A method of binding kinetics of a ligand to micropatterned proteins on a microfluidic chip

A combination of microfluidic protein patterning and quantitative microfluidic handling has been used to analyze the binding kinetics of protein-ligand interactions on the nanoliter scale. The microfluidic handling method employing hydrophobic valving and pneumatic control allowed us to control nanoliter volumes of ligand or protein on a microfluidic chip. A hydrophobic and inert fluorocarbon thin film was patterned on a silicon nitride substrate to prevent non-specific binding on the background. Selectively patterned protein patterns of various sizes were used for quantitative analysis of the kinetic parameters of immobilized proteins on the circular patterns. As a model system, a streptavidin-patterned array of the same-sized pattern, i.e. 150 microm diameter, was used to capture FITC-BSA-biotin present in solution. The fluorescence intensity was well matched with the Langmuir isotherm model results, showing a dissociation constant of 2.43x10(-8)M. Similar streptavidin arrays with different-sized spots, ranging from 50 to 200 microm, showed a consistent dissociation constant of FITC-BSA-biotin with streptavidin pattern. Therefore, the reduction of pattern size of an immobilized protein did not change the dissociation rate of the ligand.     

Development of AC microelectrophoresis for rapid protein affinity evaluation

We have developed a new method for evaluating the affinity interactions between two different proteins by applying an alternating current (AC) voltage to a micro-flow channel. An AC voltage was applied to the protein-modified microspheres in the micro-flow channel, which resulted in the oscillation of the microspheres owing to their surface charges. The oscillation amplitude showed a linear relationship with the charge density of the microspheres. As an example for protein affinity measurement, the amplitude changes of a profilin-modified microsphere were measured by the addition of actin. In the same electrical condition, the oscillation amplitude of the profilin-modified microsphere increased by ≈175% by binding with actin. Similar results in the principle were obtained for the affinity interaction between biotin and streptavidin. The results showed that the higher the charge density of the microspheres induced by binding with different proteins, the higher the oscillation amplitude of the microspheres, thus, suggesting a possible application of the micro-flow channel and AC voltage on the protein property study, as well as on the biosensor application using the oscillation amplitude changes.     

X-ray crystallographic studies of streptavidin mutants binding to biotin

On the basis of high resolution crystallographic studies of streptavidin and its biotin complex, three principal binding motifs have been identified that contribute to the tight binding. A flexible binding loop can undergo a conformational change from an open to a closed form when biotin is bound. Additional studies described here of unbound wild-type streptavidin have provided structural views of the open conformation. Several tryptophan residues packing around the bound biotin constitute the second binding motif, one dominated by hydrophobic interactions. Mutation of these residues to alanine or phenylalanine have variable effects on the thermodynamics and kinetics of binding, but they generate only small changes in the molecular structure. Hydrogen bonding interactions also contribute significantly to the binding energetics of biotin, and the D128A mutation which breaks a hydrogen bond between the protein and a ureido NH group results in a significant structural alteration that could mimic an intermediate on the dissociation pathway. In this review, we summarize the structural aspects of biotin recognition that have been gained from crystallographic analyses of wild-type and site-directed streptavidin mutants.     

Evaluation of two- and three-dimensional streptavidin binding platforms for surface plasmon resonance spectroscopy studies of DNA hybridization and protein-DNA binding

Surface plasmon resonance (SPR) spectroscopy has been used to study DNA assembly, DNA hybridization, and protein-DNA interactions on two streptavidin (SA) sensor chips. On one chip, SA molecules are immobilized on a biotin-exposed surface, forming an ordered two-dimensional (2D) SA monolayer. The other chip, BIAcore's SA chip, contains SA molecules immobilized within a three-dimensional (3D) carboxylated dextran matrix. Compared to the 2D chip, the 3D SA matrix allows for a slower immobilization rate of biotinylated DNA due to diffusion limitation in the dextran matrix, but with twice the amount of the immobilized DNA due to the greater number of reactive sites, which in turn enables a higher sensitivity for DNA hybridization detection. Interestingly, having a greater DNA probe dispersion in the 3D matrix does not induce a higher DNA hybridization efficiency. In a study of protein binding to immobilized DNA (estrogen receptor to estrogen response elements), aiming at assessing the DNA sequence dependent protein binding behavior, the 2D and 3D chips produce different binding characteristics. On the 2D chip, the protein binding exhibits a better selectivity to the specific sequences, regardless of binding stringency (e.g. salt concentration), whereas on the 3D chip, the liquid handling system needs to be optimized in order to minimize transport limitations and to detect small affinity differences. Through this study we demonstrate that the physicochemical structure of SPR chips affects the apparent binding behaviors of biomolecules. When interpreting SPR binding curves and selecting a sensor chip, these effects should be taken into account.     

Investigation of biotin-streptavidin binding interactions using microcantilever sensors

We report the investigation of biotin-streptavidin binding interactions using microcantilever sensors. A symmetric cantilever construction is employed to minimize the effects of thermal drift and the control of surface chemistry on the backside of the cantilever is demonstrated to reduce the effects of non-specific binding interactions on the cantilever. Three structurally different biotin modified cantilever surfaces are used as a model system to study the binding interaction with streptavidin. The cantilever response to the binding of streptavidin on these biotin sensing monolayers is compared. The lowest detection limit of streptavidin using biotin-HPDP is found to be between 1 and 10nM limited by the optical measurement setup. Surface characterization using quartz crystal microbalance (QCM) and high-resolution atomic force microscope (AFM) is used to benchmark the cantilever sensor response. In addition, the QCM and AFM studies reveal that the surface density of bound streptavidin on biotin modified surfaces was low, thereby implying that effects other than steric hindrance are responsible for defining cantilever response.     

Accurate measurement of avidin and streptavidin in crude biofluids with a new, optimized biotin-fluorescein conjugate

A new biotin-fluorescein conjugate with an ethylene diamine spacer was found to be the first fluorescent biotin derivative which truly mimicked d-biotin in terms of high affinity, fast association, and non-cooperative binding to avidin and streptavidin tetramers. These exceptional properties were attributed to the small size/length of the new ligand since all larger/longer biotin derivatives are known for their mutual steric hindrance and anti-cooperative binding in 4:1 complexes with avidin and streptavidin tetramers. Specific binding of the new biotin-fluorescein conjugate towards avidin and streptavidin was accompanied by 84-88% quenching of ligand fluorescence. In the accompanying study this effect was used for rapid estimation of avidin and streptavidin in a new 'single tube assay'. In the present study the strong quenching effect was utilized to accurately monitor stoichiometric titration of biotin-binding sites in samples with >/=200 pM avidin or streptavidin. The concentration was calculated from the consumption of fluorescent ligand up to the distinct breakpoint in the fluorescence titration profile which was marked by the abrupt appearance of strongly fluorescent ligands which were in excess. Due to this protocol the assay was not perturbed by background fluorescence or coloration in the unknown samples. The new fluorescence titration assay is particularly suited for quick checks on short notice because getting started only means to thaw an aliquot of a standardized stock solution of fluorescent ligand. No calibration is required for the individual assay and the ligand stock solution needs to be restandardized once per week (or once per year) when stored at -25 degrees C (or at -70 degrees C, respectively).     

Effect of microsphere binding site density on the apparent affinity of an interaction partner.

BACKGROUND: Flow cytometric microsphere-based binding assays can be used to measure molecular interactions with high sensitivity. We have used multiplexed microsphere technology to explore the effect that binding site density has on the apparent affinity of a soluble interaction partner. METHODS: The interaction of a nuclear receptor, peroxisome proliferator-activated receptor gamma ligand binding domain (PPARgamma LBD), with a synthetic peptide derived from a nuclear receptor coactivator protein, PPARgamma coactivator-1 alpha (PGC-1alpha), is the interacting system being studied. The density of this peptide coupled to fluorescently unique microsphere populations is varied by co-incubating the biotinylated peptide and avidin-coated microsphere populations with increasing the amounts of free D-biotin. The discrete-density peptide-coupled microsphere populations are combined to conduct a multiplexed binding experiment with Alexa 532-labeled PPARgamma LBD, in the absence or presence of a small molecule ligand. RESULTS: As the immobilized binding site density of PGC-1alpha peptide on fluorescent microspheres is increased the measured apparent affinity for PPARgamma LBD is increased. CONCLUSIONS: The density of binding sites immobilized to a surface has a pronounced effect on the apparent affinity for soluble binding partners. By controlling and varying the binding site density it is possible to increase the sensitivity of an interaction assay. In multiplexed assay formats it should be possible to normalize intrinsically unequal binding interactions by individually optimizing the binding site density of the immobilized interaction partner. However, to quantitatively measure intrinsic affinities of molecular interactions, low binding site densities are required and multivalent reagents must be avoided.     

Development of a tetrameric streptavidin mutein with reversible biotin binding capability: engineering a mobile loop as an exit door for biotin

A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop(3-4) functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop(3-4) keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop(7-8). This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop(7-8) is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (k(off) of 4.28×10(-4) s(-1) and K(d) of 1.9×10(-8) M) make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible.     

Colorimetric competitive inhibition method for the quantification of avidin, streptavidin and biotin.

A colorimetric competitive inhibition assay for avidin, streptavidin and biotin was developed. The method for avidin or streptavidin was based on the competitive binding between avidin or streptavidin and a streptavidin-enzyme conjugate for biotinylated dextrin immobilized on the surface of a microtitre plate. For biotin quantitation the competition is between free biotin and the immobilized biotin for the streptavidin-enzyme conjugate. The limits of detection which was determined as the concentration of competitor required to give 90% of maximal absorbency (100% inhibition) was approximately 20 ng/100 microl per assay for avidin and streptavidin and 0.4 pg/100 microl per assay for biotin. The methods are simple, rapid, highly sensitive and adaptable to high throughput analysis.     

生物素化ATP硫酸化酶的表达、固定化与应用

现代大规模焦测序技术的产生是DNA测序技术的一次革命,其关键技术之一是得到高活性的、固定于磁性微球表面的ATP硫酸化酶．生物素化的ATP硫酸化酶可以通过生物素与亲和素之间的特异结合特性固定在包被亲和素的磁性微球表面,但是利用化学修饰法将ATP硫酸化酶进行生物素化修饰很可能会影响酶的活性．利用融合表达策略,将大肠杆菌生物素酰基载体蛋白C端87个氨基酸肽段(BCCP87)与ATP硫酸化酶在大肠杆菌内融合表达,经SDS-PAGE和Western blot分析,表达的融合蛋白分子质量约为64 ku,并且能够在大肠杆菌内被生物素化．生物素化的ATP硫酸化酶能够与亲和素包被的磁珠结合,固定后的ATP硫酸化酶具有活性,并且能够用于定量检测焦磷酸盐(PPi)和焦测序,为今后建立高通量大规模焦测序系统提供了一个有效的工具酶．     

New antibody immobilization method via functional liposome layer for specific protein assays

A specific protein assay system based on functional liposome-modified gold electrodes has been demonstrated. To fabricate such assay system, a liposome layer was initially grown on top of a gold layer. The liposome layer contained two kinds of functional molecules: biotin molecules for the binding sites of streptavidin and N-(10,12-pentacosadiynoic)-acetylferrocene molecules for the facile redox probe in electrochemical detections. Then, streptavidin was attached on the functional liposme-modified layer using the interaction of streptavidin-sbiotin complex. On the streptavidin-attached surface, antibody molecules, anti-human serum albumin antibodies could be immobilized without any secondary antibodies. AFM imaging of the streptavidin-attached liposome surface revealed a uniform distribution of closely packed streptavidin molecules. In situ quartz-crystal microbalance and electrochemical measurements demonstrated that the wanted antibody-antigen reactions should occur with high specificity and selectivity. Our specific antibody assay system, based on a functional liposome modified electrode, can be developed further to yield sophisticated structures for numerous protein chips and immunoassay sensors.     

Review: Multipoint binding and heterogeneity in immobilized metal affinity chromatography

Studies carried out using engineered proteins clearly demonstrate that adsorption to derivatized surfaces involves multiple interactions between functional groups on the protein and complementary sites distributed on the surface. The fact that adsorption involves multipoint interactions has important implications for the design of separations processes and for the interpretation of heterogeneity in biological recognition phenomena. Increasing the density of surface metal sites (immobilized copper ions) is found to be functionally equivalent to increasing the number of metal-coordinating groups on the protein (histidines and deporotonated amines), m in that both processes increase the likelihood of simultaneous interactions between the protein and the surface. A consequence of multiple-site interactions is a significant in crease in protein binding affinity that depends on the arrangement of surface sites. A protein will show the highest affinity for arrangements of surface sites which best match its own pattern of functioal groups and will show lower affinity for less optimal arrangements, resulting in binding that is inherently heterogeneous. We have found that reversible protein adsorption in immobilized metal affinity chromatography (IMAC) is described by the Temikin model, which characterizes binding heterogeneity by a uniform distribution of binding energies over the population of surface binding sites. (c) 1995 John Wiley & Sons, Inc.     

Sensitivity studies for specific binding reactions using the biotin/streptavidin system by evanescent optical methods

The combination of various evanescent optical methods such as surface plasmon spectroscopy, waveguide mode spectroscopy and an integrated optical Mach-Zehnder-interferometer are used to characterize biotinylated self-assembled monolayers as well as the binding of streptavidin to these labels. The aim of designing a highly specific and sensitive, re-usable affinity sensor for antigens on the basis of an integrated optical Mach-Zehnder interferometer is based on a proper understanding of the characteristics of the entire binding matrix architecture. Therefore, a variety of biotin-derivatives immobilized in a monolayer are investigated with respect to their affinity to streptavidin and the possibility to remove the steptavidin layer specifically. The density of the streptavidin layer as well as the optical constants of the involved molecules are measured. Finally the integrated optical Mach-Zehnder interferometer is tested with respect to the sensitivity to an antigen-antibody binding reaction. An attempt to further increase the sensitivity by simultaneous detection of a fluorescence signal failed due to bleaching effects.     

A novel flow cytometric assay to quantify interactions between proteins and membrane lipids

A diverse set of experimental systems has been developed to probe protein-lipid interactions. These include measurements with the headgroups of membrane lipids in solution, immobilized membrane lipids, and analysis of protein binding to membrane lipids reconstituted in liposomes. Each of these methodologies has strengths but also substantial limitations. For example, measurements between proteins and lipid headgroups or with immobilized membrane lipids do not probe interactions in their natural environment, the lipid bilayer. The use of liposomes, however, was so far mostly restricted to biochemical flotation experiments that do not provide quantitative and/or kinetic data. Here, we present a fast and sensitive flow cytometric method to detect protein-lipid interactions. This technique allows for quantitative measurements of interactions between multiple fluorescently labeled proteins and membrane lipids reconstituted in lipid bilayers. The assay can be used to quantify binding efficiencies and to determine kinetic constants. The method is further characterized by a short sampling time of only a few seconds that allows for high-content screening procedures. Finally, using light scatter measurements, the described method also allows for monitoring changes of membrane curvature as well as tethering of liposomes evoked by binding of proteins.     

Preparation, morphological characterization, and activity of thin films of horseradish peroxidase

Active uniform films of horseradish peroxidase (HRP) have been prepared by covalent binding on Si/SiO(2) or glass supports previously activated by silanization and succinylation. Labeling by fluorescent or by Electron Spin Resonance (ESR) probes was used to quantify the surface density of active groups and of horseradish peroxidase. Atomic Force Microscopy (AFM) imaging was used to characterize the surface morphology. We observed that a non-uniform protein adsorption due to physical interactions was present when the supports were not activated for covalent binding and was, in large part, removed by washing. The enzyme deposited by covalent binding formed homogeneous layers with a height in the range 60-90 A. By using a fluorescent label, we calculated a protein density of 3.6 x 10(12) molecules cm(-2) on Si/SiO(2), corresponding to an estimated area per molecule of 2800 A(2) which is in agreement with the value expected on the basis of the crystallographic data considering the formation of a monomolecular layer. The protein density of the layer immobilized on glass was similar (1.9 x10(12) molecules cm(-2)). The enzyme immobilized on both supports showed a k(cat)/K(M) being of the order of 3-5x10(5) M(-1)s(-1) that is 1/20th of free HRP. The half-life time of the activity of the enzyme immobilized by covalent binding was longer than 40 days at 6 degrees C.     

Characterization and application of a surface modification designed for QCM-D studies of biotinylated biomolecules

The rapid development of surface sensitive biosensor technologies, especially towards nanoscale devices, requires increasing control of surface chemistry to provide reliable and reproducible results, but also to take full advantage of the sensing opportunities. Here, we present a surface modification strategy to allow biotinylated biomolecules to be immobilized to gold coated sensor crystals for quartz crystal microbalance with dissipation monitoring (QCM-D) sensing. The unique feature of QCM-D is its sensitivity to nanomechanical (viscoelastic) properties at the sensing interface. The surface modification was based on mixed monolayers of oligo(ethylene glycol) (OEG) disulfides, with terminal -OH or biotin groups, on gold. Mixtures containing 1% of the biotin disulfide were concluded to be the most appropriate based on the performance when streptavidin was immobilized to biotinylated sensors and the subsequent biotinylated bovine serum albumin (BSA) interaction was studied. The OEG background kept the unspecific protein binding to a minimum, even when subjected to serum solutions with a high protein concentration. Based on characterization by contact angle goniometry, ellipsometry, and infrared spectroscopy, the monolayers were shown to be well-ordered, with the OEG chains predominantly adopting a helical conformation but also partly an amorphous structure. Storage stability was concluded to depend mainly on light exposure while almost all streptavidin binding activity was retained when storing the sensors cold and dark for 8 weeks. The surface modification was also tested for repeated antibody-antigen interactions between BSA and anti-BSA (immobilized to biotinylated protein A) in QCM-D measurements lasting for >10h with intermediate basic regeneration. This proved an excellent stability of the coating and good reproducibility was obtained for 5 interaction cycles. With this kind of generic surface modification QCM-D can be used in a variety of biosensing applications to provide not only mass but also relevant information of the structural properties of adlayers.     

Fluorescent microplate-based analysis of protein-DNA interactions. II: Immobilized DNA

A simple protein-DNA interaction analysis has been developed using both a high-affinity/high-specificity zinc finger protein and a low-specificity zinc finger protein with nonspecific DNA binding capability. The latter protein is designed to mimic background binding by proteins generated in randomized or shuffled gene libraries. In essence, DNA is immobilized onto the surface of microplate wells via streptavidin capture, and green fluorescent protein (GFP)-labeled protein is added in solution as part of a crude cell lysate or protein mixture. After incubation and washing, bound protein is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.4 nM protein. The assay format is ideally suited to investigate the interactions of DNA binding proteins from within crude cell extracts and/or mixtures of proteins that may be encountered in protein libraries generated by codon randomization or gene shuffling.     

Drug affinity to immobilized target bio-polymers by high-performance liquid chromatography and capillary electrophoresis

This review addresses the use of high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) as affinity separation methods to characterise drugs or potential drugs-bio-polymer interactions. Targets for the development of new drugs such as enzymes (IMERs), receptors, and membrane proteins were immobilized on solid supports. After the insertion in the HPLC system, these immobilized bio-polymers were used for the determination of binding constants of specific ligands, substrates and inhibitors of pharmaceutical interest, by frontal analyses and zonal elution methods. The most used bio-polymer immobilization techniques and methods for assessing the amount of active immobilized protein are reported. Examples of increased stability of immobilized enzymes with reduced amount of used protein were shown and the advantages in terms of recovery for reuse, reproducibility and on-line high-throughput screening for potential ligands are evidenced. Dealing with the acquisition of relevant pharmacokinetic data, examples concerning human serum albumin binding studies are reviewed. In particular, papers are reported in which the serum carrier has been studied to monitor the enantioselective binding of chiral drugs and the mutual interaction between co-administered drugs by CE and HPLC. Finally CE, as merging techniques with very promising and interesting application of microscale analysis of drugs' binding parameters to immobilized bio-polymers is examined.     

A comparison of silver ion to streptavidin coated microplates

Direct comparisons are made between covalently linked streptavidin and silver ion coated microplates. Both coatings can immobilize biotinylated molecules. Silver ion coated microplate wells can immobilize 1.8 times higher amounts of biotin labeled horseradish peroxidase. The quantitation range and capacity for the capture of horseradish peroxidase using biotin labeled horseradish peroxidase are also greater for silver ion coated microplates. Approximately twice as many anti-horseradish peroxidase antibodies can be immobilized per well using silver ion coated microplates. Higher capacities are presumed to be due to the smaller footprint of silver ions as compared to streptavidin. A direct comparison between the two coatings for a beta-galactosidase ELISA showed that while the silver ion coated microplates gave higher readings, the streptavidin coated microplates exhibited smaller well-to-well variation. However, higher well to well variation for the silver microplates is attributed to the high density of anti-beta-galactosidase antibodies on the microplates and the weak binding of clone GAL-13 to beta-galactosidase, rather than the silver coating. These studies suggest silver ion coated microplates are a desirable alternative to streptavidin plates for quantitative immunoassays.