Avidin Biotin Research Articles

Immobilization of bone morphogenetic protein-2 to gelatin/avidin-modified hydroxyapatite composite scaffolds for bone regeneration.

Bone scaffold surface characterization is important for improving cell adhesion, migration, and differentiation. In this study, bone morphogenetic protein-2 (BMP-2) was immobilized to the surface of the gelatin/hydroxyapatite composite using avidin-biotin binding system to produce a bone-tissue engineering scaffold. Firstly, hydroxyapatite particles reacted with hexamethylene diisocyanate and then the terminal group was converted into a primary amine group. Avidin was then immobilized on the surfaces of hydroxyapatite particles using N-ethyl-N'-(3-(dimethylamino)propyl) carbodiimide and N-hydroxysuccinimide as coupling agents. Gelatin was blended with avidin-modified hydroxyapatite and pure hydroxyapatite to obtain gelain/hydroxyapatite composite. The composite was then cross-linked with glutaraldehyde. Finally, biotin-conjugated BMP-2 was immobilized on the surface of the composite via avidin-biotin binding. In vitro study indicated that BMP-2-immobilized composite film had a higher ALP activity than that composite film without BMP-2. The composite scaffolds were then implanted into rabbit skulls to check bone-tissue regeneration. Ultrasound and micro-CT scans demonstrated that neovascularization and new bone formation in the BMP-2-immobilized composite scaffolds were higher than those in composite scaffolds without BMP-2. Histological evaluation result was similar to that of the micro-CT. Therefore, the surface immobilization of BMP-2 could effectively improve osteogenesis in the gelatin/hydroxyapatite composite scaffold.

Immunocytochemistry of mesenteric lymph node fine-needle aspirates in the diagnosis of feline infectious peritonitis.

Immunohistochemistry (IHC) of tissue samples is considered the gold standard for diagnosing feline infectious peritonitis (FIP), and, in cats without body cavity effusion, IHC is the only method available to establish definitive antemortem diagnosis. However, IHC requires invasive tissue sample collection. We evaluated sensitivity and specificity of an immunocytochemical assay of fine-needle aspirates (FNAs) of mesenteric lymph nodes that can be obtained noninvasively by ultrasound-guided aspiration to diagnose FIP. FNAs of mesenteric lymph nodes were obtained postmortem from 41 cats suspected of having FIP based on clinical and/or laboratory findings. FIP was confirmed immunohistochemically in 30 cats. In the other 11 cats, a disease other than FIP, which explained the clinical signs, was diagnosed histopathologically. Immunocytochemistry (ICC) was performed as an avidin-biotin complex method using a monoclonal anti-FCoV IgG 2A. Sensitivity, specificity, negative and positive predictive values (NPV, PPV, respectively) including 95% confidence intervals (95% CIs) were determined. ICC was positive in 17 of 30 cats with FIP, but also in 1 of 11 control cats that was diagnosed with lymphoma. Sensitivity of ICC was 53% (95% CI: 34-72); specificity 91% (95% CI: 59-100); NPV 42% (95% CI: 22-63); and PPV 94% (95% CI: 71-100). In a lethal disease such as FIP, specificity is most important in order to avoid euthanasia of unaffected cats. Given that a false-positive result occurred and FIP was correctly detected in only approximately half of the cases of FIP, ICC of mesenteric lymph node FNA alone cannot reliably confirm or exclude FIP, but can be a helpful test in conjunction with other diagnostic measures.

Epidermal growth factor receptor as an early diagnostic marker of oral squamous cell carcinoma: Marking the line between severe dysplasia and oral squamous cell carcinoma

Context: Epidermal growth factor receptor (EGFR) and its ligands are involved in the cell growth of 70% cancer cells. EGFR overexpression or dysregulation may alter intracellular signaling pathways that affect tumor cell survival and apoptosis. It is hypothesized that only those potentially malignant lesions express high levels of EGFR progress to frank malignancies during tumorigenesis which suggests that increase in receptors might be the cause of malignancy rather than the effect of malignancy. Aims: The aim of the study was to assess and correlate the expression of EGFR in varying grades of oral epithelial dysplasia, oral squamous cell carcinoma (OSCC), and normal oral mucosa and to evaluate the early rate of malignant transformation in potentially malignant lesions. Settings and Design: It was a immunohistochemical study. Subjects and Methods: Twenty formalin-fixed, paraffin-embedded blocks of various grades of oral epithelial dysplasia, 10 formalin-fixed, paraffin-embedded blocks of OSCC, and 10 normal mucosae are stained with routine hematoxylin and eosin and immunostained with EGFR by avidin–biotin method. Statistical Analysis Used: Statistical analysis was performed by one-way ANOVA and multiple comparison tests using IBM SPSS Software 20.0 20. Results: Of 40 cases analyszd, 38 cases showed EGFR expression with varied extent and intensity of staining in the cytoplasm and cell membrane of keratinocytes in basal, parabasal, deep spinous, and superficial spinous layer. The results showed a statistically significant increase (P < 0.0001) in the staining extent and intensity of EGFR expression in severe dysplasia and OSCC. Conclusions: EGFRs may act as a marker since they reflect the early changes in dysplastic lesions – predicting the rate of malignant transformation in potentially malignant disorders and thereby aiding the oral pathologist in arresting the lesion before it enters malignancy.

Kinetic Mechanism of Metal Enhanced Fluorescence by Gold Nanoparticle with Avidin–Biotin as Spacer and by Gold–Silver Core–Shell Nanoparticle Using Fluorescence Lifetime Image Microscopy

We study the effects of metal-enhanced fluorescence (MEF) on rhodamine B fluorophore by nanoparticles of varied shapes. The avidin–biotin system was used as spacer to connect the fluorophore to the surface of nanoparticles. Fluorescence lifetime image microscopy (FLIM) was used to detect emission lifetime for dye molecules on single nanoparticles. Spherical gold particles diameter of 60 and 170 nm, respectively, cube length of 70 nm, and rhombic dodecahedron (RD) diameter of 63 nm were used. In the measured emission curves of rhodamine B, we obtained a short component with lifetime 16–26 ps attributed to the fluorophore under influence of the local electric field of gold nanoparticle and energy dissipation to nanoparticle. The second lifetime is 200, 270, 280, and 330 ps for 170 nm sphere, 70 nm cube, 63 nm RD, and 60 nm sphere, respectively. This component is referred to as the bright mode of nanoparticle which is coupled to the excited dye molecule and transfers energy back to the fluorophore. On the ba...

Avidin-Biotin Cross-Linked Microgel Multilayers as Carriers for Antimicrobial Peptides.

Herein, we report on the formation of cross-linked antimicrobial peptide-loaded microgel multilayers. Poly(ethyl acrylate- co-methacrylic acid) microgels were synthesized and functionalized with biotin to enable the formation of microgel multilayers cross-linked with avidin. Microgel functionalization and avidin cross-linking were verified with infrared spectroscopy, dynamic light scattering, and z-potential measurements, while multilayer formation (up to four layers) was studied with null ellipsometry and quartz crystal microbalance with dissipation (QCM-D). Incorporation of the antimicrobial peptide KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR) into the microgel multilayers was achieved either in one shot after multilayer formation or through addition after each microgel layer deposition. The latter was found to strongly promote peptide incorporation. Further, antimicrobial properties of the peptide-loaded microgel multilayers against Escherichia coli were investigated and compared to those of a peptide-loaded microgel monolayer. Results showed a more pronounced suppression in bacterial viability in suspension for the microgel multilayers. Correspondingly, LIVE/DEAD staining showed promoted disruption of adhered bacteria for the KYE28-loaded multilayers. Taken together, cross-linked microgel multilayers thus show promise as high load surface coatings for antimicrobial peptides.

MRNAs biotinylated within the 5' cap and protected against decapping: new tools to capture RNA-protein complexes.

The 5'-terminus of eukaryotic mRNAs comprises a 7-methylguanosine cap linked to the first transcribed nucleotide via a 5'-5' triphosphate bond. This cap structure facilitates numerous interactions with molecules participating in mRNA processing, turnover and RNA translation. Here, we report the synthesis and biochemical properties of a set of biotin-labelled cap analogues modified within the triphosphate bridge and increasing mRNA stability while retaining biological activity. Successful co-transcriptional incorporation of the cap analogues allowed for the quantification of cap-dependent translation efficiency, capping efficiency and the susceptibility to decapping by Dcp2. The utility of such cap-biotinylated RNAs as molecular tool was demonstrated by ultraviolet-cross-linking and affinity capture of protein-RNA complexes. In conclusion, RNAs labelled with biotin via the 5' cap structure can be applied to a variety of biological experiments based on biotin-avidin interaction or by means of biotin-specific antibodies, including protein affinity purification, pull-down assays, in vivo visualization, cellular delivery and many others.This article is part of the theme issue '5' and 3' modifications controlling RNA degradation'.

Enzymatically Powered Surface-Associated Self-Motile Protocells.

Cell motility is central to processes such as wound healing, immune cell surveillance, and embryonic development. Motility requires the conversion of chemical to mechanical energy. An active area of research is to create motile particles, such as microswimmers, using catalytic and enzymatic reactions. Here, autonomous motion is demonstrated in adhesive polymer-based protocells by incorporating and harnessing the energy production of an enzymatic reaction. Biotinylated polymer vesicles that encapsulate catalase, an enzyme which converts hydrogen peroxide to water and oxygen, are prepared and these vesicles are adhered weakly to avidin-coated surfaces. Upon addition of hydrogen peroxide, which diffuses across the membrane, catalase activity generates a differential impulsive force that enables the breakage and reformation of biotin-avidin bonds, leading to diffusive vesicle motion resembling random motility. The random motility requires catalase, increases with the concentration of hydrogen peroxide, and needs biotin-avidin adhesion. Thus, a protocellular mimetic of a motile cell.

GLP-1 Localisation and Proglucagon Gene Expression in Healthy and Diabetic Mouse Ileum.

IntroductionGlucagon-like peptide-1 (GLP-1) is a polypeptide that is mainly produced by intestinal L cells and is encoded by the proglucagon gene. In this study, GLP-1 localisation was investigated in the ileum of healthy and diabetic mice by immunohistochemistry and proglucagon gene expression was assayed by reverse transcription-polymerase chain reaction.Material and MethodsThis study included 18 male Balb/c mice that were divided into diabetic, sham, and control groups. Mice in the diabetic group received 100 mg/kg of streptozotocin. Immunohistochemical expression of GLP-1 was determined using the avidin–biotin–peroxidase complex technique, and proglucagon gene expression was determined by RT-PCR.ResultsAnalysis of GLP-1 immunohistochemical localisation showed that GLP-1-immunopositive cells (L cells) were present between epithelial cells in the intestinal crypts. The intensity and localisation of GLP-1 immunoreactivity were similar among the mice in all the groups. Proglucagon gene expression levels were also statistically similar among the mice in all the groups.ConclusionNo difference was demonstrated among the mice in the diabetic, sham, or control groups with respect to proglucagon gene expression and GLP-1 localisation in the ileum, suggesting that diabetes does not affect proglucagon gene expression in the ileum.

Fabrication of high-intensity electron transfer electrochemiluminescence interface for Hg2+ detection by using reduced graphene oxide-Au nanoparticles nanocomposites and CdS quantum dots

A novel electrochemiluminescence (ECL) biosensing interface for Hg2+ analysis was constructed based on the enhanced ECL signal from CdS quantum dots by reduced graphene oxide-Au nanoparticles (RGO-AuNPs) nanocomposites. RGO-AuNPs were prepared by the heat-treatment of graphene oxide and HAuCl4 solution under alkaline environment. The prepared RGO-AuNPs composites were dispersed in N,N-dimethylformamide and dropped onto the surface of glassy carbon electrode (GCE). Double-stranded DNA modified with amino at one end was then immobilized on the RGO-AuNPs/GCE surface in the presence of 1-pyrenebutyric acid N-hydroxysuccinimide linker, following which the avidin-modified CdS quantum dots were linked to double-stranded DNA labeled with biotin at another end through the formation of biotin–avidin complex. The excellent electrical conductivity of RGO-AuNPs nanocomposites and the high charge transfer efficiencies of the double-stranded DNA with T–Hg2+–T complex, which produced high-intensity electron transfer interface and improved the sensitivity of the biosensor. After Hg2+ was added into the detection solution, the increased ECL signals showed good correlation with Hg2+ concentration. The linear range of the sensor was 5.0 × 10−13–1.0 × 10−9 M with a detection limit of 2.0 × 10−13 M. This ECL biosensor showed satisfactory results when used for detecting Hg2+ in real lake water.

Transparent Protein Microtubule Motors with Controllable Velocity and Biodegradability

Slender protein microtube motors with a catalase interior surface are self-propelled in aqueous H2O2 by jetting O2 microbubbles from the open-end terminus. Immobilization of a catalase biocatalyst on the internal wall is achieved using avidin–biotin complexation. It is particularly interesting that the migration of O2 bubbles in the 1D channel and their subsequent expulsions were clearly visible because the tube walls are transparent. The microtube motor velocity reached a maximum at the optimum pH and temperature of the catalase. Furthermore, the microtubes were digested completely by proteases, showing sufficient biodegradability.

Electrochemical genosensor based on template assisted synthesized polyaniline nanotubes for chronic myelogenous leukemia detection

This work reports a facile approach to synthesize polyaniline nanotubes (PANI-NT) by using manganese oxide as sacrificial templates. This template assisted polyaniline nanotubes (t-PANI-NT) were utilized as electrode material after deposition onto the indium tin oxide (ITO) coated glass substrates by using the electrophoretic technique. The structural, morphological and electrochemical characterizations of the t-PANI-NT show relatively better results compared to chemically synthesized PANI-NT (c-PANI-NT). Moreover, the t-PANI-NT/ITO electrode exhibits improved electron transfer coefficient (α = 0.63) and charge transfer rate constant (ks = 0.05912 s−1) in comparison to c-PANI-NT/ITO electrode (α = 0.56 and ks = 0.06548 s−1). The obtained t-PANI-NT/ITO electrodes have been further immobilized with biotinylated DNA sequence, specific to chronic myelogenous leukemia (CML) by using avidin–biotin as a cross-linking agent. Electrochemical impedance spectroscopy studies revealed that the genosensor displays linearity in wide range of target DNA concentration (10−6 to 10−16 M) with an outstanding differentiation ability and low detection limit of 10−16 M. The experimental results of this highly sensitive and specific genosensor with clinical samples of CML positive patients and control negative patients indicate its potential for clinical diagnostics.

Self-assembly of tumor-targeting RNA nanoball for carrier-free delivery of therapeutic RNA

Rolling circle transcription (RCT) has been studied for enhancing the delivery efficiency of therapeutic RNA by densely packing RNA strands with particle formation. This facile fabrication method has been successfully employed to generate RNA nanoballs containing functional short interference RNA. In addition, the nanoballs produced by RCT showed high stability from the nuclease degradation. With these advantages, using biotin-labeled ribonucleoside triphosphate (rNTP), target specific and non-toxic bubble RNA nanoball (BNB) was synthesized. To achieve tumor-targeting, epidermal growth factor receptor antibody (EGFR Ab) was used to target the tumor cells by labeling the target ligands with biotin–avidin interaction. Target gene silencing by EGFR Ab-labeled RNA nanoball was confirmed in vitro. Therefore, RNA nanoball can be utilized for tumor cell-targeting delivery of therapeutic RNA.

Pretargeting in the context of theranostics and companion diagnostics in nuclear oncology

The concept of theranostics is well illustrated in nuclear oncology by radioactive iodide in thyroid diseases and by peptides or small molecular weight inhibitors in various cancers. It is also illustrated by radiolabeled antibodies, such as Zevalin, but radiolabeled antibodies have limitations that prompted the design of pretargeting. This review illustrates the achievements of pretargeting and focuses on its applications as theranostics or companion diagnostics. The review does not aim at exhaustivity. Examples from the already long history of pretargeting are selected to depict achievements but also outstanding issues. The idea of pretargeting, which separates antigen-binding from radionuclides, was proposed in the mid 1980s. Using avidin–biotin or bispecific antibodies and bivalent haptens, high contrast images and therapeutic efficacy were demonstrated in the clinic. However, the immunogenicity of streptavidin could not be abrogated, producing recombinant bispecific antibodies proved difficult and balancing between high tumor uptake and fast clearance of activity from normal tissues remains a challenge. These are the reasons for the continuing search for new approaches and new reagents. Some of these new approaches, particularly those based on biorthogonal chemistry or complementary oligonucleotides, are described and their potential as theranostics or companion diagnostics are discussed. The future of pretargeting remains uncertain, because clinical development is complex and cost of goods is high. However, pretargeting as a theranostic tool is clinical relevant, especially with short half-life radionuclides for PET imaging or targeted alpha-radionuclide therapy.

Micrometer-sized TPM emulsion droplets with surface-mobile binding groups

Colloids coated with lipid membranes have been widely employed for fundamental studies of lipid membrane processes, biotechnological applications such as drug delivery and biosensing, and more recently, for self-assembly. The latter has been made possible by inserting DNA oligomers with covalently linked hydrophobic anchors into the membrane. The lateral mobility of the DNA linkers on micrometer-sized droplets and solid particles has opened the door to creating structures with unprecedented structural flexibility. Here, we investigate micro-emulsions of TPM (3-(trimethoxysilyl)propyl methacrylate) as a platform for lipid monolayers and further functionalization with proteins and DNA oligonucleotides. TPM droplets can be produced with a narrow size distribution and are polymerizable, thus providing supports for model lipid membranes with controlled size and curvature. With fluorescence recovery after photobleaching, we observed that droplet-attached lipids, NeutrAvidin proteins, as well as DNA oligonucleotides all show mobility on the surface. We explored the assembly of micron-sized particles on TPM-droplets by exploiting either avidin–biotin interactions or double-stranded DNA with complementary single-stranded end groups. While the single molecules are mobile, the particles that are attached to them are not. We propose that this is caused by the heterogeneous nature of emulsified TPM, which forms an oligomer network that limits the collective motion of linkers, but allows the surface mobility of individual molecules.

Detection of azaspiracids in mussels using electrochemical immunosensors for fast screening in monitoring programs

Given the widespread occurrence of azaspiracids (AZAs), it is clearly necessary to advance in simple and low-cost methods for the rapid detection of these marine toxins in order to protect seafood consumers. To address this need, electrochemical immunosensors for the detection of AZAs based on a competitive direct immunoassay using peroxidase-labelled AZA as a tracer were developed. An anti-AZA polyclonal antibody was immobilised in a controlled and stable manner on protein G or avidin-coated electrodes. Experimental conditions were first optimised using colorimetric immunoassays on microtitre plates, providing intermediate products already applicable to the accurate detection of AZAs. Then, transfer of the protein G and avidin–biotin interaction-based immunoassays to 8-electrode arrays provided compact and miniaturised devices for the high-throughput detection of AZAs. The low amounts of immunoreagents required as well as the potential for reusability of the avidin–biotin interaction-based immunosensors represented significant economic savings as well as a contribution to sustainability. The electrochemical immunosensors enabled the quantification of all regulated AZAs below the regulatory limit, as well as a broad range of other toxic AZA analogues (from 63 ± 3 to 2841 ± 247 μg AZA-1 equiv./kg for the protein G-based immunosensor and from 46 ± 2 to 3079 ± 358 μg AZA-1 equiv./kg for the avidin–biotin interaction-based immunosensor). The good agreement between the results obtained by the immunosensors and LC–MS/MS in the analysis of naturally contaminated mussel samples demonstrated the easy implementation of electrochemical immunosensors for routine analysis of AZAs in food safety monitoring programs.

Dual-functionalized nanoparticles loaded microbubbles for enhancement of drug uptake

The application of microbubble (MB)-assisted ultrasound (US) can combine the advantages of real-time imaging and targeted drug delivery. However, the drug loading capacity of MB is limited restricting its application in antitumor procedure. In contrast, nanoparticles (NPs) can carry drugs more efficiently, but adverse side effect induced by unspecific accumulation can not be ignored. Herein, we developed a dual-functionalized NP loaded MB to investigate its potential feasibility for tumor-targeted drug delivery. Firstly, we prepared NPs using heparin as backbone. Targeting ligand folate and cell-penetrating ligand Tat peptide were conjugated to the backbone to deliver paclitaxel (H-F-Tat-P NPs). Subsequently, the dual-functionalized NPs were incorporated with MBs via avidin–biotin linkage to fabricate H-F-Tat-P NPs loaded MBs (NPs-loaded MBs). The combined strategy can take profit of dual functionalities from NPs and sonoporation effect from MBs triggered by US. The prepared NPs have been characterized. The excellent cellular uptake of NPs were qualitative and quantitative analysis by flow cytometry and confocal microscope, the results indicated that it was attributed to not only dual functionalities but also US effect. Foremost, the NPs-loaded MBs combined with US exhibited significant cytotoxicity on both folate receptor (FR) overexpressing and deficiency cells. The combination of dual-functionalized NPs and MBs with US is expected to be a promising strategy for targeted anticancer drug delivery and ultrasound imaging simultaneously.

Biotin–avidin interaction triggers conversion of triskelion peptide nanotori into nanochains

Triskelion biotinylated peptide is self-assembled into nanotorus structures followed by dimerization and chain formation in the presence of avidin.

Ultrasensitive detection of Bisphenol A based on an aptasensor with DNA amplification

ABSTRACT An ultrasensitive and selective method for Bisphenol A (BPA) detection using aptamer-BPA and real-time quantitative polymerase chain reaction (RT-qPCR) technology is reported, in which biotin-modified aptamer DNA (BPA) is fixed on the inner wall of a streptavidin-coated PCR tube via biotin–avidin interactions and the template is fixed through the complementary base pairing with aptamer DNA. Upon the addition of BPA, the template DNA could be off from the inner wall of PCR tube, which results from aptamer DNA interacted specifically with BPA. Thus, the number of template DNA change result from the BPA concentration is readily seen by cycle threshold (Ct) values. The sensor exhibited an excellent linear response between Ct values and BPA concentration in the range of 1–500 nM and the detection limit of this method for aqueous BPA was as low as 0.7 nM. In addition, this method showed good selectivity for BPA over its analogues.

A Quartz Crystal Microbalance Immunosensor for Stem Cell Selection and Extraction.

A cost-effective immunosensor for the detection and isolation of dental pulp stem cells (DPSCs) based on a quartz crystal microbalance (QCM) has been developed. The recognition mechanism relies on anti-CD34 antibodies, DPSC-specific monoclonal antibodies that are anchored on the surface of the quartz crystals. Due to its high specificity, real time detection, and low cost, the proposed technology has a promising potential in the field of cell biology, for the simultaneous detection and sorting of stem cells from heterogeneous cell samples. The QCM surface was properly tailored through a biotinylated self-assembled monolayer (SAM). The biotin–avidin interaction was used to immobilize the biotinylated anti-CD34 antibody on the gold-coated quartz crystal. After antibody immobilization, a cellular pellet, with a mixed cell population, was analyzed; the results indicated that the developed QCM immunosensor is highly specific, being able to detect and sort only CD34+ cells. Our study suggests that the proposed technology can detect and efficiently sort any kind of cell from samples with high complexity, being simple, selective, and providing for more convenient and time-saving operations.

Aptamer-based F0F1-ATPase biosensor for Salmonella typhimurium detection

Abstract A novel biosensor was developed by conjugating Salmonella typhimurium aptamer with the “rotator” e-subunit of F0F1–ATPase within chromatophores through an anti-e-subunit antibody–biotin–avidin–biotin–aptamer linker to capture bacteria. The bacterial load decreased the rotation rate of F0F1-ATPase, which led to the decrease in ATP synthesis. The detection of S. typhimurium was based on proton flux change driven by the F0F1-ATPase-mediated ATP-synthesis, which was indicated by fluorescence probe, monitored by a fluorescence spectrometer. Under optimal conditions, the correlation between the relative fluorescence signal intensity and the concentration of S. typhimurium was observed to be linear within the range of 101 to 104 cfu·mL−1 (R2 = 0.9944). The limit of detection for S. typhimurium was as low as 10 cfu·mL−1. The results demonstrate that the F0F1-ATPase molecular motor biosensor can specifically detect S. typhimurium at low concentration, and will be a convenient, quick, and promising tool for detecting pathogens.