Biotin-streptavidin Interaction Research Articles

Triggered Polycatenated DNA Scaffolds for DNA Sensors and Aptasensors by a Combination of Rolling Circle Amplification and DNAzyme Amplification

The concept of triggered polycatenated DNA scaffolds has been elegantly introduced into ultrasensitive biosensing applications by a combination of rolling circle amplification (RCA) and DNAzyme amplification. As compared to traditional methods in which one target could only initiate the formation of one circular template for RCA reaction, in the present study two species of linear single-stranded DNA (ssDNA) monomers are self-assembled into mechanically interlocked polycatenated nanostructures on capture probe-tagged magnetic nanoparticles (MNPs) only upon the introduction of one base mutant DNA sequence as initiator for single-nucleotide polymorphisms (SNPs) analysis. The resultant topologically polycatenated DNA ladder is further available for RCA process by using the serially ligated circular DNA as template for the synthesis of hemin/G-quadruplex HRP-mimicking DNAzyme chains, which act as biocatalytic labels for the luminol-H(2)O(2) chemiluminescence (CL) system. Notably, the problem of high background induced by excess hemin itself is circumvented by immobilizing the biotinylated RCA products on streptavidin-modified MNPs via biotin-streptavidin interaction. Similarly, a universal strategy is contrived by substitutedly employing aptamer as initiator for the construction of polycatenated DNA scaffolds to accomplish ultrasensitive detection of proteins based on structure-switching of aptamer upon target binding, which is demonstrated by using thrombin as a model analyte in this study. Overall, with two successive amplification steps and one magnetic separation procedure, this flexible biosensing system exhibits not only high sensitivity and specificity with the detection limits of SNPs and thrombin as low as 71 aM and 6.6 pM, respectively, but also excellent performance in real human serum assay with no PCR preamplification for SNPs assay. Given the unique and attractive characteristics, this study illustrates the potential of DNA nanotechnology in bioanalytical applications for both fundamental and practical research.

A novel quantum dot based nanoassay for high resolution copy number variation detection and DNA methylation quantification

Abstract Background: Detection of DNA copy number variation (CNV) is not only fundamental to study molecular etiology of cancer but also provides a potential molecular diagnostics for cancer detection. Nonetheless, current techniques are not able to reliably differentiate less than a twofold change in copy number. The hypermethylation of the cytosines at CpG islands in the promoter regions of tumor suppressor genes has been proved to be a valuable epigenetic biomarker for early cancer diagnosis. However, current ways of quantifying DNA methylation heavily rely on PCR, which is believed to have only twofold resolution. This abstract presents a novel gene quantification platform named quantum dot (QD) Electrophoretic Mobility Shift Assay (QEMSA). This nanoassay demonstrates a remarkable resolution, capable of distinguishing a 1.1-fold (~9%) difference in target copy number. QEMSA could be very useful when a precise measurement of DNA copy number is required. Methods and Results: QEMSA employs a QD to physically transform the target copy number into different electrophoretic mobility levels. DNA molecules self assemble onto the QD surface through the streptavidin-biotin interaction to form a QD-DNA nanocomplex with certain DNA to QD ratios (N). The working principle of the QEMSA is analogous to a horse wagon, of which the QD serves as a nano wagon and harnesses the horsepower from the force exerted on the DNA molecules in the electric field. Intuitively, the more DNA molecules on one QD, the more horsepower and the faster the nanocomplex migrates. We have developed a mathematic model which predicts the electrophoretic migration distance Dm is proportional to ln(N). The electrophoretic migration distances of the nanocomplex are plotted against N in logarithmic scale (Thereafter refers to as the migration curve). The linear regression of the migration curves with R2=0.99 suggests the data agree well with our model. Using QEMSA, tiny differences in copy number can be magnified hence reliably detected by enlarging the separation distance between nanocomplexes with different N values. As a result, we have successfully quantified a 1.1-fold dilution series (~9% difference). A spike-in control experiment is performed by mixing the synthetic DNA fragments from the Rsf-1 gene and the reference sequence at various ratios. A single duplication on only one allele results in a total of 3 copies per cell therefore a target/reference ratio of 1.5. The fragments are tagged with biotin using biotinylated primer. The observed ratios are plotted against the expected ratios. A good linear correlation with R2=0.99 is obtained. Using same approach, the Rsf-1 gene is found being amplified by ~8.5 fold in the ovarian cancer cell line OVCAR3, which is in good agreement with the results analyzed by SNP array but with less detection noise. Two dilution series of the methylated DNA with dilution factors of 1.5 and 1.25 respectively are spiked with 100ng normal DNA to simulate different DNA methylation levels. Both the methylated DNA and the normal DNA undergo the bisulfite treatment. The methylated p16/CDK2A promoter region is tagged with biotin using methylation specific PCR. QEMSA successfully generates the standard migration curves for both dilution series. The same dilution series are analyzed in triplicates using qPCR, the results of which show larger fluctuation. Conclusion: In summary, we have demonstrated a promising high resolution gene copy number quantification platform using a quantum dot nanotechnology. The novel platform has been successfully applied to detect CNV and quantify DNA methylation level in human cancer.

Three-dimensional directed self-assembly of Peptide nanowires into micrometer-sized crystalline cubes with nanoparticle joints.

Micro construction sites: Engineered peptides and ligand-functionalized gold nanoparticles have been assembled into micrometer-scale 3D cube-shaped crystals (see picture), creating a physical framework for a biomimetic assembly strategy. The NPs join the peptide nanowires through streptavidin–biotin interactions to create cubic unit cells.

A magnetic bead-based protein kinase assay with dual detection techniques

A novel magnetic bead-based protein kinase assay was developed using MALDI-TOF mass spectrometry (MALDI-TOF MS) and immunochemifluorescence as two independent detection techniques. Abltide substrate was immobilized onto magnetic beads via noncovalent biotin–streptavidin interactions. This noncovalent immobilization strategy facilitated peptide release and allowed MALDI-TOF MS analysis of substrate phosphorylation. The use of magnetic beads provided rapid sample handling and allowed secondary analysis by immunochemifluorescence to determine the degree of substrate phosphorylation. This dual detection technique was used to evaluate the inhibition of c-Abl kinase by imatinib and dasatinib. For each inhibitor, IC 50 (half-maximal inhibitory concentration) values determined by these two different detection methods were consistent and close to values reported in the literature. The high-throughput potential of this new approach to kinase assays was preliminarily demonstrated by screening a chemical library consisting of 31 compounds against c-Abl kinase using a 96-well plate. In this proof-of-principle experiment, both MALDI-TOF MS and immunochemifluorescence were able to compare inhibitor potencies with consistent values. Dual detection may significantly enhance the reliability of chemical library screening and identify false positives and negatives. Formatted for 96-well plates and with high-throughput potential, this dual detection kinase assay may provide a rapid, reliable, and inexpensive route to the discovery of small-molecule drug leads.

Thermally Triggered Cellular Uptake of Quantum Dots Immobilized with Poly(N-isopropylacrylamide) and Cell Penetrating Peptide

Thermally sensitive quantum dots (TSQDs) that exhibit an "on-demand" cellular uptake behavior via temperature-induced "shielding/deshielding" of cell penetrating peptides (CPP) on the surface were fabricated. Poly(N-isopropylacrylamide) (PNIPAAm) (M(w) = 11.5K) and CPP were biotinylated at their terminal ends and co-immobilized on to the surface of streptavidin-coated quantum dots (QDs-Strep) through biotin-streptavidin interaction. The cellular contact of CPP was sterically hindered due to hydrated PNIPAAm chains below the lower critical solution temperature (LCST). In contrast, above the LCST, grafted PNIPAAm chains were collapsed to make CPP moieties resurfaced, leading to increased cellular uptake of QDs. The temperature-controlled "shielding/deshielding" of CPP was further applied for a thermally triggered siRNA delivery system, where biotinylated siRNA was additionally conjugated to the surface of TSQDs. The level of gene silencing was significantly enhanced by increasing temperature above the LCST due to the surface exposure of CPP.

Convenient RNA Synthesis Using a Phosphoramidite Possessing a Biotinylated Photocleavable Group

A convenient RNA synthesis using a biotin-streptavidin interaction and a photocleavable protecting group is described. The biotinylated photocleavable group was introduced at the 2'-position of the uridine derivative. Using the phosphoramidite 12, we attempted the synthesis of a 21mer RNA, which is pure enough to show potent RNAi activity compared with a conventionally prepared and HPLC-purified 21mer RNA with the same sequence.

Targeted Cellular Delivery of Quantum Dots Loaded on and in Biotinylated Liposomes

We describe the preparation, biophysical characterization, and receptor-mediated cellular internalization of biotinylated lipid particles (BLPs) loaded on the surface and internally with two distinct (colors) of quantum dot (QD) probes. BLPs were formulated with 1.4 and 2.7 mol % PEG-lipids containing either a fusogenic or pH-sensitive lipid to promote bilayer destabilization of endosomal membranes and favor QD cytoplasmic release. The amount of PEG was chosen to provide steric stabilization of the final construct. BLPs were loaded with a red-emitting QD(655) and surface coated with a green-emitting QD(525) conjugated to the epidermal growth factor (EGF) ligand in order to target the epidermal growth factor receptor (EGFR). The targeted and QD labeled BLPs showed strong and selective binding to EGFR-expressing tumor cell line and subsequent internalization. The dual-color QD labeling strategy and colocalization analysis allow prolonged live cell imaging of BLPs and loaded cargo independently, using a single excitation wavelength and simultaneous detection of both QDs. The chemistry of bioconjugation for the EGF ligand, the QDs, and the BLPs in a single lipid particle, involves only biotin-streptavidin interaction without requiring further purification from free EGF-QDs preformed complexes. Coupled with an encapsulated drug, the targeted and QD-labeled BLPs could provide imaging and drug delivery in a single multifunctional carrier.

Genosensor for detection of four pneumoniae bacteria using gold nanostructured screen-printed carbon electrodes as transducers

This work describes the development of different formats of hybridization-based genosensors for pneumonia using disposable gold nanostructured screen-printed carbon electrodes. Gold nanoparticles, which have been formed in situ by applying a constant current intensity during a fixed time, act as an immobilization and transduction surface. Immobilization takes place through thiol–gold interaction in a relatively fast way, or through the well-known biotin–streptavidin interaction. Analytical parameters such as linearity range, reproducibility and detection limit of the genosensor were evaluated. The best results are obtained when the probe is attached to the electrode surface through the streptavidin–biotin interaction and fluorescein is used as label. In this case an anti-fluorescein antibody labelled with alkaline phosphatase is used to obtain the analytical signal.

Surface plasmon resonance instrument as a refractometer for liquids and ultrathin films

A surface plasmon resonance (SPR) setup in Kretschmann configuration is being utilized as a refractometer for both liquids as well as ultrathin films. The SPR signal detection technology used is based on a goniometer approach providing a wide angular scan range which facilitates highly accurate liquid and gas phase measurements. Attention was paid to improve sample handling and preparation. In order to avoid cross-contamination between measurements an easily removable and exchangeable molded PDMS flow cell was used during the measurements. By careful choice of components for liquid handling the dead volume of the system could be reduced down to some microliters. The angular change and thus the refractive index for sucrose, ethylene glycol (EG) and ethanol solutions with different concentrations, the thickness and refractive index of deposited Langmuir-Blodgett (LB) films, and the interaction kinetics between a biotin containing self-assembled monolayer (SAM) and streptavidin were determined. The measured refractive indices of sucrose, EG and ethanol solutions corresponded well with literature values. LB films were characterized by measuring the complete SPR curve in an angular scan range from 40 to 78°. A two-color SPR approach combined with two-media measurements was successfully employed for simultaneous and unambiguous determination of both refractive index and thickness of stearic acid monolayers. The thickness obtained for the stearic acid monolayer was 2.66 nm, and the refractive indices at 635 and 670 nm were 1.5800 and 1.4138, respectively. The developed sensor-plate holder enabled functionalization of the SPR gold chip outside the instrument, therefore making the sample handling more flexible. The affinity constant obtained for the streptavidin–biotin interaction was 1.01 × 10 −8 M. The total angle SPR method used in this study clearly shows its potential to be used as a refractometer for both liquids and ultrathin films, as well as for traditional liquid phase biomolecular kinetic studies.

Enzyme Mediated Site-Specific Surface Modification

Stable tethering of bioactive peptides like RGD to surfaces can be achieved via chemical bonding, biotin streptavidin interaction, or photocross-linking. More challenging is the immobilization of proteins, since methods applied to immobilize peptides are either not specific or versatile enough or might even compromise the protein's bioactivity. To overcome this limitation, we have employed a scheme that by enzymatic (transglutaminase) reaction allows the site-directed and site-specific coupling of growth factors and other molecules to nonfouling poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) coated surfaces under physiological conditions. By our modular and flexible design principle, we are able to functionalize these surfaces directly with peptides and growth factors or precisely position poly(ethylene glycol) (PEG)-like hydrogels for the presentation of growth factors as exemplified with vascular endothelial growth factor (VEGF).

Ultrasensitive electrochemical detection of nucleic acid based on the isothermal strand-displacement polymerase reaction and enzyme dual amplification

We describe an ultrasensitive electrochemical detection of DNA protocol based on the isothermal strand-displacement polymerase reaction (ISDPR) and enzyme dual amplifications. Target DNA triggered an ISDPR to produce numerous bi-functionalized duplex DNA complexes. Following an immuno-magnetic collection via an immunoreaction between the attached digoxin on the duplex DNA and the anti-digoxin antibody on the magnetic bead, horseradish (HRP) tracers were bound to the duplex DNA through a biotin–streptavidin interaction. The quantification of DNA was realized by square wave voltammetric detection of the enzymatic products with a screen-printed gold electrode. The voltammetric response was proportional to the concentration of DNA in the range of 0.1 fM–0.5 pM, and the limit of detection was estimated to be 0.06 fM. The new protocol showed great promise for simple, cost-effective, and quantitative gene analysis.

Biotin-directed assembly of targeted modular lipoplexes and their transfection of human hepatoma cells in vitro

The asialoglycoprotein receptor, which is abundantly and near exclusively expressed on hepatocytes, has received much attention in the design of non-viral hepatotropic DNA delivery systems. Thus, asialoglycoproteins and hexopyranosyl ligands have been coupled to DNA-binding cationic polymers and liposomes in the assembly of complexes intended for uptake by liver parenchymal cells. The aim of the study was to construct a hepatocyte-targeted multimodular liposome-based transfecting complex, in which the biotin-streptavidin interaction provides the cohesive force between the ligand asialorosomucoid and the liposome bilayer, and to evaluate its transfection capabilities in the hepatocyte-derived human transformed cell line HepG2. Dibiotinylated asialoorosomucoid was attached to cationic liposomes constructed from 3β[N-(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T):dioleoylphosphatidylethanolamine:biotinylcholesterylformylhydrazide (MSB1) (48:50:2 mole ratio) through streptavidin interposition. Liposome-pGL3 DNA interactions were studied by gel band shift and ethidium displacement assays. The cytotoxicity of assemblies was evaluated in the HepG2 cell line and transfection capabilities determined by measuring the activity of the transgene luciferase. Binding assays showed that all DNA was liposome associated at a DNA (negative):liposome (positive) charge ratio of 1:1. Accommodation of a streptavidin dibiotinylated asialoorosomucoid assembly was achieved at a DNA:liposome:streptavidin dibiotinylated asialoorosomucoid ratio of 1:4:9 (weight basis). Complexes showed optimal transfection activity at this ratio, which was reduced 10-fold by the presence of the competing ligand asialofetuin. The streptavidin-biotin interaction has been applied for the first time to the assembly of hepatocyte-targeted lipoplexes that display asialoorosomucoid and that are well tolerated by a human hepatoma cell line in which transfection is demonstrably achieved by receptor mediation. Favorable size and charge ratio characteristics suggest that this system may be suitable for in vivo application.

Reconsideration of Dynamic Force Spectroscopy Analysis of Streptavidin-Biotin Interactions

To understand and design molecular functions on the basis of molecular recognition processes, the microscopic probing of the energy landscapes of individual interactions in a molecular complex and their dependence on the surrounding conditions is of great importance. Dynamic force spectroscopy (DFS) is a technique that enables us to study the interaction between molecules at the single-molecule level. However, the obtained results differ among previous studies, which is considered to be caused by the differences in the measurement conditions. We have developed an atomic force microscopy technique that enables the precise analysis of molecular interactions on the basis of DFS. After verifying the performance of this technique, we carried out measurements to determine the landscapes of streptavidin-biotin interactions. The obtained results showed good agreement with theoretical predictions. Lifetimes were also well analyzed. Using a combination of cross-linkers and the atomic force microscope that we developed, site-selective measurement was carried out, and the steps involved in bonding due to microscopic interactions are discussed using the results obtained by site-selective analysis.

Site-selective dual modification of periplasmic binding proteins for sensing applications

We have developed three sensitive and specific amino acid sensors based on bacterial periplasmic solute binding proteins. A site-specific amino-terminal transamination reaction provides a useful complement to cysteine chemistry for the covalent modification of biomolecules in this application. We demonstrate this combination to attach two different chromophores to a single biomolecule in two locations. The periplasmic glutamine binding protein from E. coli was modified with a pair of dyes suitable for fluorescence resonance energy transfer, and this conjugate exhibited an l-glutamine dependent optical response. Two periplasmic binding proteins from the thermophilic organism Thermotoga maritima, for arginine and aliphatic amino acids, were modified and evaluated similarly. All three conjugates manifested signal changes mediated by resonant energy transfer upon binding their respective ligands, with nanomolar dissociation constants and stereochemical specificity. This represents a readily generalizable method for construction of reagentless biosensors. The double-labeling strategy was also exploited for the surface attachment of a dye-labeled glutamine binding protein via a biotin–streptavidin interaction.

Expression, purification and characterization of in vivo biotinylated dengue virus envelope domain III based tetravalent antigen

Dengue is a rapidly spreading mosquito-borne viral disease prevalent in over a hundred countries around the world. A definitive identification of dengue infection depends on reliable dengue diagnostic tests. This study describes the design, expression and purification of an in vivo biotinylated chimeric dengue antigen to exploit the high affinity of biotin–streptavidin interaction to detect anti-dengue antibodies. This chimeric antigen incorporates the envelope domain III (EDIII) of the four dengue virus serotypes. A biotin acceptor peptide was fused with the chimeric dengue antigen for in vivo biotinylation in Escherichia coli through simultaneous co-expression of the biotin ligase, BirA. Despite the localization of the chimeric dengue antigen to the insoluble fraction of induced E. coli cells, it was found to be biotinylated in vivo. It was purified to near homogeneity using affinity chromatography with final yields of 20 mg protein of ∼95% purity, from 1 L of induced E. coli shake flask culture, and the efficiency of biotinylation was estimated to be ∼85%. Mouse antibodies specific to recombinant EDIII of each of the four dengue serotypes, captured on microtiter wells sensitized with anti-mouse immunoglobulin antibodies, were recognized specifically and with high efficiency by the biotinylated antigen in conjunction with streptavidin–enzyme conjugate. An evaluation of the biotinylated antigen against a panel of pre-characterized dengue-positive and dengue-negative human sera ( n = 164), in an antibody capture ELISA format, showed that it manifested 100% specificity, but also suggested that additional epitopes may need to be included in its design to enhance sensitivity.

Label-free detection of protein-ligand interactions in real time using micromachined bulk acoustic resonators

In this paper, we present a micromachined film bulk acoustic resonator (FBAR) to detect protein-ligand interactions in real-time. The surface of the FBAR device has a thin layer of gold deposited on it to immobilize thiol-modified biotin. The resonant frequency of the biotin modified FBAR was measured to decrease by 170 ppm when exposed to streptavidin solution with a concentration of 5×10−7 M, corresponding to an added mass of 120 pg on the FBAR surface due to the biotin-streptavidin interaction. Consequently, the biotin modified FBAR can be used to observe in real time the biotin-streptavidin interaction without the use of labeling or molecular tags. The FBAR can be used in a variety of protein-ligand systems, and be designed for testing in array formats to give high throughput screening for drug discovery.

Visual detection of single-stranded target DNA using pyrroloquinoline-quinone-loaded liposomes as a tracer

The preparation of DNA-tagged liposomes containing an encapsulated prosthetic group tracer, pyrroloquinoline quinone (PQQ), and their application to the development of a sandwich-type hybridization assay for the visual detection of single-stranded DNA are described. Capture DNA is conjugated to the surface of microtiter plate wells through a biotin–streptavidin interaction. Target DNA is incubated with the plate in high salt concentrations. The reporter DNA-tagged liposomes encapsulating PQQ, the prosthetic group of the apo-enzyme glucose dehydrogenase (GDH), are used as the label to probe for bound target DNA. After washing away unbound liposomes and subsequent lysis of the bound fraction by surfactant, PQQ is released and available to activate the apo-enzyme. In the presence of glucose and a redox dye, 2,6-dichlorophenol indophenol (DCPIP), the dye is reduced to yield an optical color change from blue to colorless. This transition is observed visually or spectrophotometrically. The degree of optical change is proportional to the amount of PQQ present, which directly relates to the number of liposomes and, thus, the total amount of target DNA. An arbitrary target DNA sequence is used as a model system, and a limit of detection of 62fmol is achieved.

Ultrasensitive electrical biosensing of syphilis DNA using target-guided formation of polyaniline based on enzyme-catalyzed polymerization

An ultrasensitive assay for electrical biosensing of syphilis DNA was proposed by using target-guided formation of polyaniline (PANI) based on an enzymatically catalyzed method. The sensitive biodetection relies on the DNA hybridization and biotin–streptavidin interaction. After coupling of biotinylated catalase with streptavidin-modified hybrids, a head-to-tail structure of PANI templated by hybridized DNA was formed. The current response of PANI was linearly related to target DNA concentration between 1.0 pM and 1.0 nM with a correlation coefficient of 0.998. The detection limit was determined to be 0.5 pM with the signal-to-noise ratio of 3. The synergistic performances of DNA hybridization, strong biotin–streptavidin binding ability, and highly efficient polymerization provide a general platform for simple, highly sensitive and selective biosensor for the detection of the specific polA gene fragment of T. pallidum. It is expected that the proposed biosensor holds great promise for diagnosing disease in practice.

Size Matters: Influence of the Size of Nanoparticles on Their Interactions with Ligands Immobilized on the Solid Surface

The correlation between the size of biotinylated nanoparticles and their affinity in relation to interactions with the solid surface was investigated. The silica particles with a diameter of 50-200 nm containing amino groups on the surface were labeled with different quantities of biotin. The affinity properties of biotinylated nanoparticles were studied using a Biacore 3000 instrument equipped with a streptavidin-coated sensor chip (SA chip). It was shown that the increase in the particle size from 50 to 200 nm reduced the affinity (K(D)) of biotin-streptavidin interactions from 1.2 x 10(-12) to 1.2 x 10(-10) M. It was found that the particles with higher concentrations of immobilized biotin on particle surfaces demonstrated stronger binding with streptavidin.

Surface Plasmon Resonance on Gold and Silver Films Coated with Thin Layers of Amorphous Silicon−Carbon Alloys

The paper reports on a novel surface plasmon resonance (SPR) substrate architecture based on the coating of a gold (Au) or silver (Ag) substrate with 5 nm thin amorphous silicon-carbon alloy films. Ag/a-Si(1-x)C(x):H and Au/a-Si(1-x)C(x):H multilayers are found to provide a significant advantage in terms of sensitivity over both Ag and Au for SPR refractive index sensing. The possibility for the subsequent linking of stable organic monolayers through Si-C bonds is demonstrated. In a proof-of-principle experiment that this structure can be used for real-time biosensing experiments, amine terminated biotin was covalently linked to the acid-terminated SPR surface and the specific streptavidin-biotin interaction recorded.