Microstamping on an Activated Polymer Surface: Patterning Biotin and Streptavidin onto Common Polymeric Biomaterials (original) (raw)
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Oligonucleotide immobilization on micropatterned streptavidin surfaces
Nucleic Acids Research, 2000
We describe a simple procedure for photolithographic patterning of streptavidin on silicon substrates. Long wavelength UV (365 nm) light was used to direct the covalent attachment of photoactivatable biotin onto silylated silicon wafers. Fluorescently labeled streptavidin was found to bind only in areas exposed to the light. We used this procedure to selectively pattern streptavidin inside microwells etched in silicon, and we investigated the binding characteristics of biotinylated oligonucleotides of lengths, n = 16, 54 and 99 bases. The binding curves were found to fit the functional form of the Langmuir isotherm, with binding saturation proportional to n-3/4 .
Surface and Interface Analysis, 2004
A polystyrene biosensing chip to be used as a medical diagnostic tool has been developed. Its surface functionalization is optimized to bind the bioanalytes with high efficiency. Coatings of allyldextran monolayers were carried out on polystyrene chips activated with g-irradiation. The effect of the concentration of allyldextran solution on the coating efficiency was studied by surface-sensitive x-ray photoelectron spectroscopy (XPS) and contact-angle measurements. In a second step, sodium periodate chemistry was applied to functionalize the dextran layer, followed by coupling with streptavidin or neutravidin.
Quantification of streptavidin adsorption in microtitration wells
Analytical Biochemistry, 2004
Streptavidin-coated microtitration plates have an important role as a solid phase in clinical diagnostics. We have designed techniques for evaluating quantitative and functional aspects of streptavidin adsorbed in microtitration wells. The theoretical monolayer adsorption capacity was modeled based on the molecular dimensions of the protein. Adsorbed streptavidin was quantiWed by direct labeling of protein with terbium chelate and with a sensitive bicinchoninic acid-based protein assay. A new small molecular weight (1037 Da) reporter molecule, a europium-labeled biotin (Eu-biotin), was synthesized and used for monitoring adsorption and for determination of biotin-binding capacities of the streptavidin-coated wells. The theoretical monolayer adsorption of streptavidin yielded 6.20 pmol/cm 2 (370 ng) and consequently the theoretical adsorption capacity of a C12-format microtitration well (200 l liquid, coated area 1.54 cm 2 ) was 9.55 pmol/well (570 ng). Adsorption properties of streptavidin from two suppliers were tested, one of which yielded 350-380 ng/well while the other yielded over 500 ng/well. The biotin binding capacities were about 11 and 14 pmol/well, respectively. We managed to quantify surface-adsorbed streptavidin with sensitive Xuorescence and protein measurement methods in the microtitration well. The new Eu-biotin reporter molecule enabled an exact and convenient determination of the biotin-binding capacities of streptavidin surfaces.
Cell labeling and proximity dependent biotinylation with engineered monomeric streptavidin
Because streptavidin is a homotetramer, it can bind multiple biotinylated ligands and cause target aggregation. To allow biotin detection without clustering, we previously engineered monomeric streptavidin (mSA) that is structurally similar to a single streptavidin subunit. Introducing the S25H mutation near the binding site increases the biotin dissociation half-life t 1/2 to 83 minutes. The slowly dissociating mutant, mSA2, is useful in imaging studies because it allows stable labeling of biotinylated targets. We show that mSA2 conjugated with Alexa 488 binds biotinylated receptors on HEK293 with high specifi city, and bound mSA2–Alexa488 does not dissociate signifi cantly during an imaging study lasting 50 minutes. As a structural monomer, mSA2 can be fused to other proteins to create bifunctional molecules. We tested the use of mSA2 in proximity dependent biotinylation, in which mSA2 is fused to a peptide or a protein that binds a protein of interest (POI) and is used to recruit photoactivatable biotin (PA-biotin) to the target molecule. Once the resulting cluster of interacting proteins is subjected to UV-initiated distance-dependent biotinylation, subsequent affi nity purifi cation of biotinylated proteins on streptavidin beads can identify protein molecules that interact with POI. In addition to proteins that directly interact with the mSA2 fusion, mSA2 also induces biotinylation of other proteins that are associated through a series of noncovalent interactions. We show that mSA2 fused to an antibody recognition domain can be recruited to the kinase Erk-2 using a commercially available antibody and induce biotinylation of a known Erk-2 substrate, GST-Elk-1. Therefore, mSA2 can be used to implement proximity dependent biotinylation and detect transient enzyme-substrate interactions. INNOVATION Streptavidin is used broadly in biotechnology applications that require high-affi nity interaction with biotinylated ligands. Th e streptavidin–biotin system is versatile and easy to work with, which has contributed to its popular use. For example, biotin can be introduced using chemical and enzymatic reactions 1 , and biotinylated targets are readily detected using streptavidin. Because biotin is a small, chemically inert, hydrophilic molecule, biotinylation does not signifi cantly disrupt the native structure or stability of the target molecule or introduce a reactive group that results in non-specifi c interactions. Also, streptavidin has high thermal and aqueous stability, which are important to ensure the molecule retains function under many experimental conditions. Because streptavidin can be modifi ed with fl uorophores or enzymes without loss of function, it can be used for labeling biotinylated targets for detection and readout. Several excellent reviews on the applications of the streptavidin–biotin technology exist and may be referenced for additional information 2–5. While achieving the highest sensitivity is essential for some applications , optimizing properties other than the affi nity of interaction may be more relevant in other applications. For example, biotin binds streptavidin irreversibly, which means bound ligands can be recovered only by denatur-ing streptavidin. Since harsh elution conditions tend to disrupt the ligand structure, wild-type streptavidin cannot be used in affi nity purifi cation when the ligand structure is susceptible to perturbation. In this case, the high biotin affi nity of streptavidin becomes a liability and actually limits its utility. Also, as an obligate tetramer that requires oligomerization for stability and function 6,7 , streptavidin easily crosslinks biotinylated ligands. Since crosslinking leads to clustering of cell surface receptors on live cells, streptavidin labeling is inherently at odds with the goals of an imaging study, i.e. to measure the distribution and dynamics of cell surface proteins to draw biological insights. Labeling biotinylated cell surface receptors without crosslinking requires monovalent biotin-streptavidin interaction, which cannot be achieved with wild type streptavidin tetramer. It has been demonstrated that monovalent interaction with bioti-nylated ligands can be achieved with an engineered heterotetramer containing a single active subunit 8. However, preparation of the molecule is cumbersome and the molecule remains relatively large (~60 kDa). It also cannot be used as a fusion tag because it is still a structural tetramer. An alternative way to implement monovalent biotin binding involves the use of a monomer protein, e.g. one that corresponds to a single subunit of streptavidin. Th e monomer may be engineered from streptavidin by disrupting its subunit association with mutations at the interface. Studies have shown that disrupting the oligomer structure results in molecules that are unstable (T m = 30°C), prone to aggregation 9 , and bind biotin weakly (K d ~ 100 nM) 10 .
Controlling Multivalent Binding through Surface Chemistry: Model Study on Streptavidin
Journal of the American Chemical Society, 2017
Although multivalent binding to surfaces is an important tool in nanotechnology, quantitative information about the residual valency and orientation of surface-bound molecules is missing. To address these questions, we study streptavidin (SAv) binding to commonly used biotinylated surfaces such as supported lipid bilayers (SLBs) and self-assembled monolayers (SAMs). Stability and kinetics of SAv binding are characterized by quartz crystal microbalance with dissipation monitoring, while the residual valency of immobilized SAv is quantified using spectroscopic ellipsometry by monitoring binding of biotinylated probes. Purpose-designed SAv constructs having controlled valencies (mono-, di-, trivalent in terms of biotin-binding sites) are studied to rationalize the results obtained on regular (tetravalent) SAv. We find that divalent interaction of SAv with biotinylated surfaces is a strict requirement for stable immobilization, while monovalent attachment is reversible and, in the case ...
A Streptavidin Surface on Planar Glass Substrates for the Detection of Biomolecular Interaction
Analytical Biochemistry, 2000
Based on the requirements of biomolecular interaction analysis on direct optical transducers, a streptavidin surface is examined. A general protocol was developed allowing the immobilization of biotinylated compounds using the rife biotin-streptavidin system. This type of surface modification can be applied to all biosensors using glass surfaces as sensor devices. Reflectometric interference spectroscopy (RIfS), a labelfree, direct optical method was used to demonstrate the quality of the transducer surfaces. The surface modification is based on an aminofunctionalized polyethylene glycol layer covalently bound to the silica surface of the transducer and shows very little nonspecific binding. Biotin molecules can be easily coupled on such layers. Streptavidin followed by a biotinylated estrone derivative was immobilized by incubation of the biotinylated transducer surface. For the streptavidin layer we obtained interference signals corresponding to a protein monolayer. Finally, using a surface prepared as described above, biomolecular interaction experiments with an antibody against estrone were carried out to show the quality of the transducer surface. With RIfS all of the affinity-based surface modifications can be detected online and time resolved.
Target-activated streptavidin–biotin controlled binding probe
Chemical Science
The streptavidin–biotin controlled binding probe has several advantages for the detection of enzymes and reactive small molecules, such as minimal background, multiple signal amplification steps, and wide selection of the optimal dyes for detection.
Stable, High-Affinity Streptavidin Monomer for Protein Labeling and Monovalent Biotin Detection
The coupling between the quaternary structure, stability and function of streptavidin makes it difficult to engineer a stable, high affinity monomer for biotechnology applications. For example, the binding pocket of streptavidin tetramer is comprised of residues from multiple subunits, which cannot be replicated in a single domain protein. However, rhizavidin from Rhizobium etli was recently shown to bind biotin with high affinity as a dimer without the hydrophobic tryptophan lid donated by an adjacent subunit. In particular, the binding site of rhizavidin uses residues from a single subunit to interact with bound biotin. We therefore postulated that replacing the binding site residues of streptavidin monomer with corresponding rhizavidin residues would lead to the design of a high affinity monomer useful for biotechnology applications. Here, we report the construction and characterization of a structural monomer, mSA, which combines the streptavidin and rhizavidin sequences to achieve optimized biophysical properties. First, the biotin affinity of mSA (K d ¼ 2.8 nM) is the highest among nontetrameric streptavidin, allowing sensitive monovalent detection of biotinylated ligands. The monomer also has significantly higher stability (T m ¼ 59.88C) and solubility than all other previously engineered monomers to ensure the molecule remains folded and functional during its application. Using fluorescence correlation spectroscopy, we show that mSA binds biotinylated targets as a monomer. We also show that the molecule can be used as a genetic tag to introduce biotin binding capability to a heterologous protein. For example, recombinantly fusing the monomer to a cell surface receptor allows direct labeling and imaging of transfected cells using biotinylated fluorophores. A stable and functional streptavidin monomer, such as mSA, should be a useful reagent for designing novel detection systems based on monovalent biotin interaction.
Molecular Imaging of a Micropatterned Biological Ligand on an Activated Polymer Surface
We report here molecular characterization of a new method derived from reactive microcontact printings microstamping on an activated polymer surface (MAPS)swhich enables biological ligands and proteins to be patterned on a polymer surface with a spatial resolution of at least 5 µm and good reproducibility. MAPS is a multistep procedure: first, the surface of a polymer is modified, in one or more steps, to introduce a reactive group of interest. In a subsequent step, an elastomeric stamp, inked with a biological ligand containing a complementary terminal reactive group, is brought into contact with the activated surface of the polymer. This results in spatially resolved transfer and coupling of the biological ligand to the reactive surface of the polymer. We used MAPS to pattern biotin on carboxylic acid derivatized poly-(ethylene terephthalate) (PET), and subsequently with streptavidin, mediated by the high affinity streptavidin-biotin interaction. X-ray photoelectron spectroscopy of biotin-derivatized PET showed that approximately one in five PET repeat units in the top 50-100 Å were functionalized with biotin. Timeof-flight secondary ion mass spectrometry (TOF-SIMS) suggested an increased concentration of PET oligomers in the top 10 Å due to chain scission during modification and clearly identified the derivatization of PET with biotin. TOF-SIMS imaging mapped biotin and streptavidin to the stamped regions. TOF-SIMS also imaged the spatial distribution of residual reagents from the multistep derivatization in MAPS, such aspentafluorophenol,Tween20surfactant,aswellaspoly(dimethylsiloxane)(PDMS),whichwastransferred from the elastomeric PDMS stamp to the surface during MAPS.
Quantitative ToF-SIMS study of surface-immobilized streptavidin
Applied Surface Science, 2006
ToF-SIMS analysis with principal component analysis (PCA) has been used for quantitatively studying the interaction between streptavidin and biotin on a dendrimer surface. A poly(amidoamine) dendrimer surface was used as a model amine surface for biotinylation. The surface streptavidin density was systematically varied and independently quantified using the surface plasmon resonance (SPR) technique. A good linear correlation of streptavidin density was observed between the ToF-SIMS and SPR results.