Streptavidin Monolayer Research Articles

Streptavidin Coverage on Biotinylated Surfaces.

Biosensors and other biological platform technologies require the functionalization of their surface with receptors to enhance affinity and selectivity. Control over the functionalization density is required to tune the platform’s properties. Streptavidin (SAv) monolayers are widely used to immobilize biotinylated proteins, receptors, and DNA. The SAv density on a surface can be varied easily, but the predictability is dependent on the method by which the SAv is immobilized. In this study we show a method to quantitatively predict the SAv coverage on biotinylated surfaces. The method is validated by measuring the SAv coverage on supported lipid bilayers with a range of biotin contents and two different main phase lipids and by using quartz crystal microbalance and localized surface plasmon resonance. We explore a predictive model of the biotin-dependent SAv coverage without any fit parameters. Model and data allow to predict the SAv coverage based on the biotin coverage, in both the low- and high-density regimes. This is of special importance in applications with multivalent binding where control over surface receptor density is required, but a direct measurement is not possible.

A Stable Biotin-Streptavidin Surface Enables Multiplex, Label-Free Protein Detection by Aptamer and Aptamer-Protein Arrays Using Arrayed Imaging Reflectometry.

While label-free multiplex sensor technology enables “mixing and matching” of different capture molecules in principle, in practice this has been rarely (if ever) demonstrated. To fill this gap, we developed protocols for the preparation of mixed aptamer-protein arrays on the arrayed imaging reflectometry (AIR) sensing platform using streptavidin as a common attachment point for both biotinylated proteins and aptamers. Doing so required overcoming the noted instability of dried streptavidin monolayers on surfaces. After characterizing this degradation, stable surfaces were obtained using a commercial microarray product. Microarraying through the layer of stabilizer then provided mixed aptamer-antibody arrays. We demonstrate that sensor arrays prepared in this manner are suitable for several probes (thrombin and TGF-β1 aptamers; avi-tagged protein) and targets.

Streptavidin Affinity Grids for cryo-EM

The preparation of specimen grids for cryo-EM often proves to be difficult because of unwanted factors such as preferential orientation of particles, too few particles being seen within holes, particle disruption occurring within thin aqueous films, and unexpected aggregation of sample material. In instances where it is suspected that those difficulties might be caused by interaction with the air-water interface, a possible solution is to immobilize particles on a structure-friendly support film, thus preventing diffusion to the air-water interface of the thin aqueous film that remains after blotting away excess sample. We have developed a form of EM grid on which streptavidin-monolayer crystals serve as an affinity support-film. A thin covering of trehalose is used to embed the streptavidin monolayers during storage, thereby preserving the crystalline structure and biotin-binding function of the streptavidin. These affinity grids can thus be prepared in any desired quantity and stored for future use. Many strategies are possible for immobilizing particles by affinity-binding to the streptavidin monolayer after washing off the protective layer of trehalose. One of the simplest is to randomly biotinylate one or two surface-lysine residues on particles of interest. Advantages of these grids include: (1) a good surface density of immobilized particles is obtained rapidly at sample concentrations as low as 50 nM; (2) the streptavidin component of the image serves as an internal standard to verify sample preservation and image quality; and (3) the streptavidin “background” in the image can be removed, whenever desired, by Fourier filtering.

Wide-Field Fluorescence Microscopy of Real-Time Bioconjugation Sensing.

We apply wide-field fluorescence microscopy to measure real-time attachment of photosynthetic proteins to plasmonically active silver nanowires. The observation of this effect is enabled, on the one hand, by sensitive detection of fluorescence and, on the other hand, by plasmonic enhancement of protein fluorescence. We examined two sample configurations with substrates being a bare glass coverslip and a coverslip functionalized with a monolayer of streptavidin. The different preparation of the substrate changes the observed behavior as far as attachment of the protein is concerned as well as its subsequent photobleaching. For the latter substrate the conjugation process is measurably slower. The described method can be universally applied in studying protein-nanostructure interactions for real-time fluorescence-based sensing.

Streptavidin-coated surfaces suppress bacterial colonization by inhibiting non-specific protein adsorption.

Streptavidin is a 58 kDa tetrameric protein with the highest known affinity to biotin with a wide range of applications in bionanotechnology and molecular biology. Dissolved streptavidin is stable at a broad range of temperature, pH, proteolytic enzymes and exhibits low non-specific binding. In this study, a streptavidin monolayer was assembled directly on a biotinylated TiO2 -surface to investigate its stability against proteolytic digestion and its suppression of initial bacterial adsorption of Escherichia coli, Bacillus subtilis, and Streptococcus intermedius. In contrast to nonmodified TiO2 surfaces, streptavidin-coated substrates showed only a negligible non-specific protein adsorption at physiological protein concentrations as well as a significantly reduced bacterial adhesion. The antiadhesive properties were demonstrated to be the main reason for the suppression of bacterial adhesion, which makes this approach a promising option for future surface biofunctionalization applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 758-768, 2018.

Catalyst-free “click” functionalization of polymer brushes preserves antifouling properties enabling detection in blood plasma

Progress in biosensors for clinical detection critically relies on modifications of the transducer surface to prevent non-specific adsorption from matrix components (i.e. antifouling) while supporting biomolecular recognition elements to capture the analyte. Such combination of properties presents a significant challenge. Hierarchically structured polymer brushes comprising an antifouling polymer bottom block and a functionalizable top block are proposed as a promising strategy to achieve this goal. We employed the catalyst-free strain-promoted alkyne–azide cycloaddition (SPAAC) “click” reaction to biofunctionalize antifouling polymer brushes without impairing their resistance to fouling. The functionalization was performed on the side chains along the top polymer block or only on the end-groups of the polymer brush. The immobilized amounts of bioreceptors (streptavidin followed by biotin-conjugated proteins) and the resistance to fouling from blood plasma of the surfaces obtained were evaluated via surface plasmon resonance. The end group functionalization approach resulted in very low immobilization of bioreceptor. On the other hand, the side group modification of a top polymer block led to immobilization of 83% of a monolayer of streptavidin. Following binding of a biotin-conjugated antibody (66 ng cm−2) the functionalized layer was able to reduce the fouling from undiluted human blood plasma by 89% in comparison with bare gold. Finally, the functionalized hierarchical polymer brushes were applied to the label-free detection of a model analyte in diluted human blood plasma, highlighting the potential for translation to medical applications.

Monolayer-crystal streptavidin support films provide an internal standard of cryo-EM image quality.

Analysis of images of biotinylated Escherichia coli 70S ribosome particles, bound to streptavidin affinity grids, demonstrates that the image-quality of particles can be predicted by the image-quality of the monolayer crystalline support film. The quality of the Thon rings is also a good predictor of the image-quality of particles, but only when images of the streptavidin crystals extend to relatively high resolution. When the estimated resolution of streptavidin was 5Å or worse, for example, the ribosomal density map obtained from 22,697 particles went to only 9.5Å, while the resolution of the map reached 4.0Å for the same number of particles, when the estimated resolution of streptavidin crystal was 4Å or better. It thus is easy to tell which images in a data set ought to be retained for further work, based on the highest resolution seen for Bragg peaks in the computed Fourier transforms of the streptavidin component. The refined density map obtained from 57,826 particles obtained in this way extended to 3.6Å, a marked improvement over the value of 3.9Å obtained previously from a subset of 52,433 particles obtained from the same initial data set of 101,213 particles after 3-D classification. These results are consistent with the hypothesis that interaction with the air-water interface can damage particles when the sample becomes too thin. Streptavidin monolayer crystals appear to provide a good indication of when that is the case.

Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules.

We describe a rapid and convenient method of growing streptavidin (SA) monolayer crystals directly on holey-carbon EM grids. As expected, these SA monolayer crystals retain their biotin-binding function and crystalline order through a cycle of embedding in trehalose and, later, its removal. This fact allows one to prepare, and store for later use, EM grids on which SA monolayer crystals serve as an affinity substrate for preparing specimens of biological macromolecules. In addition, we report that coating the lipid-tail side of trehalose-embedded monolayer crystals with evaporated carbon appears to improve the consistency with which well-ordered, single crystals are observed to span over entire, 2μm holes of the support films. Randomly biotinylated 70S ribosomes are used as a test specimen to show that these support films can be used to obtain a high-resolution cryo-EM structure.

Well-defined biomimetic surfaces to characterize glycosaminoglycan-mediated interactions on the molecular, supramolecular and cellular levels

Glycosaminoglycans (GAGs) are ubiquitously present at the cell surface and in extracellular matrix, and crucial for matrix assembly, cell–cell and cell-matrix interactions. The supramolecular presentation of GAG chains, along with other matrix components, is likely to be functionally important but remains challenging to control and to characterize, both in vivo and in vitro. We present a method to create well-defined biomimetic surfaces that display GAGs, either alone or together with other cell ligands, in a background that suppresses non-specific binding. Through the design of the immobilization platform – a streptavidin monolayer serves as a molecular breadboard for the attachment of various biotinylated ligands – and a set of surface-sensitive in situ analysis techniques (including quartz crystal microbalance and spectroscopic ellipsometry), the biomimetic surfaces are tailor made with tight control on biomolecular orientation, surface density and lateral mobility. Analysing the interactions between a selected GAG (heparan sulphate, HS) and the HS-binding chemokine CXCL12α (also called SDF-1α), we demonstrate that these surfaces are versatile for biomolecular and cellular interaction studies. T-lymphocytes are found to adhere specifically to surfaces presenting CXCL12α, both when reversibly bound through HS and when irreversibly immobilized on the inert surface, even in the absence of any bona fide cell adhesion ligand. Moreover, surfaces which present both HS-bound CXCL12α and the intercellular adhesion molecule 1 (ICAM-1) synergistically promote cell adhesion. Our surface biofunctionalization strategy should be broadly applicable for functional studies that require a well-defined supramolecular presentation of GAGs along with other matrix or cell-surface components.

Hybridization of Fano and Vibrational Resonances in Surface-Enhanced Infrared Absorption Spectroscopy of Streptavidin Monolayers on Metamaterial Substrates

We present spectral hybridization of organic and inorganic resonant materials and related bio-sensing mechanism. We utilized a bound protein (streptavidin) and a Fano-resonant metasurface to illustrate the concept. The technique allows us to investigate the vibrational modes of the streptavidin and how they couple to the underlying metasurface. This optical, label-free, nonperturbative technique is supported by a coupled mode-theory analysis that provides information on the structure and orientation of bound proteins. We can also simultaneously monitor the binding of analytes to the surface through monitoring the shift of the metasurface resonance. All of this data opens up interesting opportunities for applications in biosensing, molecular electronics and proteomics.

Bimetallic gold–silver nanoplate array as a highly active SERS substrate for detection of streptavidin/biotin assemblies

The silver-modified gold nanoplate arrays as bimetallic surface-enhanced Raman scattering (SERS) substrates were optimized for the surface-enhanced Raman detection of streptavidin/biotin monolayer assemblies. The bimetallic gold–silver nanoplate arrays were fabricated by coating silver nanoparticles uniformly on the gold nanoplate arrays. Depending on silver nanoparticle coating, the localized surface plasmon resonance (LSPR) peak of the bimetallic gold–silver nanoplate arrays blue-shifted and broadened significantly. The common probe molecule, Niel Blue A sulfate (NBA) was used for testing the SERS activity of the bimetallic gold–silver nanoplate arrays. The SERS intensity increased with the silver nanoparticle coating, due to a large number of hot spots and nanoparticle interfaces. The platforms were tested against a monolayer of streptavidin functionalized over the bimetallic gold–silver nanoplate arrays showing that good quality spectra could be acquired with a short acquisition time. The supramolecular interaction between streptavidin (strep) and biotin showed subsequent modification of Raman spectra that implied a change of the secondary structure of the host biomolecule. And the detection concentration for biotin by this method was as low as 1.0nM. The enhanced SERS performance of such bimetallic gold–silver nanoplate arrays could spur further interest in the integration of highly sensitive biosensors for rapid, nondestructive, and quantitative bioanalysis, particularly in microfluidics.

Metal-Enhanced Fluorescence Lifetime Imaging and Spectroscopy on a Modified SERS Substrate

In this paper, we developed a metal-enhanced fluorescence (MEF) substrate by modification of the commercially available surface enhanced Raman spectroscopy (SERS) substrate that may meet the reproducibility and sensitivity challenge of MEF. In spite of many studies and interest on MEF from a number of research groups, application to real-world situations and its commercial use remain challenging mainly due to the difficulties in fabricating reproducible MEF substrates. Specifically, one of the challenges is achieving a standardized MEF substrate for reproducible fluorescence intensity enhancement and/or changes in lifetime. The gold standard klarite substrates for SERS were coated with a thin layer of silver nanoparticles for MEF studies. To test the newly developed MEF substrates, a monolayer of streptavidin conjugated Alexa-647 was assembled on biotinylated-glass or MEF substrates. We observed over 50-fold increase in the fluorescence intensity from a monolayer of streptavidin conjugated Alexa-647 on the biotinylated MEF substrate compared to the same on glass substrate. A significant reduction in the lifetime and increased photostability of Alexa-647 on MEF substrate was observed. Fluorescence lifetime imaging was performed on the monolayer of dye assembled on the modified SERS substrates. We expect this study will serve as a platform to encourage the future use of a standardized MEF substrate for a plethora of sensing applications.

Imidazolium-Based Ionic Liquid Surfaces for Biosensing

Ionic liquid self-assembled monolayers (SAM) were designed and applied for binding streptavidin, promoting affinity biosensing and enzyme activity on gold surfaces of sensors. The synthesis of 1-((+)-biotin)pentanamido)propyl)-3-(12-mercaptododecyl)-imidazolium bromide, a biotinylated ionic liquid (IL-biotin), which self-assembles on gold film, afforded streptavidin sensing with surface plasmon resonance (SPR). The IL-biotin-SAM efficiently formed a full streptavidin monolayer. The synthesis of 1-(carboxymethyl)-3-(mercaptododecyl)-imidazoliumbromide, a carboxylated IL (IL-COOH), was used to immobilize anti-IgG to create an affinity biosensor. The IL-COOH demonstrated efficient detection of IgG in the nanomolar concentration range, similar to the alkylthiols SAM and PEG. In addition, the IL-COOH demonstrated low fouling in crude serum, to a level equivalent to PEG. The IL-COOH was further modified with N,N'-bis (carboxymethyl)-l-lysine hydrate to bind copper ions and then, chelate histidine-tagged biomolecules. Human dihydrofolate reductase (hDHFR) was chelated to the modified IL-COOH. By monitoring enzyme activity in situ on the SPR sensor, it was revealed that the IL-COOH SAM improved the activity of hDHFR by 24% in comparison to classical SAM. Thereby, IL-SAM has been synthesized and successfully applied to three important biosensing schemes, demonstrating the advantages of this new class of monolayers.

Photonic crystal biosensor based on optical surface waves.

A label-free biosensor device based on registration of photonic crystal surface waves is described. Angular interrogation of the optical surface wave resonance is used to detect changes in the thickness of an adsorbed layer, while an additional simultaneous detection of the critical angle of total internal reflection provides independent data of the liquid refractive index. The abilities of the device are demonstrated by measuring of biotin molecule binding to a streptavidin monolayer, and by measuring association and dissociation kinetics of immunoglobulin G proteins. Additionally, deposition of PSS/PAH polyelectrolytes is recorded in situ resulting calculation of PSS and PAH monolayer thicknesses separately.

Layer-by-Layer Assembly of a Streptavidin–Fibronectin Multilayer on Biotinylated TiOX

The biomodification of surfaces, especially titanium, is an important issue in current biomedical research. Regarding titanium, it is also important to ensure a specific protein modification of its surface because here protein binding that is too random can be observed. Specific nanoscale architectures can be applied to overcome this problem. As recently shown, streptavidin can be used as a coupling agent to immobilize biotinylated fibronectin (bFn) on a TiO(X) surface. Because of the conformation of adsorbed biotinylated fibronectin on a streptavidin monolayer, it is possible to adsorb more streptavidin and biotinylated fibronectin layers. On this basis, an alternating protein multilayer can be built up. In contrast to common layer-by-layer technology, in this procedure the mechanism of layer adsorption is very specific because of the interaction of biotin and streptavidin. In addition, we showed that the assembly of this multilayer system and its stability are dependent on the degree of labeling of biotinylated fibronectin. Hence we conclude that it is possible to build up well-defined nanoscale protein architectures by varying the degree of labeling of biotinylated fibronectin.

Electron microscopy of biotinylated protein complexes bound to streptavidin monolayer crystals

Chemical biotinylation of protein complexes followed by binding to two-dimensional (monolayer) crystals of streptavidin is shown to be an effective way to prepare cryo-EM specimens from samples at low protein concentration. Three different multiprotein complexes are used to demonstrate the generality of this method. In addition, native thermosomes, purified from Sulfolobus solfataricus P2, are used to demonstrate that a uniform distribution of Euler angles is produced, even though this particle is known to adopt a preferred orientation when other methods of cryo-EM specimen preparation are used.

Cryo EM Study of Protein Complexes Bound to Streptavidin Crystal Layers

Conventional cryo-EM specimens use either continuous or holey, glow-discharge treated carbon films to prepare samples. As a result, particles are either immobilized by binding to an unnatural (i.e. carbon-film) surface, or they collide multiple times (due to rapid diffusion) with the freshly blotted air-water interface. In either case there is a risk that considerable structural heterogeneity may occur, or even that particles may undergo severe denaturation.Binding biotinylated particles to streptavidin monolayer crystals is a more structure-friendly way to immobilize particles. We have developed a protocol for preparing biotinylated protein complexes and tethering them to the surface of streptavidin monolayer-crystals. Three different multi-protein complexes have been used to demonstrate the generality of this method. The biotinylated protein complexes bind to the streptavidin crystal layer with high affinity, whereas the unbiotinylated control samples do not do so. In addition, the same technique can be used to produce a uniform distribution of Euler angles. The thermosomes, known to adopt a preferred orientation with other methods of cryo-EM specimen preparation, have been used as a test sample. Indeed, the results confirm that a uniform distribution of Euler angles is obtained for the biotinylated thermosome. We thus conclude that biotinylation, together with binding to 2-D crystals of streptavidin, is a better method to prepare protein complexes for cryo-EM study.

A comparative evaluation of microarray slides as substrates for the development of protease assay biosensors

The application of commercially available microarray slides as substrates for fluorogenic protease assays has been explored in terms of binding efficiency, stability, and activity. A fluorescent, biotinylated substrate for botulinum neurotoxin A (BoNTA) was attached via self-assembled monolayer of Streptavidin to amine-reactive aldehyde, epoxy, hydrogel, and polymer slides. Nexterion Slide P® was found to have optimal protein binding efficiency and stability of the slides examined. Addition of glycerol to the printing buffer improved spot morphology significantly and polyvinylpyrrolidone provided long-term stability, allowing chips to be stored for up to 1 month with good viability. Detection of a recombinant BoNTA light chain was then carried out at 37 °C and a sub-lethal dose was detected in 2 hours.

Adsorption and Conformation Behavior of Biotinylated Fibronectin on Streptavidin-Modified TiOX Surfaces Studied by SPR and AFM

It is well-known that protein-modified implant surfaces such as TiO(2) show a higher bioconductivity. Fibronectin is a glycoprotein from the extracellular matrix (ECM) with a major role in cell adhesion. It can be applied on titanium oxide surfaces to accelerate implant integration. Not only the surface concentration but also the presentation of the protein plays an important role for the cellular response. We were able to show that TiO(X) surfaces modified with biotinylated fibronectin adsorbed on a streptavidin-silane self-assembly multilayer system are more effective regarding osteoblast adhesion than surfaces modified with nonspecifically bound fibronectin. The adsorption and conformation behavior of biotinylated and nonbiotinylated (native) fibronectin was studied by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM). Imaging of the protein modification revealed that fibronectin adopts different conformations on nonmodified compared to streptavidin-modified TiO(X) surfaces. This conformational change of biotinylated fibronectin on the streptavidin monolayer delivers a fibronectin structure similar to the conformation inside the ECM and therefore explains the higher cell affinity for these surfaces.

Silicon-on-insulator guided mode resonant grating for evanescent field molecular sensing

We present experimental and theoretical results of label-free molecular sensing using the transverse magnetic mode of a 0.22 mum thick silicon slab waveguide with a surface grating implemented in a guided mode resonance configuration. Due to the strong overlap of the evanescent field of the waveguide mode with a molecular layer attached to the surface, these sensors exhibit high sensitivity, while their fabrication and packaging requirements are modest. Experimentally, we demonstrate a resonance wavelength shift of approximately 1 nm when a monolayer of the protein streptavidin is attached to the surface, in good agreement with calculations based on rigorous coupled wave analysis. In our current optical setup this shift corresponds to an estimated limit of detection of 0.2% of a monolayer of streptavidin.