Large Amount Of Horseradish Peroxidase Research Articles

Sensitive detection of exosomes by an electrochemical aptasensor based on double nucleic acid amplifications

Exosomes are extracellular vesicles secreted by mammalian cells and recognized as a powerful non-invasive cancer marker because they carry the cellular components specific to parent cells. The development of a convenient, rapid and sensitive exosome detection method is very important for the early diagnosis of cancer. In this study, an electrochemical biosensor based on two signal amplification methods, primer exchange reaction (PER) and rolling circle amplification (RCA), was developed for the high sensitive detection of exosomes. First, exosomes were specifically captured by CD63 aptamer and DNA strands (T) were released through a chain replacement reaction. Then the T strands initiated the PER reaction, producing a large number of short strands of DNA (P). Afterwards, RCA reaction was carried out on the electrode surface. Finally, a large amount of horseradish peroxidase (HRP) was bound to the electrode surface to catalyze o-phenylenediamine (OPD) oxidation, and the reduction current signal was obtained to realize the quantitative detection of exosomes. Under the optimal experimental conditions, there was a good linear relationship between DPV signal and the logarithm of exosome concentration in the range of 88 ∼ 8.8 × 106 particle/μL with a low limit of detection of 22 particle/μL. This strategy could realize sensitive detection of exosomes and provide a new strategy for the detection of other tumor markers.

Multiorbital DNA walker nanoprobe for portable photothermal detection based on H2S etching of cubic Prussian blue.

In the developedassay, multiorbital 3D DNA walker (MO DNA walker) was applied as signal amplified protocol for enhancing the detection signal of the photothermal biosensor, which was designed for sensitive detection of miRNA based on the H2S triggered conversation of photothermal reagent. When the target molecule is present, the DNA walking strand was released and then hybridize with track strands. The landing of walking particles (WPT) on the tracking particles (TPT) promotes the relative movement of the WPT around TPT, thus releasing large amount of horseradish peroxidase (HRP) with the aid of DNAzyme. After reacting with Na2S2O3 and H2O2, multiple H2S can be generated in situ based on the catalytic ability of HRP. Meanwhile, cubic Prussian blue (CPB) was synthesized and exhibited superior photothermal response, which can be served as efficient photothermal reagent and H2S responsive acceptor. Significantly, the photothermal signal of CPB could be obviously reduced after challenged with H2S ascribed to synchronous reaction between the ferric ion (Fe3+) and H2S. The improved walking area and freedom enable significant signal amplification, enhancing the biosensor's performance. Under ideal circumstances, the proposed photothermal assay demonstrated excellent performance for determination of miRNA-21.

A simple and sensitive turn-off biosensor for miRNA-21 detection based on tyramine signal amplification

The expression level of microRNA (miRNA) is strongly associated with a variety of diseases, and the development of new approaches for miRNA assay with high sensitivity will facilitate a deeper understanding of miRNA biological functions. Herein, a simple and sensitive turn-off biosensor for miRNA-21 detection based on tyramine signal amplification (TSA) was developed. In the absence of the target miRNA, the DNA capture probe on the surface of magnetic bead (MB) complementarily hybridizes with the DNA reporter probe; subsequently, a large amount of horseradish peroxidase (HRP) is bound by the biotin-streptavidin reaction; later, in the presence of hydrogen peroxide (H2O2), HRP catalyzes the deposition of biotinylated tyramine, and then the fluorescent phycoerythrin (PE) is successfully introduced via a biotin-streptavidin reaction, producing a robust fluorescence (FL) signal upon excitation. When the target miRNA is present, it competitively occupies the binding site of the DNA capture probes, leading to a decrease in the amount of the DNA reporter probes hybridized to the MB surface; subsequently, the effect of TSA is diminished, which ultimately leads to a weakened FL and generates the “turn-off” signal. This method utilizes the negative correlation between target miRNA concentration and FL intensity, which can achieve high sensitivity detection of miRNA with low detection limit. This method can be successfully applied to the determination of miRNA-21 in human lung cancer cells, which has great application potential for disease diagnosis.

Electrochemical H2O2 biosensor based on horseradish peroxidase encapsulated protein nanoparticles with reduced graphene oxide-modified gold electrode

In this study, an electrochemical biosensor composed of a horseradish peroxidase (HRP)-encapsulated protein nanoparticles (HEPNP) was fabricated for the sensitive and selective detection of H2O2. The HEPNP has a three-dimensional structure that can contain a large amount of HRP; therefore, HEPNP can amplify the electrochemical signals necessary for the detection of H2O2. Furthermore, reduced graphene oxide (rGO) was used to increase the efficiency of electron transfer from the HEPNP to an electrode, which could enhance the electrochemical signal. This biosensor showed a sensitive electrochemical performance for detection of H2O2 with signals in the range from 0.01–100 μM, and it could detect low concentrations up to 0.01 μM. Furthermore, this biosensor was operated against interferences from glucose, ascorbic acid, and uric acid. In addition, this fabricated H2O2 biosensor showed selective detection performance in human blood serum. Therefore, the proposed biosensor could promote the sensitive and selective detection of H2O2 in clinical applications.

Highly sensitive colorimetric immunosensor for influenza virus H5N1 based on enzyme-encapsulated liposome

Development of simple but sensitive biosensor for influenza detection is highly important in immediate and effective clinical treatment. In this study, a sensitive colorimetric immunosensor which combines the advantages of high selectivity of immunoassay and simplicity of colorimetric detection has been developed to detect influenza virus H5N1 based on enzyme-encapsulated liposome. Biotin-tagged liposome encapsulated with large amount of horseradish peroxidase (HRP) was firstly synthesized. In the presence of H5N1, H5N1 co-bound with the capture antibody and the biotinylated detection antibody to form sandwich immunocomplex. Subsequently, the HRP-encapsulated liposome was introduced to conjugate with the detection antibody through biotin-avidin-biotin linkage. Upon the addition of substrate (mixture of 3,3′,5,5′-tetramethylbenzidine (TMB) and H2O2), the liposome was directly lysed to release large amount of HRP by TMB. The released HRP catalyzed the H2O2-mediated oxidation of TMB, resulting in color change of the system, which was observed by naked eyes or UV–vis spectra. The result showed that the absorption intensity enhanced with the increase of H5N1 concentration ranging from 0.1 to 4.0 ng/mL, and the detection limit was calculated to be 0.04 ng/mL. The sensitivity of the proposed biosensor is much higher than that of conventional enzyme-linked immunosorbent assay method. The proposed immunosensor is relatively simple, low-cost, sensitive, and selective without using any sophisticated instruments, therefore it may have a promising prospect for detecting targets in clinical medicine, food safety analysis, and environmental monitoring.

An electrochemical biosensor for DNA detection based on tungsten disulfide/multi-walled carbon nanotube composites and hybridization chain reaction amplification

We report the preparation and characterization of tungsten disulfide-multi-walled carbon nanotubes (WS2-MWCNTs) composite via a simple hydrothermal process and demonstrate its application as a biosensing platform for ultrasensitive determination of hepatitis B virus genomic DNA coupled with hybridization chain reaction (HCR) signal amplification. The sensing platform includes capture DNA probe (cDNA), auxiliary DNA, two DNA hairpins, and detection signal is amplified by horse radish peroxidase (HRP) on the DNA duplex. The biosensor is fabricated with the cDNA labeled at 3′ end using thiol immobilized on the WS2-MWCNTs/Au nanoparticels modified electrode. cDNA fixed onto electrode surface then reacts with target DNA through the 3′ end of auxiliary DNA. The remaining segment on 5′ end of auxiliary DNA then takes turns to open two alternating DNA hairpins. This leads to a HCR events to produce a nicked double-helix. A large amount of HRP is then combined on DNA helix by affinity interaction of biotin-avidin, each of which amplifies the final electrochemical signal through the catalytic reaction of H2O2 and hydroquinol. Under optimal conditions, a dynamic range of 10fM–0.1nM is obtained with a detection limit of 2.5fM. Synergistic effect of WS2-MWCNTs composite and HCR have proven to greatly improve sensitivity for DNA detection.

Two-stage nicking enzyme signal amplification combined with DNAzyme amplification for the detection of bone morphogenetic protein 6 mRNA.

Highly sensitive detection of bone morphogenetic protein 6 (BMP6) mRNA is essential to monitor bone regeneration in the regenerating defects. In this work, we proposed a quantitative approach based on two-stage nicking enzyme signal amplification (NESA) and DNAzyme amplification for highly sensitive detection of BMP6 mRNA. The two-stage NESA involves two templates and two-stage amplification reactions under isothermal conditions. The first template contains two repeat sequences that could hybridize to the target RNA, triggering an exponential amplification. The amplified product was a short single-stranded DNA with the same sequence as the target RNA. The single-stranded DNA can trigger another linear NESA and produces a large amount of horseradish peroxidase (HRP)-mimicking G-quadruplex DNAzyme. This proposed assay showed a quantitative analysis of BMP6 mRNA in a wide range from 1 fM to 100 nM with a detection limit of 0.01 fM.

Sensitive Chemiluminescent Imaging for Chemoselective Analysis of Glycan Expression on Living Cells Using a Multifunctional Nanoprobe

A novel sensitive chemiluminescent (CL) imaging method was developed for in situ monitoring of cell surface glycan expression through chemoselective labeling of carbohydrate motifs and then binding to a multifunctional nanoprobe. The nanoprobe was fabricated by assembling biotin-DNA and a large amount of horseradish peroxidase (HRP) on gold nanoparticles (AuNPs). The chemoselective labeling was performed by selective oxidization of the hydroxyl sites of sialyl and galactosyl groups on cell surfaces into aldehydes by periodate and galactose oxidase, respectively, and then aniline-catalyzed hydrazone ligation with biotin hydrazide for specific recognition to avidin. With the biotin-avidin system, the multifunctional nanoprobe could conveniently be bound to the glycan sites on the cell surface. The DNA chain presenting between the AuNPs and biotin assembled on the nanoprobe could obviate the steric effect, and HRP acted to trigger the CL emission of the luminal-H(2)O(2) system. Therefore the expression of both sialyl and galactosyl groups could be selectively monitored by CL imaging with high sensitivity due to the high amount of HRP. Using human liver cancer HCCC-9810 cells as a model, this CL imaging strategy could detect HCCC cells ranging from 6 × 10(2) to 1 × 10(7) cells mL(-1) with a detection limit down to 12 cells. More importantly, this method could be used for distinguishing cancer cells from normal cells and monitoring of dynamic carbohydrate expression on living cells, providing promising application in clinical diagnosis and treatment of cancer.

Permeability for horseradish peroxidase in strial capillaries in each turn of cochlea.

The capillary permeability of the stria vascularis in each turn of cochlea was examined at intervals of 10 and 30 min and 1 and 2 h after an injection of horseradish peroxidase (HRP), using light and electron microscopic techniques. Ten minutes after HRP was administered, 18% of examined vessels showed HRP leakage. Thereafter, the level of HRP leakage increased with time. One hour after the injection, HRP had leaked from almost all vessels of the upper three turns. Two hours after the injection, the tracer in the vessel lumen became sparse with very thin staining or nonstaining, and a large amount of HRP was observed outside the vessels. It was noted that the capillary permeability to HRP at the basal turn was different from that of the upper three turns. This seems to be the result of the different pinocytic activity in the capillaries of these two regions. When the tracer disappeared from the vessels (2 h after the HRP injection), labelled vesicles in endothelial cells were frequently visible near the outside surface, whereas very few were visible near the inner surface. It is speculated, therefore, that HRP which has leaked can be reabsorbed back into the blood circulation via a similar micropinocytosis down the concentration gradient.

Analysis of horseradish peroxidase-amplified chemiluminescence produced by human neutrophils reveals a role for the superoxide anion in the light emitting reaction

When polymorphonuclear leukocytes and soluble or particulate matter interact, the cells produce chemiluminescence, which is linked to activation of the oxidative metabolism of the cells. A luminol chemiluminescence assay in which the reaction mixture contains a relatively large amount of horseradish peroxidase combined with sodium azide has been proposed to quantitate H 2O 2 produced by human neutrophils during the respiratory burst (M. P. Wymann, V. von Tscharner, D. A. Deranleau, and M. Baggiolini (1987) Anal. Biochem. 165, 371–378) . We found, when comparing the response to concanavalin A and a formylated peptide (formylmethionyl-leucyl-phenylalanine), that neutrophils produce H 2O 2 that is not detected as chemiluminescence by the horseradish peroxidase-azide-luminol system. Furthermore, the horseradish peroxidase-amplified chemiluminescence response obtained from granule-depleted neutrophil cytoplasts is inhibited by superoxide dismutase, an O 2 − scavanger. Based on these results, we question the specificity of the described technique for H 2O 2. The usefulness of the technique in the determining the extracellular and intracellular production of oxidative metabolites is discussed.

Endocytosis in the uterine luminal and glandular epithelial cells of mice during early pregnancy.

We have localized horseradish peroxidase (HRP) in the mouse uterus after intravenous administration on days 1 and 5 of pregnancy in an effort to understand how serum proteins reach the uterine lumen. Direct movement of HRP into uterine and glandular lumina was blocked by the epithelial tight junctions on both days. In luminal and glandular epithelial cells at both times, HRP was localized in endocytic vesicles along the basolateral membranes, multivesicular bodies (mvb), elongated dense bodies below the nucleus (bdb), and many small vesicles near the apical surface of the cells. The uptake of HRP was most extensive in the luminal epithelium on day 1: the number of tracer-containing apical vesicles and bdb was largest, and there were also clusters of vesicles containing the tracer above the nucleus. Acid phosphatase was localized on day 1 in mvb and bdb in both cell types, indicating that these structures are lysosomes. It appeared that HRP followed two pathways after basolateral endocytosis by the epithelial cells: it was transported to the apical region of the cells, where it was present in small vesicles that may release their contents into the uterine or glandular lumina, or it was transported to lysosomes. To investigate whether macromolecules may be transported from the uterine lumen to the stroma, we also studied endocytosis at the apical pole of luminal epithelial cells after intraluminal injection of HRP. There was no detectable uptake of HRP from the lumen on day 1, and no tracer was detected in the intercellular spaces or basement membrane region. On day 5, a large amount of HRP was taken up from the lumen into apical endocytic vesicles, mvb, and dense bodies, but tracer was not present in the Golgi apparatus, lateral intercellular spaces, or the basement membrane region at the times studied. These observations indicate that there was no transport of luminal macromolecules to the uterine stroma on day 1, while the possibility of transport on day 5 requires further study.