Search

Inspired from the funtioning and responsiveness of biological ion channels, researchers attempt to develop biosensing systems based on polymer and solid-state nanochannels. The applicability of these nanochannels for detection/sensing of any foreign analyte in the surrounding environment depends critically on the surface characteristics of the inner walls. Attaching recognition sites to the channel walls leads to the preparation of sensors targeted at a specific molecule. There are many nanochannel platforms for the detection of DNA and proteins, but only a few are capable of detecting small molecules. Here, we describe a nanochannel platform for the detection of hydrogen peroxide, H2O2, which is not only a toxic waste product in the cellular systems but also a key player in the redox signaling pathways. The sensor is based on single conical nanochannels fabricated in an ion tracked polymer membrane. The inner walls of the channel are decorated with horseradish peroxidase (HRP) enzyme using carbodiimide coupling chemistry. The success of the HRP immobilization on the channel surface is confirmed by measuring the pH-dependent current-voltage (I-V) curves of the system. The reported HRP-nanochannel system detects nanomolar concentrations of H2O2 with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as the substrate. The immobilized HRP enzyme is thus capable of inducing redox reactions in a subfemtoliter volume of single nanochannels. We demonstrate that functioning of the designed biosensor is reversible and can be used multiple times to detect H2O2 at various concentrations. [ABSTRACT FROM AUTHOR]

The hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme is assembled on Au electrodes. It reveals bioelectrocatalytic properties and electrocatalyzes the reduction of H2O2. The bioelectrocatalytic functions of the hemin/G-quadruplex DNAzyme are used to develop electrochemical sensors that follow the activity of glucose oxidase and biosensors for the detection of DNA or low-molecular-weight substrates (adenosine monophosphate, AMP). Hairpin nucleic structures that include the G-quadruplex sequence in a caged configuration and the nucleic acid sequence complementary to the analyte DNA, or the aptamer sequence for AMP, are immobilized on Au-electrode surfaces. In the presence of the DNA analyte, or AMP, the hairpin structures are opened, and the hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme stnictures are generated on the electrode surfaces. The bioelecfrocatalytic cathodic currents generated by the functionalized electrodes, upon the electrochemical reduction of H2O2, provide a quantitative measure for the detection of the target analytes. The DNA target was analyzed with a detection limit of 1 x 10-12 M, while the detection limit for analyzing AMP was 1 x 10-6 M. Methods to regenerate the sensing surfaces are presented. [ABSTRACT FROM AUTHOR]

This paper describes the construction of a mixed monolayer of ferrocenylalkanethiol and encapsulated horseradish peroxidase (HRP) at a gold electrode for amperometric detection of H2O2 at trace levels. By tuning the alkanethiol chain lengths that tether the HRP enzyme and the ferrocenylalkanethiol (FcC11SH) mediator, facile electron transfer between FcC11SH and HRP can be achieved. Unlike most HRP-based electrochemical sensors, which rely on HRP-facilitated H2O2 reduction (to H2O), the electrocatalytic current is resulted from an HRP-catalyzed oxidation reaction of H2O2 (to O2). Upon optimizing other experimental conditions (surface coverage ratio, pH, and flow rate), the electrocatalytic reaction proceeding at the electrode was used to attain a low amperometric detection level (0.64 nM) and a dynamic range spanning over 3 orders of magnitude. Not only does the thin hydrophilic porous HRP capsule allow facile electron transfer, it also enables H2O2 to permeate. More significantly, the enzymatic activity of the encapsulated HRP is retained for a considerably longer period (>3 weeks) than naked HRP molecules attached to an electrode or those wired to a redox polymer thin film. By comparing to electrodes modified with denatured HRP that are subsequently encapsulated or embedded in a poly-L-lysine matrix, it is concluded that the encapsulation has significantly preserved the native structure of HRP and therefore its enzymatic activity. The electrode covered with FcC11SH and encapsulated HRP is shown to be capable of rapidly and reproducibly detecting H2O2 present in complex sample media. [ABSTRACT FROM AUTHOR]

Protein arrays that measure multiple protein cancer biomarkers in clinical samples hold great promise for reliable early cancer detection. Herein, we report a prototype 4-unit electrochemical immunoarray based on single-wall carbon nanotube forests for the simultaneous detection of multiple protein biomarkers for prostate cancer. Immunoarray procedures were designed to measure prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), platelet factor-4 (PF-4), and interleukin-6 (IL-6) simultaneously in a single serum sample. All of these proteins are elevated in serum of patients with prostate cancer, but they have widely different relative levels of serum concentration. Horseradish peroxidase (HRP) was used as label on detection (secondary) antibodies in a sandwich immunoassay scheme. Biotinylated secondary antibodies (Ab2) that bind specifically to streptavidin-IIRP conjugates provided 14-16 labels per antibody and gave the necessary higher sensitivity required for PF-4 and IL-6 detection at physiological levels. Conventional singly labeled Ab2-HRP conjugates were sufllcient for PSA and PSMA detection. Immunoarrays were used to measure four biomarkers in clinical human serum samples of prostate cancer patients and controls with excellent correlation to referee enzyme-linked immunosorbent (EUSA) assays. [ABSTRACT FROM AUTHOR]

Engineered nucleic acid hairpin structures are used for the amplified analysis of low-molecular-weight substrates (adenosine monophosphate, AMP) or proteins (lysozyme). The hairpin structures consist of the anti-AMP or antilysozyme aptamer units linked to the horseradish per. oxidase (HRP)-mimicking DNAzyme sequence. The HRPmimicking DNAzyme sequence is protected in a "caged", inactive structure in the stem regions of the respective hairpins, whereas the loop regions include a part of the respective aptamer sequence. The opening of the hairpins by the analytes, AMP or lysozyme, through the formation of the respective analyte-aptamer complexes, results in the sell assembly of the active HRP-mimicking DNAzyme. The DNAzyme catalyzes the H2O2-mediated oxidation of 2,2'-azino-bis(3-ethylberizothiazoline-6-sulfonic acid) (ABTS2) to the colored ABTS-, thus providing the amplified optical detection of the respective analytes. The engineered aptamer-DNAzyme hairpin structures reveal significantly improved analytical performance, as compared to analogous fluorophore-quencherlabeled hairpins. [ABSTRACT FROM AUTHOR]

A new signal amplification strategy based on thionine (TH)-doped magnetic gold nanospheres as labels and horseradish peroxidase (HRP) as enhancer holds promise to improve the sensitivity and detection limit of the immunoassay for carcinoembryonic antigen (CEA), as a model protein. This immunoassay system was fabricated on a carbon fiber microelectrode (CFME) covered with a well-ordered anti-CEA/protein A/nanogold architecture. The reverse micelle method was initially used for the preparation of TH-doped magnetic gold nanospheres (nanospheres), and the synthesized nanospheres were then labeled on HRP-bound anti-CEA as a secondary antibody (bionanospheres). Sandwich-type protocol was successfully introduced to develop a new high-efficiency electrochemical immunoassay with the labeled bionanospheres toward the reduction of H2O2. Under optimized conditions, the linear range of the proposed immunoassay without HRP as enhancer was 1.2-125 ng/mL CM, whereas the assay sensitivity by using HRP as enhancer could be further increased to 0.01 ng/mL with the linear range from 0.01 to 160 ng/mL CM. The developed immunoassay method showed good precision, high sensitivity, acceptable stability and reproducibility, and could be used for the detection of real samples with consistent results in comparison with those obtained by the enzyme-linked immunosorbent assay (ELISA) method. [ABSTRACT FROM AUTHOR]

Modification of a commercial CE instrument (HP 3D-CE, or Agilent G1602A) to adapt it for chemiluminescence (CL) detection is described. The reversible modification involves interchanging the deuterium lamp (used for standard absorbance detection) with a sidearm photo- multiplier tube in the lamp housing. Power supply and amplification circuitry is added via an external bread-board, and the amplified CL signal is recorded via a recorder/integrator. The viability of the CL detector was demonstrated with both pressure rinses and electrophoresis of chemiluminescent samples. Peak area precision was 1.5% RSD for pressure rinses of a peroxyoxalate ("lightstick") sample and 4.0% RSD for electrophoresis of 100 µM luminol (catalyzed by horseradish peroxidase). The CL peak area was linear with concentration for electrophoresis of luminol (10-s injections) from at least 1.0-50 µM. [ABSTRACT FROM AUTHOR]

A methodology for the detection of protein biomarkers at picomolar concentrations that utilizes surface plasmon resonance imaging (SPRI) measurements of RNA aptamer microarrays is developed. The adsorption of proteins onto the RNA microarray is detected by the formation of a surface aptamer-protein-antibody complex. The SPRI response signal is then amplified using a localized precipitation reaction catalyzed by the enzyme horseradish peroxidase that is conjugated to the antibody. This enzymatically amplified SPRI methodology is first characterized by the detection of human thrombin at a concentration of 500 fM; the appropriate thrombin aptamer for the sandwich assay is identified from a microarray of three potential thrombin aptamer candidates. The SPRI method is then used to detect the protein vascular endothelial growth factor (VEGF) at a biologically relevant concentration of 1 pM. VEGF is a signaling protein that has been used as a serum biomarker for rheumatoid arthritis, breast cancer, lung cancer, and colorectal cancer and is also associated with age-related macular degeneration. [ABSTRACT FROM AUTHOR]

A magnetic particle-based immuno-supported liquid membrane assay (m-ISLMA) based on chemiluminescence detection of a horseradish peroxidase-labeled hapten tracer that allows sample cleanup, analyte enrichment, and detection in a single analysis unit has been developed. Antibodies were immobilized on magnetic beads, and their position in the acceptor was controlled by two alternating opposing electromagnetic fields generated by a voltage applied to either of two electromagnets placed below and above the acceptor channel of the supported liquid membrane unit. The influence of antibody bead dilution in the acceptor was investigated and found to follow the ISLM theory, that is improved enrichment and sensitivity with increasing antibody concentration. Two different extraction procedures were investigated: procedure 1 (m-ISLMA-P1), which keeps the antibody beads trapped at the bottom of the acceptor during the entire analysis process; and procedure 2 (m-ISLMA-P2), which keeps the antibody beads dispersed and in motion in the acceptor phase during the extraction process. m-ISLMA- P2 resulted in 2000 times improved enrichment of simazine and a more than 3 orders of magnitude better limit of detection (LOD10%) (1.29 × 10-5 μg L-1) than for m-ISLMA-P1 (2.00 × 10-2 μg L-1) and corresponding microtiter plate magnetic particle-based ELISA (m-ELISA, LOD10% 1.30 × 10-1 μg L-1). m-ISLMA-P2 and m-ELISA were further applied for the extraction and analysis of simazine-spiked surface water and fruit juice, finding no evidence for matrix influence for the former method; however, indications that trace amounts (nanograms per liter) of simazine or specific cross-reactants were present in both samples. [ABSTRACT FROM AUTHOR]

Enzymes are often employed as biocatalysts for the amplified detection of DNA Horseradish peroxidase (HRP) was used as biocatalytic label for the amperometric detection of DNA. The biocatalyzed precipitation of an insoluble product on electrode surfaces was reported as a means for the amplified electrochemical sensing of DNA, and the generation of redox-active DNA replica and the secondary activation of bioelectrocatalytic processes was used for the amplified amperometric analysis of DNA. Recently, researchers reported on the analysis of DNA by the HRP-mediated generation of chemiluminescence.

Focuses on the preparation of the porous titania sol-gel matrix for the immobilization of horseradish peroxidase. Application of the vapor deposition method in the sol-gel matrix; Production of amperometric hydrogen peroxide biosensor; Characterization of enzyme membranes.

Examines the use of horseradish peroxidase as marker enzymes in immunoassays. Production of recombinant fusion of a protein analyte with horseradish; Utilization of human heart fatty acid binding protein (H-FABP); Detection of H-FABP in plasma.

Applies the stability of horseradish peroxidase (HRP) in aqueous and organic solvents to develop a simple thermometric procedure to detect the binding of retinoic acid-HRP conjugate to retinol binding protein. Thermometric detection of butanone peroxide in organic phase and hydrogen peroxide in aqueous phase on a HRP column.

A new technique is reported for the enhanced colon-metric detection of multiplexed hybridization onto porous membrane-based microarrays. This approach combines the use of horseradish peroxidase (HRP) as a label together with a chromogen substrate and a local production of the hydrogen peroxide required for substrate oxidation. This in situ production of coreagent is obtained using glucose oxidase (GOx) directly immobilized within the micro-array porous membrane mesh. The oxidation of glucose by the immobilized GOx produces hydrogen peroxide which itself enables the oxidation of TMB(3,3',5,5'-tetramethylbenzidine) by HRP and yields a blue precipitate on positive spots. Thanks to a coreagent overconcentration within the membrane, this design drastically surpasses the performances of the standard TMB/H2O2 kit used for peroxidase label detection. The obtained target limit of detection is then SO times lower (20 pM) than the one obtained with the standard kit approach, and the dynamic range expands at least one decade. Furthermore, the developed method was shown to compete well with the widely used alkaline phosphatase-BClP (5-bromo-4-chIoro-3-indolyl phosphate)/NBT (nitro blue tetrazolium chloride) readout while minimizing background signal. The method was finally successfully applied to the multiplex detection of single nucleotide polymorphisms (SNPs) in complex PCR samples. The background lowering was impacted here positively on the SNPs' detection by increasing the complementary noncomplementary signal ratio. [ABSTRACT FROM AUTHOR]

The article cites a study conducted by researcher Gil Lee and colleagues who developed an array of microscale reactors on a mesoporous membrane for sensing biochemical reactions such as asymmetric reactions in membrane proteins. The device can be used for affinity, catalytic, membrane and cell-based sensing, and it has better sensitivity and time response than several existing sensors. Lee and colleagues fabricated the device by integrating alumina mesoporous membranes into polymeric microfluidic layers. They tested their new device by using a horseradish peroxidase colorimetric assay to detect enzymatic activity.