection of human MUC1 cancer biomarker using protein G-functionalized magnetic beads (MBs) and graphite-based screen-printed electrodes (SPEs) was developed. Magnetic beads were employed as the platforms for the immobilization and immunoreaction process. A pair of primary and secondary antibodies was used to capture the MUC1 protein. After labeling with a third antibody conjugated with horseradish peroxidase (HRP), the resulting conjugate was trapped at the surface of the graphite-based SPEs and MUC1 determination was carried out by differential pulse voltammetry (DPV) at 0.4 V upon H2O2addition using acetaminophen (APAP) as the redox mediator. A linear relationship was obtained for the detection of human MUC1 over a range of 0–25 ppb with the lowest detection limit of 1.34 ppb when HRP was applied as a label. Preliminary experiments were performed using disposable electrochemical sensors in order to optimize some parameters (i.e., incubation times, concentrations, and blocking agent).

A thin layer-based amperometric enzyme immunoassay for the rapid and sensitive diagnosis of respiratory syncytial virus infections

A simple electrochemical sandwich immunoassay involving a polystyrene microarray slide coated with monoclonal capture antibodies and carbon screen-printed sensors (SPS) was designed for the rapid diagnosis of respiratory syncytial virus (RSV). The detection of the antibody-antigen complex formation relied on the use of a horseradish peroxidase conjugate. Its chronoamperometric measurement detection was performed by confining a droplet of H2O2/3,3',5,5'-tetramethylbenzidine enzyme substrate/mediator solution within a thin layer between one spot of the microarray and the surface of one screen-printed electrochemical cell. The accumulation of the enzyme product in the thin film of liquid enhanced the electrochemical response which allowed the development of a rapid (25min) and sensitive thin layer-based amperometric (TLA) enzyme immunoassay. The method was successfully compared to commercially-available immunofluorescent and real-time PCR assays for RSV testing in respiratory secretion clinical samples. This suggests that owing to its rapidity, convenience, low-cost, portability and ability to provide quantified results, the reported concept could be a promising point-of-care diagnostic tool to screen patients with suspected respiratory infection or other types of infectious diseases.