Streptavidin Complex Research Articles

Dual cascade DNA walking-induced “super on” photocurrent response for constructing a novel antibiotic biosensing method

The construction of effective methods for the convenient testing of antibiotic residues in real samples has attracted considerable interest. Herein, we designed a dual cascade DNA walking amplification strategy and combined it with the controllable photocurrent regulation of a photoelectrode to develop a novel photoelectrochemical (PEC) biosensing method for antibiotic detection. The photoelectrode was prepared through the surface modification of a glassy carbon electrode with the TiO2/CdS QDs nanocomposite synthesized by an in situ hydrothermal deposition method. The strong anodic PEC response of the nanocomposite could be well inhibited by the introduction of a silver nanoclusters (Ag NCs)-labeled DNA hairpin onto its surface. Upon the target biorecognition reaction, an Mg2+-dependent DNAzyme (MNAzyme)-driven DNA walking was triggered to release another MNAzyme strand-linked streptavidin (SA) complex. As this SA complex could serve as a four-legged DNA walker, its cascade walking on the electrode surface not only released Ag NCs but also caused the linking of Rhodamine 123 with the electrode to realize the “super on” photocurrent output. By using kanamycin as the model analyte, this method showed a very wide linear range from 10 fg mL−1 to 1 ng mL−1 and a very low detection limit of 0.53 fg mL−1. Meanwhile, the simple photoelectrode preparation and the aptamer recognition-based autonomous DNA walking resulted in the convenient manipulation and excellent repeatability. These unique performances determine the great potential of the proposed method for practical applications.

Distance-controlled surface-enhanced Raman spectroscopy of nanoparticles.

Biological particles, e.g., viruses, lipid particles, and extracellular vesicles, are attracting significant research interest due to their role in biological processes and potential in practical applications, such as vaccines, diagnostics, and therapies. Their surface and interior contain many different molecules including lipids, nucleic acids, proteins, and carbohydrates. In this Letter, we show how distance-controlled surface-enhanced Raman spectroscopy (SERS) is a promising method to extract essential information from the spatial origin of the signal. This is a highly important parameter in the analysis of these biological particles. The principle of the method is demonstrated by using polystyrene (PS) beads as a biological particle model conjugated with gold nanospheres (AuNSs) functioning as distance-controlled SERS probes via biotin-streptavidin binding. By tuning the size of AuNSs, the Raman signal from the PS beads can be weakened while the signal from the biotin-streptavidin complex is enhanced.

Specific Binding and Endocytosis of Liposomes to HEK293T Cells via Myrisoylated Pre-S1 Peptide Bound to Sodium Taurocholate Cotransporting Polypeptide.

(1) Background: Sodium taurocholate cotransporting polypeptide (NTCP) functions as a key receptor for the hepatitis B virus (HBV) infection. Analyzing HBV and NTCP interaction is an important issue not only for basic research but also for the development of anti-HBV therapeutics. We developed here a novel model system to analyze the interaction of NTCP with liposomes instead of HBV. (2) Methods: Liposomal binding and endocytosis through NTCP in HEK293T cells were achieved by serial treatments of HEL293T cells transiently expressing NTCP-green fluorescence protein (GFP) fusion protein with a synthetic biotinylated pre-S1 peptide (Myr47-Bio) and streptavidin (SA) complex (i.e., Myr47-Bio+SA) followed by biotinylated liposomes. By this procedure, binding of [biotinylated liposomes]-[Myr47-Bio+SA]-[NTCP-GFP] was formed. (3) Results: Using this model system, we found that liposomal binding to NTCP on the cell surface via Myr47-Bio+SA was far more efficient than that to scavenger receptor class B type 1 (SR-B1). Furthermore, liposomes bound to cell surface NTCP via Myr47-Bio+SA were endocytosed into cells after cells were cultured at 37 °C. However, this endocytosis was suppressed by 4 °C or cytochalasin B treatment. (4) Conclusions: This model system will be useful for not only analyzing HBV entry mechanisms but also screening substances to prevent HBV infection.

(Invited, Digital Presentation) Carbon Nanotube Near-Infrared Fluorescent Labels, and Biodistribution of Carbon Nanotubes after Intravenous Injection

Single-walled carbon nanotubes (CNTs) are promising candidates for near-infrared (NIR) fluorescent labels in diagnostic fields. We here report a complex of oxygen-doped CNT (o-CNT) and streptavidin (SA) to prepare CNT-based NIR labels with a high reaction efficiency in immunoassays [1]. This complex specifically binds to biotin molecules by conjugating a linker molecule of phospholipid polyethylene glycol (PL-PEG) to SA (o-CNT-SA). The immunoprecipitation (IP) reaction efficiency between o-CNT-SA and biotin reaches 79.3% by uniformly covering the surface of o-CNTs with SA conjugated PL-PEG (SA-PEG). Further, we also report the biodistribution and toxicity of oxidized super-growth single-wall carbon nanotubes (oxSG) injected intravenously into mice over 3months [2].[1] K. Kojima, Y. Iizumi, M. Zhang, T. Okazaki, submitted.[2] M. Zhang, Y. Xu, M. Yang, M. Yudasaka, T. Okazaki, 15, 798-811 (2021).

Binding feasibility and vibrational characteristics of single-strand spacer-added DNA and protein complexes

Two of the most important features in the field of nanotechnology are self-assembly with nanometre-scale precision, and the self-alignment of functionalised nanomaterials. Here, we discuss the binding feasibility of single-strand spacer-added DNA building blocks to biotin–streptavidin (SA) complexes. We use atomic force microscopy, photoluminescence (PL) spectroscopy, and dynamic simulation to study the topological, optical, and vibrational characteristics of DNA lattices. To construct the DNA lattices, we use two distinct DNA building blocks, i.e. a double-crossover tile with a biotin (DXB), and a double-crossover tile with a flexible single-strand spacer containing a biotin (DXSB). Biotinylated DXB and DXSB lattices grown on the substrate eventually attracted streptavidins (SA, a tetramer protein) and formed DXB + SA, and DXSB + SA lattices, respectively. Furthermore, we examine the feasibility of alignments of an individual DXB (DXSB) tile on SA-bound DXB (DXSB) lattices, and a SA-conjugated Au nanoparticle (NP) on DXB (DXSB) lattices. To use more than two binding sites of biotins on SA (to serve as a connector between biotinylated tiles), the introduction of flexible single-strand spacers in DX tiles helped to overcome geometrical hindrance. In addition, the PL spectra of DXB and DXSB lattices with SA–Au conjugates are analysed to understand the periodic bindings of Au NPs on DXB (DXSB) lattices. We also conduct dynamic simulations of modal analysis and molecular dynamics simulation, which provide the vibrational characteristics and evidence of the importance of single-strand spacer-added DNA samples. Patterning of nanomaterials with specific functionalities with high precision using a simple method would be useful for the manufacture of high-density nanoelectronic devices and extreme-sensitivity biosensors.

Streptavidin-Conjugated Oxygen-Doped Single-Walled Carbon Nanotubes as Near-Infrared Labels for Immunoassays.

Single-walled carbon nanotubes (CNTs) are promising candidates for near-infrared (NIR) fluorescent labels in diagnostic fields. We report a complex of oxygen-doped CNT (o-CNT) and streptavidin (SA) for preparing CNT-based NIR labels with a high reaction efficiency in immunoassays. This complex specifically binds to biotin molecules by conjugating a linker molecule of phospholipid polyethylene glycol (PL-PEG) to SA (o-CNT-SA). The immunoprecipitation reaction efficiency between o-CNT-SA and biotin reaches 79.3% when the surface of o-CNTs is uniformly covered with SA-conjugated PL-PEG. The strong affinity between SA and biotin is useful for preparing CNT-based sensitive NIR fluorescent labels.

Electrochemical Aptasensor Employing Enzyme-Aptamer One-to-One Complex Showing Quasi-Direct Electron Transfer Ability

1. Introduction In this study, we report an innovative electrochemical aptasensor based on the labeling aptamer with quasi-direct electron transfer (quasi-DET)-type redox-enzyme, by developing a breakthrough technological platform to prepare one-to-one complex of enzyme and aptamer.The chronoamperometric-based electrochemical aptasensor is an ideal sensor principle using aptamers for biomolecular recognition considering signal amplification by the labelled redox-enzyme. However, conventional methods to label aptamers with enzymes are chemical modification using cross-linking reagents, which often result in uncontrollable multimer-complexes. Therefore, the method to prepare enzyme-aptamer one-to-one complexes which yields in reproducible and designed enzyme-aptamer complex is technologically challenging. We here report the development of a novel technological platform to prepare enzyme-aptamer one-to-one complex, based on biomolecular engineering concept. 2. Methods Enzyme-aptamer one-to-one complex was constructed utilizing monomeric streptavidin (mSA) and biotin complex formation [1] [2]. We used FAD-dependent glucose dehydrogenase (GDH) from Aspergillus flavus as an aptamer labelling enzyme, and anti-vascular endothelial growth factor (VEGF) aptamer, as to proof of this conceptional study. VEGF is a biomarker for cancer diagnosis. We created the recombinant fusion protein of GDH and mSA (GDH-mSA). The combination of GDH-mSA with biotinylated anti-VEGF aptamer results in their one-to-one complex. To evaluate the formation of the complex and detect VEGF using GDH activity, we performed enzyme-linked aptamer assay (ELAA). Next, GDH-mSA was evaluated as a label enzyme for electrochemical aptasensor, by chronoamperometry analysis using one-to-one complex of GDH-mSA and thiolated and biotinylated aptamer on the gold electrode. Before the analysis, we modified GDH-mSA covalently with phenazine ethosulfate (PES) which was a redox probe enabling redox-enzymes to possess quasi-DET ability [3]. The formation of the enzyme-aptamer one-to-one complex on the electrode and quasi-DET ability of PES-modified GDH-mSA was evaluated. 3. Results GDH-mSA showed dye-mediated GDH activity, indicating the fusion of mSA did not negatively affect the catalytic activity. As a result of ELAA, GDH-mSA showed remarkable GDH activity in the presence of VEGF and biotinylated anti-VEGF aptamer. This indicated one-to-one complex of GDH-mSA and biotinylated anti-VEGF aptamer was formed as we designed, and showed specific recognition and binding with VEGF. Chronoamperometry analysis was carried out, and the signal was increased strikingly upon the addition of glucose only with the gold electrode immobilizing PES-modified GDH-mSA with biotinylated aptamer. Unmodified GDH-mSA did not show quasi-DET. These results indicated our success in the construction of a novel technological platform to prepare enzyme-aptamer one-to-one complex, based on biomolecular engineering concept. 4. Conclusions In this study, to circumvent the formation of uncontrollable multimer-complexes of enzymes and aptamers, we created enzyme-aptamer one-to-one complex based on the mSA-biotin interaction. Thus designed enzyme-aptamer one-to-one complex was prepared without losing both characteristics and acknowledging both benefits. These results suggested that GDH-mSA and biotinylated aptamer complex can be applied for the development of quasi-DET-type aptasensor by simply modifying GDH-mSA with PES. These benefits of utilizing GDH-mSA combined with aptamer will lead to the further application of aptamer-based biosensing of various targets. 5.

Investigation of streptavidin-ligand biochemical interactions by IGZO thin film transistors integrated with microfluidic channels

In this work, we demonstrate an IGZO TFT-based biosensor integrated with a microfluidic channel that can be used to monitor biochemical reactions between proteins and small molecules. The target biomaterials are applied to a Y-shaped microfluidic chamber while the reactant signal is readout by the TFT. Using Biotin-Streptavidin reaction as an example, the specific diffusion time of biotin, streptavidin, or streptavidin-biotin complex across the fluidic channel are benchmarked. By intentionally delaying the application of one specie to another, one can study properties of biochemical reactions, such as excess and exhaust conditions of the reactants, and the formation of the products while diffusing in the channel. The proposed TFT sensor provides an excellent platform in understanding the biochemical functions for the advance of biological science.

Synthesis of biotinylated bivalent zanamivir analogs as probes for influenza viruses

Abstract The synthesis of a biotinylated bivalent zanamivir analog as a probe for influenza viruses is reported. The compound was used in a ‘glycan’ based sandwich assay; where glycans were immobilized on glass slides to capture strains of influenza A H1N1, A/Brisbane/59/2007 virus; the biotinylated bivalent zanamivir analog-labeled streptavidin complex was used as reporter. This research strongly suggests that glycans can be used for capturing and reporting influenza viruses and the biotinylated compounds can be used as probes for capturing and isolating influenza viruses from complex mixtures.

Purification of Biotinylated Proteins Using Single Walled Carbon Nanotube-Streptavidin Complexes

In this study, Single walled carbon nanotube (SWNT)-streptavidin complexes were used to capture and purify biotinylated proteins, including bio-GFP and bio-DBS using a pull-down method. The purification conditions were systematically studied, including surface blocking of SWNT using chicken egg albumin (CEA), the ratio of SWNT-streptavidin complexes to the cell lysate, as well as the centrifugation speed. Optimization of the protein purification using SWNT-streptavidin complexes shows the possibility of carbon nanotubes as a promising candidate for protein purification applications. The SWNT-streptavidin could be used as a scaffold to analyze protein structure directly by cryo-transmission electron microscopy, which provides better understanding in protein–protein interactions and biological processes.

A Programmable DNA Double-Write Material: Synergy of Photolithography and Self-Assembly Nanofabrication.

We demonstrate a DNA double-write process that uses UV to pattern a uniquely designed DNA write material, which produces two distinct binding identities for hybridizing two different complementary DNA sequences. The process requires no modification to the DNA by chemical reagents and allows programmed DNA self-assembly and further UV patterning in the UV exposed and nonexposed areas. Multilayered DNA patterning with hybridization of fluorescently labeled complementary DNA sequences, biotin probe/fluorescent streptavidin complexes, and DNA patterns with 500 nm line widths were all demonstrated.

A Hyaluronidase-Responsive Nanoparticle-Based Drug Delivery System for Targeting Colon Cancer Cells.

The ability of nanoparticles to target tumors and to enable site-specific drug release provides a unique system for the delivery of effective therapy with reduced toxic side effects. In this study, we used mesoporous silica nanoparticles (MSN) to fabricate a targeted drug delivery system that is responsive to hyaluronidase (HAase). Following engraftment of desthiobiotin onto the surface of MSN, a streptavidin complex was generated, which was functionalized with biotin-modified hyaluronic acid (HA) to enable controlled drug release at cancer cells expressing HAase. Various technologies were used to confirm the successful fabrication of this MSN-based nanocarrier system for targeted drug delivery. In vitro analyses showed that the release of doxorubicin hydrochloride (Dox) was accelerated significantly in the presence of biotin or HAase and accelerated further in the presence of biotin and HAase. Uptake by cancer cells was mediated efficiently by CD44 receptor-mediated endocytosis and the MSN exhibited good biocompatibility in vitro and in vivo MSN-HA/Dox nanoparticles induced apoptosis in cancer cells more efficiently than free doxorubicin and inhibited tumor growth with minimal systemic toxicity in vivo Collectively, our findings offered a preclinical proof of concept for a novel targeted drug delivery carrier system for cancer therapy. Cancer Res; 76(24); 7208-18. ©2016 AACR.

Nanophotonic Waveguide Enhanced Raman Spectroscopy of Biological Submonolayers

Characterizing a monolayer of biological molecules has been a major challenge. We demonstrate nanophotonic waveguide enhanced Raman spectroscopy (NWERS) of monolayers in the near-infrared region, enabling real-time measurements of the hybridization of DNA strands and the density of submonolayers of biotin–streptavidin complex immobilized on top of a photonics chip. NWERS is based on enhanced evanescent excitation and collection of spontaneous Raman scattering near nanophotonic waveguides, which for a 1 cm silicon nitride waveguide delivers a signal that is more than 4 orders of magnitude higher in comparison to a confocal Raman microscope. The reduced acquisition time and specificity of the signal allows for a quantitative and real-time characterization of surface species, hitherto not possible using Raman spectroscopy. NWERS provides a direct analytic tool for monolayer research and also opens a route to compact microscopeless lab-on-a-chip devices with integrated sources, spectrometers, and detectors fa...

A Streptavidin Binding Site Mutation Yields an Unexpected Result: An Ionized Asp128 Residue Is Not Essential for Strong Biotin Binding.

We report a detailed study of a point mutation of the crucial binding site residue, D128, in the biotin-streptavidin complex. The conservative substitution, D128N, preserves the detailed structure observed for the wild-type complex but has an only minimal impact on biotin binding, even though previous experimental and computational studies suggested that a charged D128 residue was crucial for high-affinity binding. These results show clearly that the fundamental basis for streptavidin's extremely strong biotin binding affinity is more complex than assumed and illustrate some of the challenges that may arise when analyzing extremely strong ligand-protein binding interactions.

Application of ADA1 as a new marker enzyme in sandwich ELISA to study the effect of adenosine on activated monocytes.

Enzyme-linked immunosorbent assay (ELISA) is a valuable technique to detect antigens in biological fluids. Horse radish peroxidase (HRP) is one of the most common enzymes used for signal amplification in ELISA. Despite new advances in technology, such as a large-scale production of recombinant enzymes and availability of new detection systems, limited research is devoted to finding alternative enzymes and their substrates to amplify the ELISA signals. Here, HRP-avidin was substituted with the human adenosine deaminase (hADA1)-streptavidin complex and adenosine as a detection system in commercial ELISA kits. The hADA1 ELISA was successfully used to demonstrate that adenosine, bound to A1 and A3 adenosine receptors, increases cytokine secretion by LPS activated monocytes. We show that hADA1-based ELISA has the same sensitivity, and also provides identical results, as HRP ELISA. In addition, the sensitivity of hADA1-based ELISA could be easily adjusted by changing the adenosine concentration and the incubation time. Therefore, hADA1 could be used as a detection enzyme with any commercial ELISA kit with a wide range of concentration of antigens.

A novel tag-free probe for targeting molecules interacting with a flavonoid catabolite

3,4-Dihydroxyphenylacetic acid (DOPAC) is one of the colonic microflora-produced catabolites of quercetin 4′-glucoside (Q4′G). Although the interaction of DOPAC with cellular proteins might be involved in its biological activity, the actual proteins have not yet been identified. In this study, we developed a novel tag-free DOPAC probe to label the targeted proteins by the copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) and verified its efficacy. Various labeled proteins were detected by the DOPAC probe with the azide labeled biotin and a horseradish peroxidase (HRP)-streptavidin complex. Furthermore, a pulldown assay identified Keap1 and aryl hydrocarbon receptor (AhR) as the target proteins for the phase 2 enzyme up-regulation.

Achievement of linear response for competitive bioaffinity assays of ligands: criteria of optimized interaction systems

For a linear response, an optimized competitive bioaffinity assay of a ligand requiresCRT> 3 ×CPT,CPT> 50 ×KdR, andKdR> 260 ×KdX(CRTandCPTare concentrations of the probe and protein whileKdXandKdRareKdfor the ligand and probe, respectively).

Piezoelectric tuning fork biosensors for the quantitative measurement of biomolecular interactions

The quantitative measurement of biomolecular interactions is of great interest in molecular biology. Atomic force microscopy (AFM) has proved its capacity to act as a biosensor and determine the affinity between biomolecules of interest. Nevertheless, the detection scheme presents certain limitations when it comes to developing a compact biosensor. Recently, piezoelectric quartz tuning forks (QTFs) have been used as laser-free detection sensors for AFM. However, only a few studies along these lines have considered soft biological samples, and even fewer constitute quantified molecular recognition experiments. Here, we demonstrate the capacity of QTF probes to perform specific interaction measurements between biotin–streptavidin complexes in buffer solution. We propose in this paper a variant of dynamic force spectroscopy based on representing adhesion energies E (aJ) against pulling rates v (nm s–1). Our results are compared with conventional AFM measurements and show the great potential of these sensors in molecular interaction studies.

Critical link between glycogen synthase kinase 3β and forkhead box P3 in patients with chronic rhinosinusitis with nasal polyps

Critical link between glycogen synthase kinase 3β and forkhead box P3 in patients with chronic rhinosinusitis with nasal polyps

Artificial Ligands of Streptavidin (ALiS): Discovery, Characterization, and Application for Reversible Control of Intracellular Protein Transport.

Artificial ligands of streptavidin (ALiS) with association constants of ∼10(6) M(-1) were discovered by high-throughput screening of our chemical library, and their binding characteristics, including X-ray crystal structure of the streptavidin complex, were determined. Unlike biotin and its derivatives, ALiS exhibits fast dissociation kinetics and excellent cell permeability. The streptavidin-ALiS system provides a novel, practical compound-dependent methodology for repeated reversible cycling of protein localization between intracellular organella.