Streptavidin Linker Research Articles

A novel mutant allele specific amplification and electrochemiluminescence method for the detection of point mutation in clinical samples

A novel mutant allele specific amplification (MASA) and electrochemiluminescence (ECL) method for point mutation detection is proposed. Briefly, the target gene was amplified by a biotinylated mutant specific sense primer and a Ru(bpy) 3 2+ (TBR)-labeled universal antisense primer. Only the mutant allele can be selectively amplified by the mutant specific primer pair. Then, the MASA product was captured onto the streptavidinylated magnetic beads through biotin–streptavidin linkage and detected by measuring the ECL emission of TBR. The method was applied to detect a possible point mutation at codon 12 of K-ras oncogene in 30 colorectal cancer (CAC) clinical samples. The experimental results show that the method can detect K-ras mutant in a 5000-fold excess of wild-type allele. Furthermore, different kinds of mutations can be clearly discriminated. The point mutation was found in 15 (50%) out of 30 CAC samples. This novel MASA–ECL method could potentially become a sensitive, specific, simple, rapid and safe approach for point mutation detection.

Cytoplasmic and Nuclear Accumulation of Antisense Oligomers Incubated as Streptavidin Delivery Nanoparticles

The cytoplasmic accumulation and nuclear targeting of antisense oligomers were evaluated in cell culture following incubation with the cell penetrating peptide tat or polyargenine as a two-component delivery nanoparticle using streptavidin as linker. The fluorescent labeled uniform phosphodiester (PO), phosphorothioate (PS) and MORF oligomers were antisense to the mdr1 mRNA and were studied in KB-G2 (Pgp++) and KB-31 (Pgp+/-) cells. Fluorescence microscopy showed much higher cellular accumulation using the antisense compared to the sense PS/tat nanoparticle. The distribution in cells receiving either the PS or MORF, but not the PO, antisense oligomers in all of their forms showed definite migration to the nucleus. Incubations in KB-G2 cells of all three antisense oligomers with streptavidin as the onecomponent or including tat as the two-component nanoparticles led to increases in Rhodamine 123 accumulation by 200∼250% compared to free oligomers, almost certainly due to antisense interference with Pgp function. Thus presence of the streptavidin linker had no obvious detrimental influence on the functions of the carriers or the antisense oligomers. Since nuclear migration appears to be a characteristic property of oligomers, delivery via the nanoparticle may be a useful method of increasing the accumulation into the nucleus of antisense oligomers radiolabeled for Auger emittingbased radiotherapy.

Optimization detection of plant pathogenic bacteria by electrochemiluminescence polymerase chain reaction method

Plant pathogenic bacteria spread all over the world, causing a great deal of economic loss. This study has developed a novel method for rapid detection of plant pathogenic bacteria by electrochemiluminescence polymerase chain reaction (ECL-PCR) using two universal probes, a biotin-probe and a Ru(bpy) 3 2+ (TBR)-probe. Biotin-probe sequence is complementary to the universal sequence of anti-sense primer, and TBR-probe sequence is the same as the universal sequence of sense primer. So the amplified PCR products can hybridize with TBR-probe and biotin-probe. After hybridizing, PCR products are captured by streptavidin coated magnetic bead through the biotin–streptavidin linkage, and then TBR reacts with tripropylamine to emit light for detection of plant pathogenic bacteria. This proposed method is applied to detect Fusarium oxysporum f. sp. Cubense and Xanthomonas oryzae pv. Oryzae. The results show that the method can successfully identify the plant pathogenic bacteria in the infected samples and these results are consistent with the results of gel electrophoresis. Importantly, this study can be used as an illustration for detecting various plant pathogenic bacteria and provides a feasible approach on developing ECL sensors to meet the demand of rapid detection of pathogenic bacteria, fungi, and viruses.

Dynamic Conformation Fluctuations of λ-Phage DNA in an Optical Trap

Dynamic fluctuations in the conformation of λ-phage DNA have been observed in a fiber optic based light trap. The light force is used to confine a polystyrene bead (diameter, 0.5μm - 2.8μm) in three dimensions at a fixed distance from a flat surface coated with anti-digoxygenin antibody. The DNA is fixed to this bead through a biotin - streptavidin linkage on the 3′end, whereas a digoxygenin label is present on the opposing 5′. Through this simple assay, the dynamics of the free ends have been quantified for various times and separation distances. The dynamics have been observed using a high frame rate camera and the intercalating fluorophore, YOYO-1. In contrast to a conventional optical trap, this trap lacks a highly focused beam, greatly reducing localized heating and the associated risk to the biological specimen which could potentially cause error in this type of measurement.

Particle flow assays for fluorescent protein microarray applications

Microarray technology has brought a paradigmatic change in bioanalytics. However, highly sensitive and accurate assays are still needed for a real breakthrough. We present a simple and generic approach for fluorescent signal amplification with fluorescent microparticle labels. The assay principle was demonstrated using a reverse array model consisting of spots of bovine serum albumin with a small fraction of the proteins biotinylated. Specific binding of streptavidin coated fluorescent microparticles to the spots was promoted by applying a controlled continuous microparticle flow. The surface bound beads were visualized and quantified with confocal microscopy images. Comparison with standard fluorescent and flow discrimination assays has revealed several advantages of our approach. First, non-specific particle binding could be reduced to less than 1 particle/spot making therefore the visualization of single biomolecular bonds possible. Second, the amplification scheme presented here is generic and can be applied to any fluorescent microarray. Furthermore, this assay makes use of a biotin–streptavidin linkage and can therefore be applied to all kind of assays. Finally, single fluorescent microbeads can be easily visualized with standard optical equipments, so that no high performance equipment is required.

Cell Culture and Xenograft-Bearing Animal Studies of Radiolabeled Antisense DNA Carrier Nanoparticles with Streptavidin as a Linker

Transmembrane transfectors (carriers) are increasingly being viewed as helpful or even necessary to improve cellular delivery in connection with antisense tumor targeting and other applications requiring cell membrane transport of DNAs, RNAs, and other oligomers. We are investigating streptavidin as a convenient linker for biotinylated carriers and oligomers because it requires only simple mixing for preparation. The goal of this study was to evaluate antisense DNA-streptavidin-carrier nanoparticles for accumulation in cell culture and in xenograft-bearing mice. The 3 carriers were cholesterol, a 10-mer Tat peptide, and a 10-mer polyarginine peptide. A 20-mer DNA targeting the mdr1 messenger RNA coding for Pgp expression was used as the phosphodiester (PO) DNA as well as the phosphorothioate (PS) DNA. In all cases, the (99m)Tc radiolabel was on the DNA. The 8 nanoparticles were first tested in mdr1(++) KB-G2 and TCO-1 cells and in mdr1(+/-) KB-31 cells in culture for evidence of improved accumulation and antisense targeting. Thereafter, the PS DNA-streptavidin-Tat, PO DNA-streptavidin-Tat, and PS DNA-streptavidin-cholesterol nanoparticles were administered intravenously to KB-G2 xenograft-bearing mice, and tissue distributions were measured. In culture, the PO nanoparticles showed increased accumulation compared with the corresponding nanoparticles without the carrier in all 3 cell types; in contrast, with the PS nanoparticles, any similar carrier-mediated increase may have been obscured by the much higher protein-binding affinity of PS DNA. As evidence of antisense targeting, the Tat and cholesterol PS nanoparticles showed statistically significant accumulation at 23 h in cells in the descending order TCO-1, KB-G2, and KB-31, although there were no significant differences among the PO nanoparticles. In xenograft-bearing mice, the tissue accumulation of both forms of the PS nanoparticles greatly exceeded that of the PO nanoparticles and, including in the tumor, were similar to that obtained previously for naked PS DNA. The presence of the streptavidin linker had no obvious detrimental effect on the functions of the carriers and antisense DNAs. The higher protein-binding affinity of the PS nanoparticles than the PO nanoparticles was still apparent both in vitro and in vivo, the pharmacokinetics of the PS nanoparticles were similar to that of naked PS DNA, and the carriers improved cellular accumulation, at least for the PO nanoparticles. These observations, taken together with the higher accumulation of both forms of the antisense PS nanoparticles in mdr1(++) KB-G2 and TCO-1 cells than in mdr1(+/-) KB-31 cells, suggest that further effort is justified to confirm that the antisense properties of the DNAs were not compromised by the presence of streptavidin.

Directing Placement, Alignment, and Transport of Magnetic Nanoparticle-Labeled Microtubules

In this report, we describe the functionalization of biotinylated microtubules with biotinylated cobalt ferrite (CoFe2O4) nanoparticles using a streptavidin linkage. These are exposed to kinesin-covered glass surfaces, where they bind and are monitored by epifuorescence microscopy. Placement of a magnet below the cell introduces an externally controlled magnetic field to manipulate microtubule placement, orientation, and transport on the kinesin-covered surfaces.

Analysis of a non-labeling protein array on biotin modified gold surfaces using atomic force microscopy and surface plasmon resonance

Streptavidin was used as a linker protein for binding a second biotinylated protein. To efficiently combine streptavidin without non-specific binding, a well-controlled array of biotin was prepared on a gold surface by a mixed self-assembly method. Two thiol derivatives containing hydroxyl and biotin functional groups, respectively, were used to fabricate the mixed self-assembled monolayer. The antibody was fragmented, modified with biotin, and added to the array on the streptavidin linker layer. Topographical changes to the array surface caused by protein immobilization were demonstrated by applying atomic force microscopy and ellipsometry. The resonance wavelengths of SPR spectra were red shifted according to the increase in thickness of the dielectric layer by protein immobilization.

Magnetic Beads With High Saturation Magnetization

Magnetic beads with the core-shell structure of an iron core encapsulated by titanium oxide and silica shell layers were prepared. The beads, HMM-beads, exhibited soft magnetic properties with high magnetization of 105 Amiddotm2/kg and low coercitivity of ~4 kA/m. HMM-beads with a diameter of 3 mum showed DNA isolation performance comparable to that of conventional beads composed of iron oxides. HMM-beads with a diameter of 1 mum coupled with biotinylated anti-EpCAM antibody through Streptavidin linkers also exhibited comparable yields to conventional beads of the same diameter in respect to capturing tumor cells. The response time of the HMM-beads to a magnetic field at magnetic separation was less than one third that of conventional beads in both cases.

Site-directed biotinylation of antibodies for controlled immobilization on solid surfaces

Site-directed biotinylation of antibodies at the hinge region was developed to immobilize antibodies in an oriented manner via biotin–streptavidin linkage. When intact antibody was biotinylated with maleimide-activated biotin after reduction, the reaction preferentially occurred at the sulfhydryl groups between the C H1 and the C L domains and, provided that the reagent concentration exceeded a certain level, at those between the C H2 and the C H2 domains at the hinge. Based on this result, we devised an approach in which free maleimide was added to compete with the activated biotin for the preferential sites between the C H1 and the C L domains. Since the smaller molecular size of free maleimide made it more accessible for the reaction than biotin, maleimide bound to the groups between the C H1 and the C L domains first and thus conceded the groups between the C H2 and the C H2 domains to biotin under optimal conditions. In an alternative approach, selective biotinylation at the hinge was also achieved by reacting activated biotin with F(ab′) 2 fragment prepared by enzymatic cleavage. This result indicated that, when free of Fc, the hinge structure, which contains the functional groups, of the fragment was open, allowing easy access to the biotin derivative from the aqueous medium. Both site-directed biotinylation preparations were tested as capture antibodies in sandwich-type immunoassays and compared to whole antibody randomly biotinylated at amino groups on the molecule. Preparations of both the intact antibody and the F(ab′) 2 showed consistently enhanced detection capabilities that were 2.6 and 20 times that of the control, respectively.

Real-Time Detection of Antigen–Antibody Reactions by Imaging Ellipsometry

In the field of proteomics the quantification of the affinity of an antibody to its partners and the evaluation of its specific binding is an important issue. With an imaging ellipsometer the interaction of an antibody with immobilized antigens on a model microarray is observed in a time-resolved and label-free manner. Imaging ellipsometry was developed for real-time monitoring of the biomolecule interaction between an antigen in solution and an antibody immobilized on a silicon surface. Proteins were immobilized by the formation of carboxy-alkyl monolayers on silicon substrates, where a biotin-labelled antibody was immobilized by a biotin–streptavidin linkage. Anti-human IgG bound specifically to human antibody and protein A, similarly anti-goat IgG bound to goat antibody. No binding was observed between anti-rabbit IgG and goat antibody. All stages of the formation of the antigen–antibody complex were imaged by imaging ellipsometry. By monitoring changes in y, the mole fraction θ of the antigen–antibody binding was determined. Immunological reactions of two different antigen–antibody combinations were fitted by the Langmuir adsorption equation, and affinity constants for two reactions were calculated.

Two modes of microtubule sliding driven by cytoplasmic dynein

Dynein is a huge multisubunit microtubule (MT)-based motor, whose motor domain resides in the heavy chain. The heavy chain comprises a ring of six AAA (ATPases associated with diverse cellular activities) modules with two slender protruding domains, the tail and stalk. It has been proposed that during the ATP hydrolysis cycle, this tail domain swings against the AAA ring as a lever arm to generate the power stroke. However, there is currently no direct evidence to support the model that the tail swing is tightly linked to dynein motility. To address the question of whether the power stroke of the tail drives MT sliding, we devised an in vitro motility assay using genetically biotinylated cytoplasmic dyneins anchored on a glass surface in the desired orientation with a biotin-streptavidin linkage. Assays on the dyneins with the site-directed biotin tag at eight different locations provided evidence that robust MT sliding is driven by the power stroke of the tail. Furthermore, the assays revealed slow MT sliding independent of dynein orientation on the glass surface, which is mechanically distinct from the sliding driven by the power stroke of the tail.

Charge Patterns as Templates for the Assembly of Layered Biomolecular Structures

Electric fields are used to guide the assembly of biomolecules in predefined geometric patterns on solid substrates. Local surface charges serve as templates to selectively position proteins on thin-film polymeric electret layers, thereby creating a basis for site-directed layered assembly of biomolecular structures. Charge patterns are created using the lithographic capabilities of an atomic force microscope, namely by applying voltage pulses between a conductive tip and the sample. Samples consist of a poly(methyl methacrylate) layer on a p-doped silicon support. Subsequently, the sample is developed in a water-in-oil emulsion, consisting of a dispersed aqueous phase containing biotin-modified immunoglobulinG molecules, and a continuous nonpolar, insulating oil phase. The electrostatic fields cause a net force of (di)electrophoretic nature on the droplet, thereby guiding the proteins to the predefined locations. Due to the functionalization of the immunoglobulinG molecules with biotin-groups, these patterns can now be used to initiate the localized layer-by-layer assembly of biomolecules based on the avidin-biotin mechanism. By binding 40 nm sized biotin-labelled beads to the predefined locations via a streptavidin linker, we verify the functionality of the previously deposited immunoglobulinG-biotin. All assembly steps following the initial deposition of the immunoglobulinG from emulsion can conveniently be conducted in aqueous solutions. Results show that pattern definition is maintained after immersion into aqueous solution.

Electrically Controlled Drug Delivery from Biotin‐Doped Conductive Polypyrrole

A platform for controlled drug delivery using a conductive-polymer substrate has been created. Through the incorporation of biotin into the conductive polymer polypyrrole (PPy) and the subsequent attachment of the desired drug molecule (such as nerve growth factor, NGF) via a streptavidin linker, an applied potential can trigger release of the drug from the polymer surface (see figure).

Sequence-specific electrochemical recognition of multiple species using nanoparticle labels

In this work, we report an electrochemical methodology that enables the rapid identification of different DNA sequences on the microfabricated electrodes. Our approach starts with an electropolymerization process on a patterned indium tin oxide (ITO)-coated glass electrode, followed by a selective immobilization of biotin-tagged probes on individually addressable spots via the biotin–streptavidin linkage. An exemplary target mixture containing E. coli and Stachybotrys Chartarum, an airborne pathogen, is then introduced. Recognition of the DNA hybridization event of the immobilized probes with the target pathogen PCR products or synthetic oligonucleotides is achieved by a novel electrochemistry-based technique utilizing the preferential catalytic silver electrodeposition process on the DNA-linked nanogold shells. The ability to selectively immobilize different oligonucleotide probes together with a sensitive electrochemistry-based detection for multiple species, as demonstrated in this study, is an important step forward for the realization of a portable bioanalytical microdevice for the rapid detection of pathogens.

A Streptavidin Linker Layer That Functions after Drying

The ability of streptavidin (SA) to simultaneously bind four biotins is often used in linker layers, where a biotinylated molecule is linked to a biotin-functionalized surface via SA. For biosensor and array applications, it is desirable that the SA linker layer be stable to drying and rehydration. In this study it was observed that a significant decrease in binding capacity of a SA layer occurred when that layer was dried. For this study a SA linker layer was constructed by binding SA to a biotin-containing alkylthiolate monolayer (BAT/OEG) self-assembled onto gold. Its stability after drying was investigated using surface plasmon resonance (SPR). Approximately a quarter of the SA layer was removed from the BAT/OEG surface upon drying and rehydration, suggesting disruption of SA-biotin binding when dry. This resulted in the dried SA layer losing approximately 40% of its biotinylated ferritin (BF) binding capacity. Coating the layer with trehalose before drying was found to inhibit the loss of SA from the BAT/OEG surface. SPR showed that the trehalose-protected SA linker layer retained approximately 91% of its original BF binding capacity after drying and rehydration. Atomic force microscopy, which was used to image individual surface-bound SA and BF molecules, qualitatively confirmed these observations.

Parallel, quantitative measurement of protein binding to a 120-element double-stranded DNA array in real time using surface plasmon resonance microscopy.

Quantitative, real-time measurement of kinetics of sequence-specific binding of DNA-binding proteins to double-stranded DNA (dsDNA) immobilized in a 10 x 12 array on a planar gold surface is demonstrated using surface plasmon resonance (SPR) microscopy. This binding of the yeast transcription factor Gal4 to a 120-spot dsDNA array made with alternating 200-microm spots of its dsDNA operator sequence and an unrelated DNA sequence proves that this method could be used to simultaneously monitor the kinetics of binding of proteins to 120 different dsDNA sequences with a sensitivity to approximately 0.5 pg (<2 x 10(7) molecules) of bound protein in each array spot at a time resolution of 1 s. The method is label free and also allows absolute quantitative determination of the binding stoichiometry (i.e., the number of proteins bound per dsDNA) at each time. The dsDNA array was fabricated using a robotic microspotting system to deliver nanoliter droplets of biotinylated dsDNA solutions onto a streptavidin linker layer immobilized with biotinylated alkylthiols on a thin gold film. Simultaneous monitoring of binding to the many array elements allows the use of reference spots (i.e., array elements with unrelated dsDNA sequences) to correct the signal for nonspecific protein-DNA binding and changes in bulk refractive index of the solutions in the SPR microscope's flow cell. This allows high-throughput analyses of the kinetics and equilibrium of protein-dsDNA binding.

A streptavidin-biotin-based microarray platform for immunoassays.

The authors describe a microarray system for disease diagnosis based on antibody-antigen interactions. Either biotinylated antibodies or antigens are coupled via streptavidin linkers onto a gold surface. This platform has been used to establish recombinant antibody-antigen interactions and to detect specific IgM antibodies in sera of patients suspected of Lyme borreliosis. Therefore, this microarray system can be adapted for further applications.

DNA probe chip system for multiple detection of food poisoning microorganisms

A DNA probe chip system that can simultaneously analyze multiple genes at the place where a specimen is offered has been developed. Genes to be detected were extracted from food-contaminating microorganisms ( Escherchia coli O157:H7 and Salmonella typhimurium in this study) and then amplified by multiplex polymerase chain reaction (PCR) (utilizing the reverse primers with fluorescein at each 5′-end). These DNA products were measured in a chip system containing capture probes specific to the respective product. The probes were synthesized to contain biotin at each 5′-end and bound onto spatially separated glass surfaces via biotin–streptavidin linkage. Upon addition of a PCR mixture after denaturation, hybridization between the capture probe and the DNA product occurred, which was sequentially monitored utilizing an immune reaction network. Provision of such simplified analytical protocol for multiple PCR products was achieved without a significant sacrifice in the detection sensitivities.

Engineering Issues in the Fabrication of a Hybrid Nano-Propeller System Powered by F 1 -ATPase

A hybrid nanoscale device consisting of nanofabricated structures integrated with a biomolecular motor was constructed on a 25-mm diameter round glass cover slip. Glass substrates were patterned using electron beam lithography and reactive ion etching to define elevated nickel anchor points with a minimum feature size of 60 nm in diameter. Subsequent additions of histidine-tagged F1-ATPase biomolecular motor resulted in selective attachment of the biomotors to the nanofabricated substrates. Electron beam lithography also was employed to fabricate 1 μm long nickel propellers, which subsequently were released through wet etching, collected, and concentrated in buffer solution. Biotin-Cys-6x-His peptides were attached to nickel propellers for interfacing with a biotinylated biomolecular motor through a streptavidin linker. Successful assembly of the hybrid system was demonstrated with observation of rotating nickel propellers. This engineered hybrid nanomechanical system demonstrates the potential application of a new class of devices with enhanced functionality in biological environments.