Interferometric biosensor based on planar optical waveguide sensor chips for label-free detection of surface bound bioreactions

A label free optical biosensor based on a free-space Young interferometer configuration is presented. Commercial planar Ta(2) O(5) waveguides are used as sensing elements and allow the investigation of surface bound bioreactions like immunoreactions or biological affinity systems. Design criteria are discussed and a detailed characterization of the sensor performance is presented. The developed interferometer yields an effective refractive index resolution of 9 x 10(-9), corresponding to a surface coverage of approximately 13 fg/mm(2). The performance of the system is characterized by two different affinity systems: the antibody-antigen complex protein G-immunoglobulin G is used as a model system for monitoring reaction kinetics. Further measurements on a silanized surface show the formation of a streptavidin monolayer on a biotinylated surface.     

A streptavidin surface on planar glass substrates for the detection of biomolecular interaction

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 label-free, 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.     

Screening scheme based on measurement of fluorescence lifetime in the nanosecond domain

The authors demonstrate that the fluorescence lifetime of certain fluorescent labels is a useful parameter to detect affinity binding between biotin and streptavidin, as well as between biotinylated bovine serum albumin and streptavidin. The assay is performed in a microplate format, and lifetimes are determined using dye laser-induced fluorescence. Four fluorescent labels are presented that undergo a significant change in their lifetime upon affinity binding. The scheme, referred to as the fluorescence lifetime affinity assay, has several attractive features in that it requires single labeling only, represents a homogeneous assay, allows each of the 2 binding partners to be labeled, and is compatible with the standard microwell formats used in high-throughput screening.     

Construction of a high sensitive Escherichia coli alkaline phosphatase reporter system for screening affinity peptides

An enzyme-linker-peptide fusion protein reporter system was constructed for sensitive analysis of affinity of peptide ligands to their receptor. An E. coli alkaline phosphatase (EAP) mutant enzyme with high catalytic activity was selected as the reporter protein. Interaction of affinity peptide and streptavidin was applied as demonstration of the method. Three affinity peptides, strep-tag I (SI), strep-tag II (SII) and streptavidin binding peptide (SBP) were genetically fused to the C-terminal of EAP respectively, with an insertion of a flexible linker peptide in between. The enzyme activity of the EAP fusions showed no obvious change. After expression and purification, the EAP-affinity peptide fusions were applied to the streptavidin modified surface. Binding of the fusions to the surface through interaction of affinity peptides to streptavidin was indicated by color generated from conversion of the substrate by EAP. The relative affinity and specificity of each affinity peptides to the immobilized streptavidin were then evaluated with high sensitivity and broad detection range. This method may be used for effective high-throughput screening of high affinity peptide from the peptide pool.     

Construction of a high sensitive Escherichia coli alkaline phosphatase reporter system for screening affinity peptides

An enzyme-linker-peptide fusion protein reporter system was constructed for sensitive analysis of affinity of peptide ligands to their receptor. An E. coli alkaline phosphatase (EAP) mutant enzyme with high catalytic activity was selected as the reporter protein. Interaction of affinity peptide and streptavidin was applied as demonstration of the method. Three affinity peptides, strep-tag I (SI), strep-tag II (SII) and streptavidin binding peptide (SBP) were genetically fused to the C-terminal of EAP respectively, with an insertion of a flexible linker peptide in between. The enzyme activity of the EAP fusions showed no obvious change. After expression and purification, the EAP-affinity peptide fusions were applied to the streptavidin modified surface. Binding of the fusions to the surface through interaction of affinity peptides to streptavidin was indicated by color generated from conversion of the substrate by EAP. The relative affinity and specificity of each affinity peptides to the immobilized streptavidin were then evaluated with high sensitivity and broad detection range. This method may be used for effective high-throughput screening of high affinity peptide from the peptide pool.     

Porous gold surfaces for biosensor applications

The sensitivity of optical biosensors where the detection takes place on a planar gold surface can be improved by making the surface porous. The porosity allows a larger number of ligands per surface area resulting in larger optical shifts when interacting with specifically binding analyte molecules. The porous gold was deposited as a thin layer on a planar gold surface by electrochemical deposition in a solution of tetrachloroaurate and lead acetate. A protein, streptavidin, was adsorbed into the formed porous layer and the time course of the adsorption was monitored by in-situ ellipsometry. When the porous layer was 500 nm in thickness a six-fold increase of the ellipsometric response was obtained compared with a planar gold surface. The dependency of porosity and layer thickness was explained with a mathematical model of the gold/porous gold/protein/solution system.     

Dual-polarization interferometry: an analytical technique to measure changes in protein structure in real time, to determine the stoichiometry of binding events, and to differentiate between specific and nonspecific interactions

The study of solution-phase interactions between small molecules and immobilized proteins is of intense interest, especially to the pharmaceutical industry. An optical sensing technique, dual polarization interferometry, has been employed for the detailed study of a model protein system, namely, d-biotin interactions with streptavidin immobilized on a solid surface. Changes in thickness and density of an immobilized streptavidin layer as a result of the binding of d-biotin have been directly measured in solution and in real time. The results obtained from this approach are in excellent agreement with X-ray crystallographic data for the structural changes expected in the streptavidin-D-biotin system. The mass changes measured on binding d-biotin also agree closely with anticipated binding capacity values. Determination of the density changes occurring in the protein adlayer provides a means for differentiation between specific and nonspecific interactions.     

Genetic engineering of streptavidin,a versatile affinity tag

Streptavidin, a tetrameric protein produced by Streptomyces avidinii, has been used as a useful, versatile affinity tag in a variety of biological applications. The efficacy of streptavidin is derived from its extremely high binding affinity for the vitamin biotin. For the last several years, we have used genetic engineering as a primary means to enhance the properties of streptavidin and to expand the application of streptavidin as an affinity tag. In this review, we describe several genetically engineered streptavidin variants, which include a streptavidin with a reduced biotin-binding affinity, a dimeric streptavidin, and a fusion protein between streptavidin and protein A, along with their potential applications in biological science.     

Love–Hate ligands for high resolution analysis of strain in ultra-stable protein/small molecule interaction

The pathway of ligand dissociation and how binding sites respond to force are not well understood for any macromolecule. Force effects on biological receptors have been studied through simulation or force spectroscopy, but not by high resolution structural experiments. To investigate this challenge, we took advantage of the extreme stability of the streptavidin–biotin interaction, a paradigm for understanding non-covalent binding as well as a ubiquitous research tool. We synthesized a series of biotin-conjugates having an unchanged strong-binding biotin moiety, along with pincer-like arms designed to clash with the protein surface: ‘Love–Hate ligands’. The Love–Hate ligands contained various 2,6-di-ortho aryl groups, installed using Suzuki coupling as the last synthetic step, making the steric repulsion highly modular. We determined binding affinity, as well as solving 1.1–1.6 Å resolution crystal structures of streptavidin bound to Love–Hate ligands. Striking distortion of streptavidin’s binding contacts was found for these complexes. Hydrogen bonds to biotin’s ureido and thiophene rings were preserved for all the ligands, but biotin’s valeryl tail was distorted from the classic conformation. Streptavidin’s L3/4 loop, normally forming multiple energetically-important hydrogen bonds to biotin, was forced away by clashes with Love–Hate ligands, but Ser45 from L3/4 could adapt to hydrogen-bond to a different part of the ligand. This approach of preparing conflicted ligands represents a direct way to visualize strained biological interactions and test protein plasticity.     

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 = 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 = 59.8°C) 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. Biotechnol. Bioeng. 2013; 110: 57–67. © 2012 Wiley Periodicals, Inc.     

Immobilization of biomolecules on polyurethane membrane surfaces.

Lipophilic polymer membranes incorporating binding sites are widely used in various potentiometric, amperometric, and optical sensors. Here, we report on the biofunctional modification of the surface of a Ca(2+)-selective membrane. A photoactivatable biotin derivative was synthesized and covalently immobilized on a soft polyurethane membrane. The modified polymer was characterized by X-ray photoelectron spectroscopy (XPS) as well as by potentiometric measurements. The selective binding of streptavidin by the photo-cross-linked biotin derivative was demonstrated. The surface coverage obtained with different experimental protocols was analyzed by autoradiography using [(35)S]-streptavidin. The new approach may significantly extend the scope of applicability of potentiometric sensors.     

A review of specialty fiber biosensors based on interferometer configuration

Optical fiber biosensors have attracted extensive research attention in fields such as public health research, environmental science, bioengineering, disease diagnosis and drug research. Accurate detection of biomolecules is essential to limit the extent of disease outbreaks and provide valuable guidance for regulatory agencies to take timely measures. Among many optical fiber sensors, optical fiber biosensors based on specialty fibers have the advantages of biocompatibility, small size, high measurement resolution, high stability and immunity to electromagnetic interference. In this paper, four types interferometer biosensors based on specialty fiber, namely Mach-Zehnder interferometer, Michelson interferometer, Fabry - Perot interferometer and Sagnac interferometer, are reviewed in terms of operating principles, sensing structure and application fields. The fiber types are further divided into micro-nano optical fiber, thin core fiber, polarization maintaining fiber, polymer fiber, microstructure optical fiber. Furthermore, this paper evaluates the advantages and disadvantages of these interferometer biosensors. Finally, main challenging problems and expectational development direction of specialty fiber interferometer biosensors are summarized. This text clearly shows the huge development potential of optical fiber biosensors in biomedical.     

Engineered streptavidin monomer and dimer with improved stability and function

Although streptavidin's high affinity for biotin has made it a widely used and studied binding protein and labeling tool, its tetrameric structure may interfere with some assays. A streptavidin mutant with a simpler quaternary structure would demonstrate a molecular-level understanding of its structural organization and lead to the development of a novel molecular reagent. However, modulating the tetrameric structure without disrupting biotin binding has been extremely difficult. In this study, we describe the design of a stable monomer that binds biotin both in vitro and in vivo. To this end, we constructed and characterized monomers containing rationally designed mutations. The mutations improved the stability of the monomer (increase in T(m) from 31 to 47 °C) as well as its affinity (increase in K(d) from 123 to 38 nM). We also used the stability-improved monomer to construct a dimer consisting of two streptavidin subunits that interact across the dimer-dimer interface, which we call the A/D dimer. The biotin binding pocket is conserved between the tetramer and the A/D dimer, and therefore, the dimer is expected to have a significantly higher affinity than the monomer. The affinity of the dimer (K(d) = 17 nM) is higher than that of the monomer but is still many orders of magnitude lower than that of the wild-type tetramer, which suggests there are other factors important for high-affinity biotin binding. We show that the engineered streptavidin monomer and dimer can selectively bind biotinylated targets in vivo by labeling the cells displaying biotinylated receptors. Therefore, the designed mutants may be useful in novel applications as well as in future studies in elucidating the role of oligomerization in streptavidin function.     

Streptavidin contains an RYD sequence which mimics the RGD receptor domain of fibronectin

Streptavidin binds at low levels and high affinity to cell surfaces, the cause of which can be traced to the occurrence of a sequence containing RYD (Arg-Tyr-Asp) in the protein molecule. This binding is enhanced in the presence of biotin. Cell-bound streptavidin can be displaced by fibronectin, as well as by RGD- and RYD-containing peptides. In addition, streptavidin can displace fibronectin from cell surfaces. The RYD sequence of streptavidin thus mimics RGD (Arg-Gly-Asp), the universal recognition domain present in fibronectin and other adhesion-related molecules. The observed adhesion to cells has no relevance to biotin-binding since the RYD sequence is not part of the biotin-binding site of streptavidin. Since the use of streptavidin in avidin-biotin technology is based on its biotin-binding properties, researchers are hereby warned against its indiscriminate use in histochemical and cytochemical studies.     

A monovalent streptavidin with a single femtomolar biotin binding site

Streptavidin and avidin are used ubiquitously because of the remarkable affinity of their biotin binding, but they are tetramers, which disrupts many of their applications. Making either protein monomeric reduces affinity by at least 10(4)-fold because part of the binding site comes from a neighboring subunit. Here we engineered a streptavidin tetramer with only one functional biotin binding subunit that retained the affinity, off rate and thermostability of wild-type streptavidin. In denaturant, we mixed a streptavidin variant containing three mutations that block biotin binding with wild-type streptavidin in a 3:1 ratio. Then we generated monovalent streptavidin by refolding and nickel-affinity purification. Similarly, we purified defined tetramers with two or three biotin binding subunits. Labeling of site-specifically biotinylated neuroligin-1 with monovalent streptavidin allowed stable neuroligin-1 tracking without cross-linking, whereas wild-type streptavidin aggregated neuroligin-1 and disrupted presynaptic contacts. Monovalent streptavidin should find general application in biomolecule labeling, single-particle tracking and nanotechnology.     

High resolution structure of streptavidin in complex with a novel high affinity peptide tag mimicking the biotin binding motif

A novel peptide was designed which possesses nanomolar affinity of less than 20 nM for streptavidin. Therefore it was termed Nano-tag and has been used as an affinity tag for recombinant proteins. The minimized version of the wild type Nano-tag is a seven-amino acid peptide with the sequence fMDVEAWL. The three-dimensional structure of wild type streptavidin in complex with the minimized Nano-tag was analyzed at atomic resolution of 1.15 A and the details of the binding motif were investigated. The peptide recognizes the same pocket of streptavidin where the natural ligand biotin is bound, but the peptide requires significantly more space than biotin. Therefore the binding loop adopts an "open" conformation in order to release additional space for the peptide. The conformation of the bound Nano-tag corresponds to a 3(10) helix. However, the analysis of the intermolecular interactions of the Nano-tag with residues of the binding pocket of streptavidin reveals astonishing similarities to the biotin binding motif. In principle the three-dimensional conformation of the Nano-tag mimics the binding mode of biotin. Our results explain why the use of the Nano-tag in fusion with recombinant proteins is restricted to their N-terminus and we describe the special significance of the fMet residue for the high affinity binding mode.     

Structure-Guided Design of an Engineered Streptavidin with Reusability to Purify Streptavidin-Binding Peptide Tagged Proteins or Biotinylated Proteins

Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop_3–4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (K_d) from 4×10⁻¹⁴ M to 4.45×10⁻¹⁰ M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible.     

Optimisation of a multivalent Strep tag for protein detection

The Strep tag is a peptide sequence that is able to mimic biotin's ability to bind to streptavidin. Sequences of Strep tags from 0 to 5 have been appended to the N-terminus of a model protein, the Stefin A Quadruple Mutant (SQM) peptide aptamer scaffold, and the recombinant fusion proteins expressed. The affinities of the proteins for streptavidin have been assessed as a function of the number of tags inserted using a variety of labelled and label-free bioanalytical and surface based methods (Western blots, microarray assays and surface plasmon resonance spectroscopy). The binding affinity increases with the number of tags across all assays, reaching nanomolar levels with 5 inserts, an observation assigned to a progressive increase in the probability of a binding interaction occurring. In addition a novel interfacial FRET based assay has been developed for generic Strep tag interactions, which utilises a conventional microarray scanner and bypasses the requirement for expensive lifetime imaging equipment. By labelling both the tagged StrepX-SQM(2) and streptavidin targets, the conjugate is primed for label-free FRET based displacement assays.     

Conservative secondary structure motif of streptavidin-binding aptamers generated by different laboratories

Aptamers that are selected in vitro from random pools of DNA or RNA molecules by SELEX (Systematic evolution of ligands by exponential enrichment) technique have been extensively explored for analytical and biomedical applications. Although many aptamers with high affinity and specificity against specific ligands have been reported, there is still a lack of well characterized DNA aptamers. Here we report the selection of a group of aptamer candidates (85 mer) against streptavidin. Through comparing the predicted secondary structures of all the candidates, a conservative bulge-hairpin structure section (about 29 mer) was found, and then it was determined to be the binding motif to streptavidin. This binding motif was further discovered to also exist in streptavidin-binding aptamers (SBAs) selected by three other laboratories using different methods. The primary sequences of this secondary structure motif are very different, only several nucleotides in the loop and bulge area are critical for binding and other nucleotides are variable. The streptavidin binding of all the SBAs could be competed by biotin implying that they bind to the same site on streptavidin. These results suggest that the evolution of SBA is predominated by specific groups on streptavidin. The highly variable sequence composition of streptavidin-binding aptamer would make the design of aptameric sensor or device based on streptavidin more flexible and easy.     

Amplified surface plasmon resonance immunosensor for interferon-gamma based on a streptavidin-incorporated aptamer

Interferon-gamma (IFN-γ) is associated with susceptibility to tuberculosis, which is a major public health problem worldwide. Although significant progress has been made with regard to the design of enzyme immunoassays for IFN-γ, this assay is still labor-intensive and time-consuming. We therefore designed a DNA aptamer hairpin structure for the detection of IFN-γ with high sensitivity and selectivity. A streptavidin DNA aptamer was incorporated into the IFN-γ binding aptamer probe for the amplified detection of the target molecules. Initially, the probe remained in the inactive configuration. The addition of IFN-γ induced the rearrangement of the aptamer structure, allowing the self-assembly of the active streptavidin aptamer conformation for the streptavidin molecular recognition. Under optimized conditions, the detection limit was determined to be 33 pM, with a dynamic range from 0.3 to 333 nM, both of which were superior to those of corresponding optical sensors. Because combined aptamers are composed of nucleic acids, this optical aptasensor provided the advantages of high sensitivity, simplicity, reusability, and no further labeling or sample pre-treatment.