Overcoming the adverse effects of crosslinking in biosensors via addition of PEG: Improved sensing of hydrogen peroxide using immobilized peroxidase

Glutaraldehyde (GA) is widely used as a crosslinker to immobilize enzymes, for examples in biosensors, but often causes partial denaturation. We find that the proper use of poly(ethylene glycol) (PEG) during the crosslinking process can fully preserve the native state and activity of horseradish peroxidase (HRP). An amperometric biosensor was developed based on these findings for the direct determination of hydrogen peroxide. UV-Vis and FTIR spectroscopy reveal that the HRP entrapped in a polypyrrole matrix retains its native structure. The addition of PEG increases the sensitivity and stability of the biosensor and prevents many of effects caused by intra-crosslinking via GA. The biosensor was operated at a potential of ?350?mV (vs Ag/AgCl) without any mediator and gave a linear response to H₂O₂ in the 5 to 190???M concentration range. The apparent Michaelis-Menten constant is 3.37?mM, and maximal current is as high as 3.43???A. The surface of the biosensor was characterized by atomic force microscopy operated in the tapping mode.

Quality assurance in immunoassay performance—comparison of different enzyme immunoassays for the determination of caffeine in consumer products

Enzyme immunoassays with optical detection are amongst the most widely used bioanalytical tools. We defined seven parameters for the quality assessment of immunoassays that were addressed in a systematic study of direct and indirect immunoassays, using the enzymes horseradish peroxidase (HRP) and alkaline phosphatase (AP), the chromogenic substrates 3,3′,5,5′-tetramethylbenzidine (TMB) and para-nitrophenyl phosphate, and the fluorescent substrates 3-(4-hydroxyphenyl)propionic acid and 4-methylumbelliferyl phosphate. The same monoclonal antibody against caffeine was used throughout the study. The four quality parameters regarding the standard curve were the test midpoint (sensitivity), the measurement range, the relative dynamic range of the signal, and the goodness of fit of the adjusted four-parameter logistic function. All HRP immunoassays showed a higher sensitivity compared to the AP assays. On the basis of all four criteria, it was established that the direct assay format is superior to the indirect format, the immunoassay using HRP TMB fulfilling all requirements best. In a second step, caffeine concentrations in 24 beverage and cosmetics samples were determined and three more quality parameters were assessed with this application. The direct HRP TMB assay showed one of the best intra- and inter-plate precisions and the best accuracy, defined by the correlation of results with those from the chosen reference method liquid chromatography tandem mass spectrometry (LC-MS/MS). Considering all criteria, HRP TMB seems to be the enzyme substrate system of choice preferably used in the direct assay format.

Mediatorless amperometric bienzyme glucose biosensor based on horseradish peroxidase and glucose oxidase cross-linked to multiwall carbon nanotubes

We report on a bienzyme-channeling sensor for sensing glucose without the aid of mediator. It was fabricated by cross-linking horseradish peroxidase (HRP) and glucose oxidase (GOx) on a glassy carbon electrode modified with multiwalled carbon nanotubes (MWNTs). The bienzyme was cross-linked with the MWNTs by glutaraldehyde and bovine serum albumin. The MWNTs were employed to accelerate the electron transfer between immobilized HRP and electrode. Glucose was sensed by amperometric reduction of enzymatically generated H₂O₂ at an applied voltage of ?50 mV (vs. Ag/AgCl). Factors influencing the preparation and performance of the bienzyme electrode were investigated in detail. The biosensor exhibited a fast and linear response to glucose in the concentration range from 0.4 to 15 mM, with a detection limit of 0.4 mM. The sensor exhibited good selectivity and durability, with a long-term relative standard deviation of <5 %. Analysis of glucose-spiked human serum samples yielded recoveries between 96 and 101 %.

Direct electrochemistry of horseradish peroxidase based on hierarchical porous calcium phosphate microspheres

Biomorphic calcium phosphate (CaP) microspheres with hierarchical porous structure were synthesized using natural cole pollen grains as templates and were further employed for the immobilization of horseradish peroxidase (HRP). Scanning electron microscopy and Fourier transform infrared spectroscopy revealed (a) the porous structure of the CaP microspheres, (b) the effective immobilization, and (c) the retention of the conformation of HRP on CaP. The immobilized HRP was placed on a glassy carbon electrode where it underwent a direct, fully reversible, and surface-controlled redox reaction with an electron transfer rate constant of 1.96 s^?1. It also exhibits high sensitivity to the reduction of H₂O_2. The response to H₂O₂ is linear in the 5.00 nM to 1.27 μM concentration range, and the sensitivity is 30357 μA?mM^?1?cm^?2. The detection limit (at an SNR of 3) is as low as 1.30 nM. The apparent Michaelis–Menten constant (K _M ^app ) of the immobilized enzyme is 0.92 μM. This new CaP with hierarchical porous structure therefore represents a material that can significantly promote the direct electron transfer between HRP and an electrode, and is quite attractive with respect to the construction of biosensors.

Mixed immunoassay design for multiple chemical residues detection

In this research, a mixed immunoassay design for multiple chemical residues detection based on combined reverse competitive enzyme-linked immunosorbent assay (ELISA) procedure was developed. This method integrated two reverse ELISA reactions in one assay by labeling horseradish peroxidase to deoxynivalenol (DON) and orbifloxacin. Within this method, IC50 of the two mAbs for each analyte we produced ranged from 23?～?68 ng?mL^?1 for DONs and 4.1?～?49 ng?mL^?1 for quinolones (QNs). The limit of detection measured by IC10 was achieved at 0.45–1.3 ng?mL^?1 for DONs and 0.59–6.9 ng?mL^?1 for QNs, which was lower than the maximum residue levels. Recoveries in negative samples spiked at concentrations of 100, 200, and 500 ng?mL^?1 ranged from 91.3 to 102.2 % for DONs and 88.7–98.05 % for QNs with relative standard deviation less than 9.88 and 12.67 %. The results demonstrated that this developed immunoassay was suitable for screening of low molecular weight contaminants.

Application of tubular tetrapod magnesium oxide in a biosensor for hydrogen peroxide

Tubular tetrapod magnesium oxide (tt-MgO) can be synthesized by thermal evaporation of Mg metal powder with a pre-grown tetrapod ZnO template. The morphology and structure of the tt-MgO were characterized by X-ray diffraction, scanning electron microscopy and transmission electron microscopy. A composite prepared from tt-MgO, nafion and horseradish peroxidase was employed to modify a gold electrode to result in an electrochemical biosensor for hydrogen peroxide that displays excellent sensitivity and rapid response in the presence of hydroquinone as a mediator. Its sensitivity is 335.4 μA mM^-1 cm^-2, its response is linear in the range from 1.0 to 450 μM, and the detection limit is 0.3 μM. These results demonstrate that tt-MgO provides a promising material for the designs of biosensors.

Amperometric hydrogen peroxide biosensor based on a glassy carbon electrode modified with polythionine and gold nanoparticles

We report on a novel hydrogen peroxide biosensor that was fabricated by the layer-by-layer deposition method. Thionine was first deposited on a glassy carbon electrode by two-step electropolymerization to form a positively charged surface. The negatively charged gold nanoparticles and positively charged horseradish peroxidase were then immobilized onto the electrode via electrostatic adsorption. The sequential deposition process was characterized using electrochemical impedance spectroscopy by monitoring the impedance change of the electrode surface during the construction process. The electrochemical behaviour of the modified electrode and its response to hydrogen peroxide were studied by cyclic voltammetry. The effects of the experimental variables on the amperometric determination of H₂O₂ such as solution pH and applied potential were investigated for optimum analytical performance. Under the optimized conditions, the biosensor exhibited linear response to H₂O₂ in the concentration ranges from 0.20 to 1.6?mM and 1.6 to 4.0?mM, with a detection limit of 0.067?mM (at an S/N of 3). In addition, the stability and reproducibility of this biosensor was also evaluated and gave satisfactory results.

One-step electrochemical immunosensing for simultaneous detection of two biomarkers using thionine and ferrocene as distinguishable signal tags

We report on a new kind of electrochemical immunosensors for simultaneous determination of the biomarkers carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). Thionine and ferrocene were applied as distinguishable electrochemical tags (and mediators) which were covalently conjugated on anti-AFP and anti-CEA antibodies, respectively, via carboxy groups. The resulting conjugates were co-immobilized on a glassy carbon electrode functionalized with gold nanoparticles. Finally, horseradish peroxidase (HRP) was immobilized onto the modified electrode. Labeled thionine and ferrocene, respectively, act as distinguishable tags for simultaneous determination of AFP and CEA due to the difference in the location of their voltammetric peaks. With a one-step immunoassay format, the analytes in the sample produced transparent immunoaffinity reaction with the corresponding antibodies on the electrode. Once the immunocomplex is formed, it partially inhibits the active center of the immobilized HRP, and this decreased the activity of HRP in terms of reduction of hydrogen peroxide. This immunosensor enables the simultaneous determination of AFP and CEA in a single run and within the same dynamic range (0.01–50?ng?mL^?1) and the same lower detection limit (0.01?ng?mL^?1). The reproducibility and stability of the immunosensors are acceptable. The dual immunosensor was applied to evaluate several specimens, and the assay results are in acceptable agreement with clinical data.

Amperometric biosensor for hydrogen peroxide based on the co-electrodeposition of horseradish peroxidase and methylene blue on an ITO electrode modified with an anodic aluminum oxide template

We report on a nano-array sensor for hydrogen peroxide (H₂O₂) that is based on a nanoporous anodic aluminum oxide template. This was used as a matrix for the co-immobilization of horseradish peroxidase (HRP) and methylene blue (MB) on the surface of an indium tin oxide electrode. The immobilized HRP retained its natural activity and MB is capable of efficiently shuttle electrons between HRP and the electrode. The new electrode was characterized by SEM and electrochemical methods. It exhibits fast response, long-term stability, high sensitivity and good selectivity to H₂O₂. Under optimized conditions, it linearly responds to H₂O₂ in the concentration range from 1.0?μM to 26?mM, with a detection limit of 0.21?μM (at S/N?=?3).

A silica-dextran nanocomposite as a novel matrix for immobilization of horseradish peroxidase, and its application to sensing hydrogen peroxide

We report on a novel matrix of solgel organic–inorganic nanocomposite that was fabricated from silica sol gel and dextran. It was used for the immobilization of horseradish peroxidase (HRP) to give a biosensor for hydrogen peroxide (H₂O₂). The sensor film was characterized by Fourier transform infrared and UV–vis spectroscopy with respect to structural features and the conformation of the enzyme. The topographies of the surface of the electrode were investigated by field emission scanning electron microscopy. The biosensor was used to determine H₂O₂ quantitatively in the presence of Methylene blue as a mediator with high electron transfer efficiency. A pair of stable and well defined quasi-reversible redox peaks of the HRP [Fe (III)]/HRP [Fe (II)] redox couple was observed at pH 7.0. The biosensor responds to H₂O₂ in the 0.5 mM to 16.5 mM concentration range, and the limit of detection is 0.5 mM.

An immunochemical test for rapid screening of zearalenone and T-2 toxin

An immunochemically based test for non-instrumental simultaneous detection of zearalenone (ZEA) and T-2 toxin (T2) in feed was developed. The method combines clean-up of sample extract, pre-concentration of analytes by immunoextraction and immunodetection through the enzymatic reaction of horseradish peroxidase (HRP). The test is housed inside a standard 1-mL solid-phase extraction column and consists of three layers: two test layers (one for ZEA and another for T2) with immobilised specific antibodies and one control layer with bound anti-HRP antibodies. Feed extract was passed through an additional column with clean-up layer, which was disconnected after extract application. Total assay time was about 15 min for six samples and detection time was 4 min after chromogenic substrate application. Under optimised conditions a cut-off level for ZEA and T2 of 100 µg/kg was established. Different feed types were analysed for ZEA and T2 contamination by the proposed method and results were confirmed by LC-MS/MS.

Enzyme-linked immunosorbent assay and immunochromatographic strip for rapid detection of atrazine in water samples

We have developed a heterologous direct competitive enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-based immunochromatographic (ICG) strip for the determination of the herbicide atrazine in water samples. The ELISA had a half-maximum inhibition concentration (IC₅₀) of 0.12 ng mL^?1 and a limit of detection (LOD, calculated as the IC₁₅ value) of 0.01 ng mL^?1. The average of recoveries for all spiked water samples was 96.5%. There was a good correlation between the data determined by this ELISA and those obtained by high performance liquid chromatography (HPLC) (r ² ?=?0.996). The visual LOD of the ICG strip assay was 2 ng mL^?1. The assay process only took 10 min, and no sample pretreatment was required. Its high specificity, sensitivity and fast detection made the strip well suited for on-site screening of atrazine in water samples. Both the ELISA and the ICG strip assay are useful for rapid analysis of a large number of water samples at low cost.

Titanium dioxide nanorod-based amperometric sensor for highly sensitive enzymatic detection of hydrogen peroxide

Titanium dioxide nanorods (TNR) were grown on a titanium electrode by a hydrothermal route and further employed as a supporting matrix for the immobilization of nafion-coated horseradish peroxidase (HRP). The strong electrostatic interaction between HRP and TNR favors the adsorption of HRP and facilitates direct electron transfer on the electrode. The electrocatalytic activity towards hydrogen peroxide (H₂O₂) was investigated via cyclic voltammetry and amperometry. The biosensor exhibits fast response, a high sensitivity (416.9 μA·mM^?1), a wide linear response range (2.5 nM to 0.46 mM), a detection limit as low as 12 nM, and a small apparent Michaelis-Menten constant (33.6 μM). The results indicate that this method is a promising technique for enzyme immobilization and for the fabrication of electrochemical biosensors.

Highly sensitive FRET-based fluorescence immunoassay for aflatoxin B1 using cadmium telluride quantum dots

We report on a competitive immunoassay for the determination of aflatoxin B1 using fluorescence resonance energy transfer (FRET) from anti-aflatoxin B1 antibody (immobilized on the shell of CdTe quantum dots) to Rhodamine 123 (Rho 123-labeled aflatoxin B1 bound to albumin). The highly specific immunoreaction between the antibody against aflatoxin B1 on the QDs and the labeled-aflatoxin B1 brings the Rho 123 fluorophore (acting as the acceptor) and the QDs (acting as the donor) in close spatial proximity and causes FRET to occur upon photoexcitation of the QDs. In the absence of unlabeled aflatoxin B1, the antigen-antibody complex is stable, and strong emission resulting from the FRET from QDs to labeled aflatoxin B1 is observed. In the presence of aflatoxin B1, it will compete with the labeled aflatoxin B1-albumin complex for binding to the antibody-QDs conjugate so that FRET will be increasingly suppressed. The reduction in the fluorescence intensity of the acceptor correlates well with the concentration of aflatoxin B1. The feasibility of the method was established by the detection of aflatoxin B1 in spiked human serum. There is a linear relationship between the increased fluorescence intensity of Rho 123 with increasing concentration of aflatoxin B1 in spike human serum, over the range of 0.1–0.6 μmol·mL^?1. The limit of detection is 2?×?10^?11 M. This homogeneous competitive detection scheme is simple, rapid and efficient, and does not require excessive washing and separation steps.

Automated regenerable microarray-based immunoassay for rapid parallel quantification of mycotoxins in cereals

An automated flow-through multi-mycotoxin immunoassay using the stand-alone Munich Chip Reader 3 platform and reusable biochips was developed and evaluated. This technology combines a unique microarray, prepared by covalent immobilization of target analytes or derivatives on diamino-poly(ethylene glycol) functionalized glass slides, with a dedicated chemiluminescence readout by a CCD camera. In a first stage, we aimed for the parallel detection of aflatoxins, ochratoxin A, deoxynivalenol, and fumonisins in cereal samples in a competitive indirect immunoassay format. The method combines sample extraction with methanol/water (80:20, v/v), extract filtration and dilution, and immunodetection using horseradish peroxidase-labeled anti-mouse IgG antibodies. The total analysis time, including extraction, extract dilution, measurement, and surface regeneration, was 19 min. The prepared microarray chip was reusable for at least 50 times. Oat extract revealed itself as a representative sample matrix for preparation of mycotoxin standards and determination of different types of cereals such as oat, wheat, rye, and maize polenta at relevant concentrations according to the European Commission regulation. The recovery rates of fortified samples in different matrices, with 55–80 and 58–79 %, were lower for the better water-soluble fumonisin B1 and deoxynivalenol and with 127–132 and 82–120 % higher for the more unpolar aflatoxins and ochratoxin A, respectively. Finally, the results of wheat samples which were naturally contaminated with deoxynivalenol were critically compared in an interlaboratory comparison with data obtained from microtiter plate ELISA, aokinmycontrol® method, and liquid chromatography–mass spectrometry and found to be in good agreement.

Two kinds of electrochemical immunoassays for the tumor necrosis factor α in human serum using screen-printed graphite electrodes modified with poly(anthranilic acid)

We present two kinds of electrochemical immunoassays for the tumor necrosis factor α (TNF-α) which is a protein biomarker. The antibody against TNF-α was immobilized on a graphite screen-printed electrode modified with poly-anthranilic acid (ASPE). The first is based on impedimetry (and thus label-free) and the target antigen (TNF-α) is captured by the surface of the modified electrode via an immunoreaction upon which impedance is changed. This sensing platform has a detection limit of 5.0 pg mL^?1. In the second approach, the monoclonal antibodies on the modified electrode also bind to the target antigen (TNF-α), but detection is based on a sandwich immunoreaction. This is performed by first adding secondary anti-TNF-α antibodies labeled with horseradish peroxidase, and then detecting the response of the sandwich system by adding hydrogen peroxide and acetaminophen as a probe system for HRP activity. This immunosensor also has a very low detection limit (3.2 pg mL^?1). The experimental conditions of both assays were studied and optimized via electrochemical impedance spectroscopy and differential pulse voltammetry. The method was then applied to the determination of TNF-α in serum samples where it displayed high sensitivity, selectivity and reproducibility.

Novel chemiluminescent assay for staphylococcal enterotoxin B

An enzyme-linked immunosorbent assay, a horseradish peroxidase-catalyzed fluorogenic reaction, and chemiluminescence (CL) analysis have been combined to develop a sandwich ELISA for Staphylococcal enterotoxin B (SEB) using monoclonal antibodies for different epitopes of SEB. The enzyme catalyzed reaction of 3-(4-hydroxyphenyl propionate) with the urea complex of hydrogen peroxide produced a fluorescent dimer which was detected by chemiluminescence analysis. The CL response to SEB is linear in the range from 6.0 to 564?pg?mL^?1 (r?=?0.9993), and the detection limit is 3.3?pg?mL^?1 (S/N?=?3). Intra- and interassay coefficients of variation are <7.0% at three concentrations (24, 96 and 384?pg?mL^?1). The method was applied to the analysis of SEB in serum, lake water and milk samples. The results compared well with those obtained by conventional ELISAs.

Developing new materials for paper-based diagnostics using electrospun nanofibers

The use of electrospun nanofibers as functional material in paper-based lateral flow assays (LFAs) was studied. Specific chemical features of the nanofibers were achieved by doping the base polymer, poly(lactic acid) (PLA), with poly(ethylene glycol) (PEG) and polystyrene_8K-block-poly(ethylene-ran-butylene)_25K-block-polyisoprene_10K-Brij76 (K3-Brij76) (KB). The LFAs were assembled such that the sample flowed through the nanofiber mat via capillary action. Initial investigations focused on the sustainable spinning and assembly of different polymer structures to allow the LFA format. Here, it was found that the base polymer poly(vinyl alcohol) (PVA), which was shown to function well in microfluidic biosensors, did not work in the LFA format. In contrast, PLA-based nanofibers enabled easy assembly. Three relevant features were chosen to study nanofiber-based functionalities in the LFA format: adsorption of antibodies, quantification of results, and nonspecific binding. In particular, streptavidin-conjugated sulforhodamine B (SRB)-encapsulating liposomes were captured by anti-streptavidin antibodies adsorbed on the nanofibers. Varying the functional polymer concentration within the PLA base enabled the creation of distinct capture zones. Also, a sandwich assay for the detection of Escherichia coli O157:H7 was developed using anti-E. coli antibodies as capture and reporter species with horseradish peroxidase for signal generation. A dose–response curve for E. coli with a detection limit of 1.9?×?10⁴ cells was achieved. Finally, functional polymers were used to demonstrate that nonspecific binding could be eliminated using antifouling block copolymers. The enhancement of paper-based devices using functionalized nanofibers provides the opportunity to develop a broad spectrum of sensitive and specific bioassays with significant advantages over their traditional counterparts.

Double layer enzyme modified carbon nanotubes as label for sandwich-type immunoassay of tumor markers

An immunosensor was prepared for the determination of carcinoembryonic antigen (CEA). It is based on the use of multiwalled carbon nanotubes (MWCNTs) along with horseradish peroxidase-labeled antibody. The enzyme was assembled onto MWCNTs templates using the layer-by-layer technique and then conjugated to carcinoembryonic secondary antibodies (Ab₂) as the enzyme label. The resulting assembly results in a largely amplified sensitivity. The response is linear in the range of 0.05 to 45?ng?mL^-1, with a detection limit of 16.0?pg?mL^-1. The immunosensor possesses good stability and good reproducibility.

Ultra-sensitive electrochemical DNA biosensor based on signal amplification using gold nanoparticles modified with molybdenum disulfide,graphene and horseradish peroxidase

We have developed an ultra-sensitive electrochemical DNA biosensor by assembling probe ssDNA on a glassy carbon electrode modified with a composite made from molybdenum disulfide, graphene, chitosan and gold nanoparticles. A thiol-tagged DNA strand coupled to horseradish peroxidase conjugated to AuNP served as a tracer. The nanocomposite on the surface acts as relatively good electrical conductor for accelerating the electron transfer, while the enzyme tagged gold nanoparticles provide signal amplification. Hybridization with the target DNA was studied by measuring the electrochemical signal response of horseradish peroxidase using differential pulse voltammetry. The calibration plot is linear in the 5.0?×?10^?14 and 5.0?×?10^?9 M concentration range, and the limit of detection is 2.2?×?10^?15 M. The biosensor displays high selectivity and can differentiate between single-base mismatched and three-base mismatched sequences of DNA. The approach is deemed to provide a sensitive and reliable tool for highly specific detection of DNA.