Avidin-horseradish Peroxidase Research Articles

Immune Cells Activating Biotin-Decorated PLGA Protein Carrier.

Nanoparticle formulations have long been proposed as subunit vaccine carriers owing to their ability to entrap proteins and codeliver adjuvants. Poly(lactic-co-glycolic acid) (PLGA) remains one of the most studied polymers for controlled release and nanoparticle drug delivery, and numerous studies exist proposing PLGA particles as subunit vaccine carriers. In this work we report using PLGA nanoparticles modified with biotin (bNPs) to deliver proteins via adsorption and stimulate professional antigen-presenting cells (APCs). We present evidence showing bNPs are capable of retaining proteins through the biotin-avidin interaction. Surface accessible biotin bound both biotinylated catalase (bCAT) through avidin and streptavidin horseradish peroxidase (HRP). Analysis of the HRP found that activity on the bNPs was preserved once captured on the surface of bNP. Further, bNPs were found to have self-adjuvant properties, evidenced by bNP induced IL-1β, IL-18, and IL-12 production in vitro in APCs, thereby licensing the cells to generate Th1-type helper T cell responses. Cytokine production was reduced in avidin precoated bNPs (but not with other proteins), suggesting that the proinflammatory response is due in part to exposed biotin on the surface of bNPs. bNPs injected subcutaneously were localized to draining lymph nodes detectable after 28 days and were internalized by bronchoalveolar lavage dendritic cells and macrophages in mice in a dose-dependent manner when delivered intranasally. Taken together, these data provide evidence that bNPs should be explored further as potential adjuvanting carriers for subunit vaccines.

Study of Thitonia Diversifolia Extract in Histomorphology of Pancreas and Interleukin-1beta expression on Aloxan Induced Wistar Rats

The purpose of this study is to determine effect of antiinflammation of Thitonia diversifolia (TD) extracts in histopathology liver and pancretic tissue on alloxan-induced in Wistar rat. Thirty two male Wistar rats (Rattus norvegicus) with a body weight (bw)150-200 g in 3 month of age, were allocated into four groups, with eight animals per group. The control group received normal saline (P0), positive control group received CMC Na 0.01% (P1), the treatment group were received 100 mg/kg bw of the TD extracts (P2) and received 100 mg/kg bw of the metformin (P3) respectively for 7 days. Bloods serum collected for measuring of blood glucose (BG). cholesterol and insulin (INS) concentrations. The levels of insulin (INS) concentrations were analysed by Avidin-Horseradish Peroxidase (HRP) Sandwich-ELISA. All animal were sacrified for tissue staining with haematoxylin eosin. Thitonia diversifolia extract significantly decreased the level of blood glucoseand choelsterol concentrations compared to the postive control group (p<0.05). The level of INS could be detected by its level significantly increased in TD treatment group (p<0.05). Histopathological showed that induction of alloxan could effect severe multifocal degeneration, vacuolisation, cell inflammation, acute langerhan cells injury with destruction cells. Immunohistochemical staining labeled with IL-1β monoclonal antibodies (Mab) showed lowering expression of IL-1β reveal as brownish color aggregates on the langerhans cells in the TD treatment group. This study was concluded that TD extract is inhibited hepatic tissue injuries and decreased destruction in langerhan cells by decreasing IL-1β expression on alloxan-induced rats.

Effect of Tithonia diversifolia Leaf Extract on Leptin, Adiponectin, and Insulin Receptor Levels in Diabetic Rats

This study aimed to evaluate the effect of Tithonia diversifolia extract (TDE) on leptin, adiponectin, and insulin receptor (IR) concentrations in diabetic rats. Twenty-four Wistar rats were divided into a control and treatment groups (n=6 per group). The control group received normal saline, and the treatment groups received 0.25% sodium carboxymethyl cellulose, TDE at 100 mg/kg body weight (bw), and catechin at 10 mg/kg bw for 7 days. On day 8, the rats were sacrificed, blood samples were obtained, and leptin, adiponectin, and insulin concentrations were measured using avidinhorseradish peroxidase sandwich-enzyme-linked immunosorbent assay. A calorimetric method was used to measure blood glucose (BG) and total serum cholesterol concentrations. The pancreas and kidneys were collected for the measurement of renal IR and macrophage cluster of differentiation (CD)14 levels using immunohistochemical staining. Acute type 2 diabetes mellitus (T2DM) with elevated BG and total serum cholesterol concentrations was observed in the treatment groups administered streptozotocin. The administration of TDE at 100 mg/kg bw significantly decreased leptin and increased adiponectin concentrations (P≤0.05). Furthermore, TDE treatment significantly increased renal IR and decreased macrophage CD14 levels (P<0.05). Therefore, TDE decreased leptin and BG concentrations by increasing IR levels. TDE also suppressed the necrosis of pancreatic tissues by inhibiting macrophage CD14 expression in diabetic rats. However, further research is necessary to determine the effect of TDE on interleukin and IR levels in the related tissues of patients with T2DM.

Electrochemical micro-aptasensors for exosome detection based on hybridization chain reaction amplification

Exosomes are cell-derived nanovesicles that have recently gained popularity as potential biomarkers in liquid biopsies due to the large amounts of molecular cargo they carry, such as nucleic acids and proteins. However, most existing exosome-based analytical sensing methods struggle to achieve high sensitivity and high selectivity simultaneously. In this work, we present an electrochemical micro-aptasensor for the highly sensitive detection of exosomes by integrating a micropatterned electrochemical aptasensor and a hybridization chain reaction (HCR) signal amplification method. Specifically, exosomes are enriched on CD63 aptamer-functionalized electrodes and then recognized by HCR products with avidin-horseradish peroxidase (HRP) attached using EpCAM aptamers as bridges. Subsequently, the current signal that is generated through the enzyme reaction between the HRP enzyme and 3,3’,5,5’-tetramethylbenzidine (TMB)/H2O2 directly correlates to the amount of bound HRP on the HCR products and thus to the number of target exosomes. By introducing anti-EpCAM aptamers, micro-aptasensors can detect cancerous exosomes with high specificity. Due to the micropatterned electrodes and HCR dual-amplification strategy, the micro-aptasensors achieve a linear detection response for a wide range of exosome concentrations from 2.5×103 to 1×107 exosomes/mL, with a detection limit of 5×102 exosomes/mL. Moreover, our method successfully detects lung cancer exosomes in serum samples of early-stage and late-stage lung cancer patients, showcasing the great potential for early cancer diagnosis.

Roles of Averrhoa bilimbi Extract in Increasing Serum Nitric Oxide Concentration and Vascular Dilatation of Ethanol-Induced Hypertensive Rats.

The considerably high incidence of cardiovascular disease in Indonesia has attracted scientists to investigate various plant and fruit extracts as preventive agents. Averrhoa bilimbi (AB) is rich in bioactive constituents that may be effective in preventing indicators of hypertension. This study evaluated the roles of AB extract in increasing serum nitric oxide (NO) concentration and vascular dilatation in ethanol-induced hypertensive rats. A total of 24 male Wistar rats (Rattus norvegicus) were divided equally into 4 treatment groups (n=6): P0 (control group, administered placebo); P1 [administered captopril 3 mg/kg body weight (BW) orally]; P2 (administered AB extract at 20 g/kg BW); and P3 (administered AB extract at 40 g/kg BW). The AB extract was obtained from fresh AB macerated in 96% ethanol and was subjected to bioactive compounds identification using thin layer chromatography. After pretreatment with ethanol for 15 days, treatments were administered daily for 14 days. All rats were measured for tail blood pressure by the tail-cuff method and NO concentrations by avidin-horseradish peroxidase sandwich-enzyme-linked immunosorbent assays. All rats were sacrificed to collect blood vessels for histopathology. The results showed that AB extracts contained flavonoids, saponins, polyphenols, essential oils, and anthraquinone. Treatment with AB extract at a dose of 40 mg/kg BW significantly increased NO concentrations (P<0.05). Histopathological analysis showed that AB extracts inhibited endothelial pyknosis, intimal body, and adventitial leukocyte infiltration of posterior vena cava blood vessels. These results suggest that the protective effect of AB extracts is associated with NO concentration in the blood by inhibiting blood vessel dysfunction.

Study of doking molecule flavonoid Coleus amboinicus in TGF-1b receptor and lowering MDA concentration on Cisplatin-induce Wistar Rats

The aims of this study is to evaluate molecular docking of flavonoid Coleus amboinicus (CA) extracts in transforming growth factor-1b and lowering MDA concentration on cisplatin-induced in Wistar rat. Eighteen male Wistar rats (Rattus norvegicus), 3 months of age with a body weight (BW) of 150-200 g, were allocated into three groups, with six animals per group. The control group received aquadest (P0), the treatment group were treated with single doses of cisplatin (5 mg/kg bw., ip) (P1) and received 100 mg/kg bw of the CA extracts (P2) respectively for 7 days. Bloods collected for analysis of serum alkaline phospatase (AP), Blood Nitrogen Urea (BUN) and Malondialdehid (MDA) concentrations. The levels of Malondialdehid (MDA) concentrations were analysed by Avidin-Horseradish Peroxidase (HRP) Sandwich-ELISA. All groups were sacrified for histopathology. Coleus amboinicus extract significantly decreased the level of AP, BUN and MDA concentrations compared to the control group (p&lt;0.05). The level of MDA could be detected by its level significantly decreased in CA treatment group (p&lt;0.05). Coleus amboinicus (CA) extract has a flavonoid as a marker compound of CA extract has stronger bind to the TGF-β1receptor than its of 3WA_601 ligand in silico analyzed. In histopathological examination showed that cisplatin-induced could alter severe multifocal hemorrhage, interstitial congestion, cell inflammatory, acute glomerular and tubular injury with necrotic cells. Immunohistochemical staining labeled with TGF-1β monoclonal antibodies (Mab) showed marked expression of brownish color aggregates on the surface of tubular epithelial cells and around glomerular mesangial cells in the CA treatment group. This study was concluded that CA extract is inhibited renal tissue injuries by lowering MDA and increasing TGF-1b expression on cisplatin-induced rats. Flavonoid as marker of CA extract has stronger bind to TGF-1b receptor by in silico.

STUDY OF Coleus amboinicus STEM EXTRACT IN INHIBITING MACROPHAGE CD-68 EXPRESSION IN WISTAR RATS WITH URIC ACID-INDUCED NEPHROPATHY

The purpose of this study was to evaluate effect of Coleus amboinicus (CA) stem extracts on uric acid-induced nephropathy by comparingthe levels of Macrophage CD-68 expression and concentration of serum Cystatine C (CYS C ) in Wistar rats. Twenty-four male Wistar rats(Rattus norvegicus) with a body weight (bw) of 200-250 g, were allocated into three groups, with eight animals per group. The rats in controlgroup (PO) received 0.1% carboxymethyl cellulose (CMC) solution orally The rats in group 2 (P1) were orally induced with uric acid (UA) (500mg/kg) and oxonic acid (OA) (750 mg/kg.) and the rats in group 3 (P2) received uric acid (500 mg/kg), oxonic acid (750 mg/kg), and 500 mg/kgof the CA stem extracts for 35 days. Bloods were collected for analysis of serum CYS C expression and concentration of serum creatinine andblood nitrogen urea (BUN). The rats in all groups were sacrificed for kidney tissue extractions for macrophage CD-68 identification andhistopathology analysis. The levels of CYS C concentrations were analyzed by Avidin-Horseradish Peroxidase (HRP) Sandwich-ELISA. Theresults showed that Coleus amboinicus stem extract at dose of 500 mg/kg bw can significantly reduce BUN and creatinine levels (P≤0.05), whileCys C levels were not different. In the treatment group (P2) compared with group (P1). CD-68 (ED-1) macrophage activity decreasedsignificantly (P≤0.05) in the treatment group (P2) compared to the control group and (P1). Nephrophaty induction using UA and OA causessevere kidney lesions characterized by degeneration, necrosis and inflammation of the renal tubules and glomerulus in the treatment group.

An Electrochemical Sense Array Based on Aptamer and Biotin-Avidin System for the Selective Detection of Glucagon-Like Peptide-1.

GLP-1 as an incretin, has the ability to decrease blood sugar levels in a glucose-dependent manner by enhancing the secretion of insulin. Besides the insulinotropic effects, GLP-1 has been associated with numerous regulatory and protective effects. Thus, the action of GLP-1 is preserved in patients with type 2 diabetes and substantial pharmaceutical research has therefore been directed towards the development of GLP-1-based treatment. In this work, we reported an electrochemical sense array based on the aptamer and biotin-avidin system for the detection of glucagon-like peptide-1 (GLP-1). The sense array employed a "stem-loop" conformation ap-tamer which was immobilized on the electrode of the 16-unit gold array via pre-labeled thiol group (-SH). Pre-labeled biotin serves as an affinity tag for the binding of avidin-horseradish peroxidase (avidin-HRP). The stem-loop structure of the aptamer kept the biotin from being approached by a bulky avidin-HRP by means of the steric hindrance. After the interaction of the target (GLP-1) and the aptamer, the aptamer would undergo a significant conformational change to force biotin away from the electrode, giving the avidin-HRP easy access to the labeled biotin. The HRP in the substrate could sensitively transduce the concentration of GLP-1 into the electrical signals, which were then measured by electrochemical technology of cyclic voltammetry and amperometric i-t curve. Under the optimal experimental conditions, the proposed sense array for GLP-1 had a good linear relationship from 0.1 pmol/L to 20 pmol/L with a detection limit of 0.05 pmol/L and can be used to accurately detect the GLP-1 in serum. The experimental results show that GLP-1 could be selectively detected by the electrochemical sense array, indicating that the proposed sense array based on the biotin-avidin system and the stem-loop aptamer has great potential in the detection of GLP-1.

Multi‐binding biotinylated iron oxide nanoparticles as a promising versatile material for magnetic biomedical applications

The tetrameric streptavidin and avidin both have four sites that can specifically bind with biotin. Taking advantage of this high affinity binding, it is possible to realise multi-binding of biotinylated nanoparticles to avidin or streptavidin molecules. The iron oxide nanoparticles (IONPs) have been actively investigated in biomedical applications. In this work, biotinylated IONPs (biotin-IONPs) with around 10.5 nm magnetic core size were synthesised. They exhibited long-term hydrophilic stability, and the activity of the biotin content was verified by avidin-horseradish peroxidase induced chromogenic experiment. These biotin-IONPs were then studied as magnetic labels in substrate-based and substrate-free magnetic bio-labelling scheme of alpha-fetoprotein. The multi-binding ability of these biotin-IONPs with streptavidin functionalised detection antibody (streptavidin-detection antibody) was proved both on microplate well surfaces and in solution. Consequently, biotin-IONPs are promising magnetic label materials for the improvement of magnetic biodetection. In addition, the avidin-induced biotin-IONP assemblies were produced in MilliQ water solution and in water-in-oil microemulsion system. In summary, based on their multi-binding nature, the biotin-IONPs can be considered as a versatile material to improve the sensitivity of magnetic biodetection and to be further built into assemblies for future biomedical applications such as magnetic resonance imaging and target drug delivery.

Electrochemical aptasensor based on a novel flower-like TiO2 nanocomposite for the detection of tetracycline

Abstract Herein, we developed a sensitive sensing platform for tetracycline detection by combining the specific DNA aptamers with a novel TiO2 nanocomposite. In this composite, cylinder-shaped TiO2 nanorods were synthesized on MoS2 nanospheres by a facile hydrothermal method to produce a MoS2-TiO2 composite, which was then aminated. Following that, gold nanoparticles (Au) were deposited on the aminated MoS2-TiO2 to form MoS2-TiO2@Au composite, on which thiolated DNA aptamers were applied to construct an electrochemical aptasensor. The tetracycline aptamer could well hybridize to its complementary biotin-DNA oligonucleotide (bio-cDNA), and then avidin-horseradish peroxidase (avidin-HRP) mediated by H2O2 was introduced to produce amplified electrochemical response. The competition between tetracycline and bio-cDNA for binding of the aptamer would decrease the electrochemical signal, which was used for quantitative detection of tetracycline. The prepared aptasensor showed a wide linear range from 1.5 × 10−10 to 6.0 × 10−6 M, and a low detection limit of 5 × 10−11 M.

Electrochemical DNA Biosensor Based on a Tetrahedral Nanostructure Probe for the Detection of Avian Influenza A (H7N9) Virus.

A DNA tetrahedral nanostructure-based electrochemical biosensor was developed to detect avian influenza A (H7N9) virus through recognizing a fragment of the hemagglutinin gene sequence. The DNA tetrahedral probe was immobilized onto a gold electrode surface based on self-assembly between three thiolated nucleotide sequences and a longer nucleotide sequence containing complementary DNA to hybridize with the target single-stranded (ss)DNA. The captured target sequence was hybridized with a biotinylated-ssDNA oligonucleotide as a detection probe, and then avidin-horseradish peroxidase was introduced to produce an amperometric signal through the interaction with 3,3',5,5'-tetramethylbenzidine substrate. The target ssDNA was obtained by asymmetric polymerase chain reaction (PCR) of the cDNA template, reversely transcribed from the viral lysate of influenza A (H7N9) virus in throat swabs. The results showed that this electrochemical biosensor could specifically recognize the target DNA fragment of influenza A (H7N9) virus from other types of influenza viruses, such as influenza A (H1N1) and (H3N2) viruses, and even from single-base mismatches of oligonucleotides. Its detection limit could reach a magnitude of 100 fM for target nucleotide sequences. Moreover, the cycle number of the asymmetric PCR could be reduced below three with the electrochemical biosensor still distinguishing the target sequence from the negative control. To the best of our knowledge, this is the first report of the detection of target DNA from clinical samples using a tetrahedral DNA probe functionalized electrochemical biosensor. It displays that the DNA tetrahedra has a great potential application as a probe of the electrochemical biosensor to detect avian influenza A (H7N9) virus and other pathogens at the gene level, which will potentially aid the prevention and control of the disease caused by such pathogens.

Neu3 Activity Enhances Egfr Activation Without Affecting Egfr Expression

ABSTRACT Aim: The plasma membrane associated sialidase NEU3 is often deregulated in colorectal cancer (CRC) and was demonstrated to coimmunoprecipitate in HeLa cells with the epidermal growth factor receptor (EGFR), the molecular target of the recent therapies based on monoclonal antibodies. Elucidating this correlation may provide novel understandings in cancer biology and may represent a new basis to develop innovative therapeutic approaches. Therefore, our aim was to evaluate the effect of NEU3 deregulation on EGFR expression and activation in CRC cell lines. Methods: NEU3 and EGFR expression was evaluated by Real-time PCR (Q-PCR) in 13 human CRC cell lines, as well as in DiFi cells. Regulation of EGFR pathway by NEU3 activity was studied using NEU3 wild-type (wt) and double-mutant (encoding for NEU3 inactivated protein) vectors transiently transfected in DiFi and SW480 cell lines. EGFR gene and mutational status were assessed by FISH and direct sequencing. Western Blot analysis was performed to study expression and phosphorylation status of EGFR downstream proteins. EGFR sialylation levels were evaluated by lecting binding assay using biotinilated SNA (Sambucus nigra agglutinin) and avidin-horseradish peroxidase. Cell viability was assessed by MTT assay. Results: We observed up-regulation of NEU3 mRNA levels and EGFR activation in all the investigated cell lines, independent of EGFR mRNA/protein expression or gene status. Transfection assays confirmed that only NEU3 wt enhanced EGFR activation without affecting its expression. Moreover, the level of EGFR sialylation was reduced in cells overexpressing the NEU3 wt sialidase, strongly suggesting that EGFR sialylation is regulated by NEU3. MTT test's results showed a significant increase of cell viability (p Conclusions: Since NEU3 reveals to be overexpressed in the totality of cancer cell lines analyzed and its activity regulates the EGFR pathway, we assumed this sialidase as a good and innovative marker for diagnosis and therapy of CRC. Disclosure: All authors have declared no conflicts of interest.

Determination of zearalenone in corn based on a biotin-avidin amplified enzyme-linked immunosorbent assay

A biotin-avidin amplified enzyme-linked immunosorbent assay (BA-ELISA) method was developed to detect zearalenone (ZEN) residues in corn. The concentration of coating antigen, biotinylated monoclonal antibody specific to ZEN, avidin-horseradish peroxidase and reaction time in the system of BA-ELISA method were optimised. Under the optimum conditions, a good linear relationship between binding ratio and ZEN concentration in the range of 0.54 to 7.99 ng/mL was obtained. The regression equation was y=−21.26 ln(x)+66.89 (R2=0.9989). The limit of detection and limit of quantification were 0.35 ng/mL and 0.812 ng/mL. The IC50 was calculated to be 2.071 ng/mL. In comparison with the traditional ELISA method, the sensitivity of BA-ELISA method developed has been elevated by six times. The recovery and coefficient of variation with the spiked corn samples were in the range of 86.6% to 93.7% and less than 7.8%, respectively. It promises a bright future for the sensitive and rapid detecting method of ZEN residues in corn.

Enzyme-based E-RNA sensor array with a hairpin probe: Specific detection of gene mutation

An enzyme-based E-RNA sensor for the sequence-specific detection of nucleic acids was reported. The RNA sensor employed a “stem-loop” DNA probe which was immobilized on a 16-unit gold array surface via pre-labeled with thiol group (–SH), and pre-labeled biotin serves as an affinity tag for the enzyme (avidin-horseradish peroxidase) binding. The stem-loop structure of the sensor probes kept the biotin from being approached by a bulky avidin-horseradish peroxidase (avidin-HRP) by means of the steric hindrance. However, the probes would undergo a significant conformational change after DNA/RNA hybridization by forcing biotin away from the electrode, resulting the avidin-HRP access to the labeled biotin. Thus, target DNA/RNA hybridization event could be sensitively transduced via the enzymatically amplified electrochemical voltage and current signals, which were then measured by means of cyclic voltammetry and amperometric detection. The results showed that a single-base mismatch in target RNA could be selectively detected by the E-RNA sensor array, indicating that this E-RNA sensing strategy based on enzyme amplification is versatile with great potential in the construction of stem-loop based biosensors for the detection of nucleic acid mutations.

Simultaneous Immunoassay Analysis of Plasma IL-6 and TNF-α on a Microchip

Sandwich enzyme-linked immunosorbant assay (ELISA) using a 96-well plate is frequently employed for clinical diagnosis, but is time-and sample-consuming. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. The microchip was made of cyclic olefin copolymer with 4 straight microchannels. For the construction of the sandwich ELISA for interleukin-6 (IL-6) or tumor necrosis factor-α (TNF-α), we used a piezoelectric inkjet printing system for the deposition and fixation of the 1st anti-IL-6 antibody or 1st anti-TNF-α antibody on the surface of the each microchannel. After the infusion of 2 µl of sample to the microchannel and a 20 min incubation, 2 µl of biotinylated 2nd antibody for either antigen was infused and a 10 min incubation. Then 2 µl of avidin-horseradish peroxidase was infused; and after a 5 min incubation, the substrate for peroxidase was infused, and the luminescence intensity was measured. Calibration curves were obtained between the concentration and luminescence intensity over the range of 0 to 32 pg/ml (IL-6: R2 = 0.9994, TNF-α: R2 = 0.9977), and the detection limit for each protein was 0.28 pg/ml and 0.46 pg/ml, respectively. Blood IL-6 and TNF-α concentrations of 5 subjects estimated from the microchip data were compared with results obtained by the conventional method, good correlations were observed between the methods according to linear regression analysis (IL-6: R2 = 0.9954, TNF-α: R2 = 0.9928). The reproducibility of the presented assay for the determination of the blood IL-6 and TNF-α concentration was comparable to that obtained with the 96-well plate. Simultaneous detection of blood IL-6 and TNF-α was possible by the deposition and fixation of each 1st antibody on the surface of a separate microchannel. This assay enabled us to determine simultaneously blood IL-6 and TNF-α with accuracy, satisfactory sensitivity, time saving ability, and low consumption of sample and reagents, and will be applicable to clinic diagnosis.

Portable chemiluminescence multiplex biosensor for quantitative detection of three B19 DNA genotypes

A miniaturized multiplex biosensor exploiting a microfluidic oligonucleotide array and chemiluminescence (CL) lensless imaging detection has been developed for parvovirus B19 genotyping. The portable device consists of a reaction chip, comprising a glass slide arrayed with three B19 genotype-specific probes and coupled with a polydimethylsiloxane microfluidic layer, and a charge-coupled device camera modified for lensless CL imaging. Immobilized probes were used in DNA hybridization reactions with biotin-labeled targets, and then hybrids were measured by means of an avidin-horseradish peroxidase (HRP) conjugate and CL detection. All hybridization assay procedures have been optimized to be performed at room temperature through the microfluidic elements of the reaction chip, with sample and reagents delivery via capillary force exploiting adsorbent pads to drive fluids along the microchannels. The biosensor enabled multiplex detection of all B19 genotypes, with detectability down to 80 pmol L(-1) for all B19 genotype oligonucleotides and 650 pmol L(-1) for the amplified product of B19 genotype 1, which is comparable with that obtained in traditional PCR-ELISA formats and with notably shorter assay time (30 min vs. 2 h). The specificity of the assay has been evaluated by performing DNA-DNA hybridization reactions among sequences with different degrees of homology, and no cross hybridizations among B19 genotypes have been observed. The clinical applicability has been demonstrated by assaying amplified products obtained from B19 reference serum samples, with results completely consistent with the reference PCR-ELISA method. The next crucial step will be integration in the biosensor of a miniaturized PCR system for DNA amplification and for heat treatment of amplified products.

Single-nucleotide polymorphism genotyping using a novel multiplexed electrochemical biosensor with nonfoulingsurface

A novel electrochemical DNA biosensor for single-nucleotide polymorphism (SNP) analysis was developed. In this work, an oligonucleotide-incorporated nonfouling surface (ONS) was constructed to resist nonspecific absorption. The biosensor was developed using a 16-electrode array for high-throughput SNP analysis. The proposed strategy was primarily based on specific oligonucleotide ligation. Fully matched target DNA templated the ligation between a capture probe assembled on gold electrodes and a tandem signal probe with a biotin moiety that could capture avidin–horseradish peroxidase and sequentially generate a catalysed amperometric signal. A pre-core mutation in the hepatitis B virus (HBV) genome at G1896A and two adjacent polymorphisms in the human CYP2C19 genome at C680T and G681A were analysed. Polymerase chain reaction (PCR) products were used as real-life samples and analysed. Our results showed that 10% of a single-mismatched mutant gene was clearly distinguished with a current signal 16 times higher than that of the blank sample, demonstrating the selectivity and practicability of the multiplexed electrochemical DNA biosensor.

A surface-initiated enzymatic polymerization strategy for electrochemical DNA sensors

In this work, we report a novel strategy of electrochemical DNA (E-DNA) sensor based on surface initiated enzymatic polymerization (SIEP). This DNA sensor employs a capture DNA probe labeled with thiol at its 3′ terminal to be immobilized at gold electrode via gold–thiol chemistry. Oligo (ethylene glycol) -terminated thiols (SH-OEGs) are then used to prepare an oligonucleotide-incorporated nonfouling surface (ONS). After the sequence-specific recognition of target DNA, terminal deoxynucleotidyl transferase (TdT) is employed to catalyze the sequential addition of deoxynucleotides (dNTPs) at the 3′-OH group of target DNA without template. During the TdT-mediated extension reaction, by using biotinlated 2′-deoxyadenosine 5′-triphosphate (biotin-dATP), biotin labels are incorporated into the SIEP-generated long single-stranded DNA (ssDNA). Specific binding of avidin-horseradish peroxidase (Av-HRP) to the biotin label leads to enzyme turnover-based signal transduction. By using this new strategy, we demonstrated the high picomolar sensitivity and a broad detection range of six orders of magnitude.

The Hunt for 5-Hydroxymethylcytosine: The Sixth Base

transfers a glucose from UDP-glucose to 5-hmC in T4 bacteriophages. Two groups independently reported the use of b-GT to detect and quantify 5-hmC. Szwagierczak et al. used H radioactive glucose to label and quantify 5-hmC by scintillation counting [10]. Song et al. used b-GT to transfer an azide-labeled glucose to 5-hmC and subsequently attached a biotin group through the azido group via copper-free click reaction [11]. This strategy allows for the detection and quantification of 5-hmC in a simple dot-blot assay using avidin-horseradish peroxidase. The approach of b-GT labeling greatly improved the detection limit of 5-hmC from 0.004–0.006% of total nucleotide, and displayed a linear response to 5-hmC since the b-GT-based method is able to label every single 5-hmC in the genome. With these highly sensitive methods, extremely low levels of 5-hmC in other cultured cells, especially cancer cells, can be measured [11]. This is of particular interest since Tet enzymes may play important roles in various cancers; however, cancer cell lines tend to have low levels of 5-hmC [11,12]. The b-GTbased labeling approaches represent promising diagnostic tools for specific cancers. Compared to simple 5-hmC detection, sequencing methods amenable for wholegenome, next-generation sequencing of 5-hmC are much more desirable, especially if 5-hmC-containing genomic DNA fragments can be enriched first. We generated the first genome-wide 5-hmC distribution map using the same b-GT/azide–glucose/biotin-labeling method [11]. The high specificity of the click chemistry and the highly selective and strong biotin-avidin/streptavidin interaction enabled the robust and efficient pull-down of 5-hmCcontaining DNA for deep sequencing. The biotin–avidin/streptavidin enrichment approach proved superior to the antibody-based pull down After its rediscovery in 2009, 5-hydroxymethylcytosine (5-hmC) and its natural creators, the Tet dioxygenases, have received a tremendous amount of attention from the epigenetics and other related communities [1,2]. 5-hmC is now widely accepted as the sixth base in the mammalian genome, following its precursor, 5-methylcytosine (5-mC), the fifth base. To elucidate the exact biological roles of this base modification, the development of new analytical technologies, including detection and sequencing methods for 5-hmC, has become essential. The f irst two reports in 2009 used P-radioactive labeling 1D or 2D thin layer chromatography to detect 5-hmC [1,2]. With a detection limit of approximately 0.08% of total nucleotide, the thin layer chromatography method is able to detect 5-hmC in genomic DNA from brain tissue and embryonic stem cells; however, it fails to yield a signal with genomic DNA isolated from other cultured cells, especially cancer cells. More recently, a more accurate HPLC-mass spectroscopy method has been developed with isotope-labeled 5-hmC as an internal standard [3]. This method allows a complete quantification of the 5-hmC in all mouse tissues; however, the detection limit still needed to be improved. Since then anti-5hmC antibodies have become available and are widely used to detect 5-hmC [4–7]. However, this approach is not very quantitative owing to its density-dependent recognition of 5-hmC, as expected [8]. Anti-5-methylenesulfonate – generated from 5-hmC after a bisulfite treatment – antibodies have been demonstrated to be more quantitative than anti-5-hmC antibodies [8]. However, these antibody-based methods c annot improve the detection limit. In 2010, enzymatic labeling of 5-hmC began to emerge [9]. T4-b-glucosyltransferase (b-GT) is the first enzyme of choice, since it naturally

Immunoperoxidase detection of neuronal antigens in full-thickness whole mount preparations of hollow organs and thick sections of central nervous tissue

Immunofluorescently stained whole mounts have proved useful for defining the innervation of the gut and large blood vessels. Nerves supplying other hollow organs are usually studied in sections, which provide much less information. Aiming to describe the entire innervation of rat uterus, we developed a method for immunoperoxidase staining of full-thickness whole mounts that allowed us to visualize all immunoreactive axons. Uterine horns were dissected out, slit open, stretched, pinned flat and fixed. Entire horns were treated with methanol/peroxide, buffered Triton X-100 and normal serum and then incubated in primary antibodies, biotinylated secondary antibodies and avidin-horseradish peroxidase (HRP), each for at least 3 days. Peroxidase reactions revealed immunoreactivity. Immunostained horns were dehydrated, infiltrated with epoxy resin, mounted on slides under Aclar coverslips and polymerized. We treated bladders, gut, major pelvic ganglia and thick sections of perfused medulla oblongata similarly to assess the applicability of the method. Using this method, we could map the entire uterine innervation provided by axons immunoreactive for a variety of antigens. We could also assess the entire tyrosine hydroxylase-immunoreactive innervation in all layers of bladder, gut and ganglia whole mounts and throughout 300 μm sections of medulla. These observations show that this method for immunoperoxidase staining reliably reveals the complete innervation of full-thickness whole mounts of hollow organs and thick sections of central nervous tissue. The method has several advantages. The resin-embedded tissue does not degrade; the immunostaining is non-fading and permanent and neurochemically defined features can be mapped at large scale without confocal microscopy.