Single probe nucleic acid immobilization on chemically modified single protein by controlling ionic strength and pH

In an effort toward determining the feasibility of single molecule analysis, we describe a case whereby the binding of one biotinylated DNA to one streptavidin molecule via electrostatic interactions was controlled by altering in pH 4.0-9.0 and 0.16 of the ion strength. The quantitative analysis of immobilized probe ssDNA was realized in real-time via a quartz crystal microbalance (QCM) and electrochemical (EC) measurement in the range 100 pM to 50 μM of probe oligonucleotide concentration. The variation amount of biotinylated ssDNA immobilized on the streptavidin-modified surface at pH 7.5 was about 0.16 pmol, giving a ratio of streptavidin to biotinylated ssDNA of about 1:1.1. On the other hand, at pH 4.9, it was immobilized about 0.29 pmol. From the shape of the Langmuir plot and QCM, the immobilization efficiency of biotinylated DNA via streptavidin at pH 4.9 was approximately twofold that at pH 7.5. In view points of the reaction velocity, it was increased with decreasing buffer solution pH, indicating a strong interaction of negatively charged probe DNA with the positively charged streptavidin. And also the EC response value of ΔI/I_streptavidin for the immobilized biotinylated ssDNA in pH 4.9 was about 49%, while the corresponding value for the pH 7.5 was approximately 34%. As DNA molecules possess negative charges, electrostatic repulsion occurred between streptavidin and biotinylated ssDNA at pH 7.5. At pH 4.9, the attraction between the biotinylated ssDNA and streptavidin resulted in increased adsorption which has an isoelectric point of about 5.9. It was deduced that the binding of biotinylated ssDNA to one or two of the four binding sites of streptavidin can be controlled by adjusting the pH-controlled electrostatic interaction.     

A native, affinity-based protein blot for the analysis of streptavidin heterogeneity: consequences for the specificity of streptavidin mediated binding assays

Commercial preparations of streptavidin, a bacterial biotin-binding protein, were analyzed by isoelectric focusing combined with an affinity-based protein blot using biotinylated, protein-saturated nitrocellulose. The colorimetrical detection of streptavidin with biotinylated alkaline phosphatase allows the selective visualization of streptavidin molecules with at least two active biotin-binding sites. Dependent on the preparation, seven to sixteen streptavidin forms were found with isoelectric points ranging from 5 to 8. Molecular weight analysis of the subunits of streptavidin showed that the observed heterogeneity was mainly due to limited proteolysis, which does not destroy the biotin-binding activity. The preparations differed also in the nonspecific reactivity of streptavidin with single-stranded DNA, bovine serum albumin and Tween 20. No relationship was observed between heterogeneity and non-specific binding activity. Data obtained from protein blots onto nitrocellulose saturated with single-stranded DNA showed that it cannot be excluded that streptavidin with only a single active biotin-binding site is mainly responsible for the nonspecific reactivity of some streptavidin preparations.     

The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures

The biotin-streptavidin system is the strongest noncovalent biological interaction known, having a dissociation constant, K(d), in the order of 4x10(-14) M. The strength and specificity of the interaction has led it to be one of the most widely used affinity pairs in molecular, immunological, and cellular assays. However, it has previously been impossible to re-use any streptavidin solid support, since the conditions needed to break the interaction with biotin has led to the denaturation of the streptavidin. Here, we show that a short incubation in nonionic aqueous solutions at temperatures above 70 degrees C can efficiently break the interaction without denaturing the streptavidin tetramer. Both biotin and the streptavidin remain active after dissociation and both molecules can therefore be re-used. The efficiency of the regeneration allowed solid supports with streptavidin to be used many times, here exemplified with the multiple re-use of streptavidin beads used for sample preparation prior to automated DNA sequencing. The results suggest that streptavidin regeneration can be introduced as an improvement in existing methods and assays based on the streptavidin system as well as emerging solid phase applications in fields, such as microfluidics and nanotechnology.     

Evaluation of Binding Between Electroactive Biotin Derivative and Streptavidin Immobilized on Chitin Film

Evaluation of the streptavidin‐biotin binding at the surface of chitin film was carried out with voltammetry. Immobilization of streptavidin was attempted to the protonated chitin film, based on an electrostatic interaction that hardly causes any change in the protein structure. The streptavidin‐biotin binding was estimated from changes in the electrode response of biotin labeled with an electroactive compound. Although the response of daunomycin as an electroactive compound did not change at an electrode covered with streptavidin/chitin film, the response of the labeled biotin decreased. This observation shows that streptavidin is immobilized on the chitin film and the biotin binds with immobilized streptavidin. Consequently, it was clear that the chitin film is useful as a reaction field for protein‐ligand binding. Generally, a binding event between protein and its ligand in the living body occurs on the cell surface. The electrochemical evaluation of protein‐ligand binding on a natural polysaccharide like chitin membrane surface is important.     

Drop-shape analysis of receptor-ligand binding at the oil/water interface

Drop-shape analysis was used to study the binding of streptavidin to biotin at the interface between water and a pendant chloroform droplet. Polyethylene oxide molecules were synthesized with a hydrophobic tail at one end of the molecule and a hydroxyl or biotin group at the other end. The interfacial tension of the water/chloroform interface was measured before and after addition of these amphiphiles to the chloroform phase and before and after addition of streptavidin to the aqueous phase. The hydroxyl-terminated amphiphiles eliminate nonspecific adsorption of the streptavidin to the interface, while streptavidin binds irreversibly to the biotin-terminated molecules. Molecular interactions within this bound layer were studied by measuring changes in the interfacial pressure as the layer is contracted and expanded by changing the volume of the chloroform droplet. A picture of the interfacial structure was obtained from quantitative comparisons between the experimental results and a molecular theory of protein binding to tethered ligands. These comparisons suggest that protein binding is controlled by the extension of the PEO tethers away from the interface.     

Temperature sensitive dopamine-imprinted (N,N-methylene-bis-acrylamide cross-linked) polymer and its potential application to the selective extraction of adrenergic drugs from urine

A temperature sensitive dopamine-imprinted polymer was prepared in 80% aqueous methanol solution by free-radical cross-linking co-polymerisation of methacrylic acid and acrylamide at 60 degrees C in the presence of N,N-methylene-bis-acrylamide as the cross-linker and dopamine hydrochloride as template molecule. The resulting molecularly imprinted polymer (MIP) formed temperature responsive materials, which could be used for the selective separation of appropriate dopamine and adrenergic compounds from a liquid matrix at ambient temperatures. The thermoresponsive MIP exhibited a swelling-deswelling transition in 80% aqueous methanol solution at about 35 degrees C. The capacity of the thermoresponsive MIP to recognise the template molecule when present in aqueous methanol solution changed with temperature, with the highest selectivity found at 35 degrees C. Additionally, binding parameters obtained from Scatchard analyses indicate that increasing temperature resulted in an increased affinity and binding capacity of specific binding sites, but had less effect on non-selective binding sites. Subsequently, the thermoresponsive MIP was tested for its application as a sorbent material, utilisable in the selective solid-phase extraction (SPE) of dopamine and other adrenergic compounds (epinephrine, isoproterenol, salbutamol and serotonin) from urine samples. It was shown that the compounds that were structurally related to dopamine could be removed by elution, while dopamine and serotonin, the analytes of interest, remained strongly adsorbed to the adsorbent during SPE applications. The thermoresponsive MIP displayed different efficiency in clean-up and enrichments using the SPE protocol at different temperatures.     

Synthesis of monodisperse biotinylated p(NIPAAm)-coated iron oxide magnetic nanoparticles and their bioconjugation to streptavidin

We describe here the synthesis of 10 nm, monodisperse, iron oxide nanoparticles that we have coated with temperature-sensitive, biotinylated p(NIPAAm) (b-PNIPAAm). The PNIPAAm was prepared by the reversible addition fragmentation chain transfer polymerization (RAFT), and one end was biotinylated with a PEO maleimide-activated biotin to form a stable thioether linkage. The original synthesized iron oxide particles were stabilized with oleic acid. They were dispersed in dioxane, and the oleic acid molecules were then reversibly exchanged with a mixture of PNIPAAm and b-PNIPAAm at 60 degrees C. The b-PNIPAAm-coated magnetic nanoparticles were found to have an average diameter of approximately 15 nm by dynamic light scattering and transmission electron microscopy. The ability of the biotin terminal groups on the b-PNIPAAm-coated nanoparticles to interact with streptavidin was confirmed by fluorescence and surface plasmon resonance. It was found that the b-PNIPAAm-coated iron oxide nanoparticles can still bind with high affinity to streptavidin in solution or when the streptavidin is immobilized on a surface. We have also demonstrated that the binding of the biotin ligands on the surface of the temperature-responsive magnetic nanoparticles to streptavidin can be turned on and off as a function of temperature.     

Microcalorimetric study on the influence of temperature on bacterial coaggregation

Binding isotherms and heats of interaction have been determined at 15, 25, and 40 degrees C for a coaggregating and a non-coaggregating oral bacterial pair. Heats of interaction were measured upon three consecutive injections of streptococci into an actinomyces suspension using isothermal titration calorimetry. After each injection, the number of streptococci injected remaining free in suspension was quantified microscopically and the degree of binding between the two bacterial strains was established. The coaggregating pair shows positive cooperative binding. The highest cooperativity, at 25 degrees C, correlates with a strong, macroscopically visible coaggregation. The non-coaggregating pair shows low cooperativity and lacks macroscopically visible coaggregation. Interactions between the coaggregating partners seem to be mainly due to specific, enthalpically saturable and favorable binding sites. Even though the enthalpic part of the interaction is saturated, cooperativity increases with consecutive injections, implying that the coaggregation phenomenon is driven by entropy gain. The change in heat capacity (DeltaC(p)) is positive for the non-coaggregating pair from 15-40 degrees C as well as for the coaggregating pair beyond 25 degrees C. At lower temperatures the coaggregating pair causes a negative DeltaC(p). The decrease in heat capacity together with an increase in entropy is considered to be indicative of hydrophobic interactions playing an important role in the formation of large coaggregates as observed for the coaggregating pair at 25 degrees C.     

Supported lipopolymer membranes as nanoscale filters: simultaneous protein recognition and size-selection assays

A technique for size-selective discrimination of protein analytes was developed by incorporating poly(ethylene glycol) (PEG) lipopolymers into supported lipid bilayers. The membranes also contained biotinylated lipids, which recognized both streptavidin and anti-biotin IgG. By employing various PEG lipopolymer concentrations, clear discrimination against anti-biotin (Mw = 150 000 Da) binding could be observed, which became more pronounced at higher polymer densities. On the other hand, streptavidin (Mw = 52 800) binding to the membrane remained unaffected even at PEG concentrations that were well into the mushroom-to-brush phase transition. These observations were exploited to create an on-chip ligand-receptor binding assay that favored streptavidin binding over anti-biotin by several orders of magnitude in the presence of the lipopolymer. Control experiments revealed that the two proteins are bound to similar extents from a multi-protein analyte solution in the absence of PEG.     

Chiroptical properties of 2-chloropropionitrile

(S)-(-)-2-chloropropionitrile has been prepared from (S)-(+)-alanine, and the ORD curves have been obtained in several solvents and in the gas phase. A reaction field extrapolation of the solution data to the gas phase led to an estimated value of [alpha]D = -21 degrees, whereas the interpolated gas phase value is -8 degrees. The specific rotation was found to be temperature dependent in ethylcyclohexane solution over the range 0-100 degrees C. Although rotation of the methyl group leads to large calculated effects on the specific rotation, it does not lead to the temperature dependence. Rather, a low frequency mode at 224 cm(-1) was found to be responsible. This is a mixed mode involving methyl torsion and C-C[triple bond]N bending. The specific rotations calculated at the B3LYP/aug-cc-pVDZ level including electric field dependent functions are in very good agreement with the measured gas phase values.     

Directed evolution of streptavidin variants using in vitro compartmentalization

We have developed and implemented an in vitro compartmentalization (IVC) selection scheme for the identification of streptavidin (SA) variants with altered specificities for the biotin analog desthiobiotin. Wild-type SA and selected variants bind desthiobiotin with similar affinities ( approximately 10(-13) M), but the variants have off rates almost 50 times slower and a half-life for dissociation of 24 hr at 25 degrees C. The utility of streptavidin variants with altered specificities and kinetic properties was shown by constructing protein microarrays that could be used to differentially organize and immobilize DNAs bearing these ligands. The methods we have developed should prove to be generally useful for generating a variety of novel SA reagents and for evolving other extremely high-affinity protein:ligand couples.     

Protective effects of small amounts of bis(2-ethylhexyl)sulfosuccinate on the helical structures of human and bovine serum albumins in their thermal denaturations

The protective effect of an anionic double-tailed surfactant, sodium bis(2-ethylhexyl)sulfosuccinate (AOT), on the structures of human serum albumin (HSA) and bovine serum albumin (BSA) in their thermal denaturations was examined by means of circular dichroism measurements. The structural changes of these albumins were reversible in the thermal denaturation below 50 degrees C, but became partially irreversible above this temperature. The effect was observed in the thermal denaturation above 50 degrees C. Although the helicity of HSA decreased from 66% to 44% at 65 degrees C in the absence of the surfactant, the decrement of it was restrained in the coexistence of AOT of extremely low concentrations. When the HSA concentration was 10 muM, the maximal protective effect appeared at 0.15 mM AOT. In the coexistence of the surfactant of this concentration, the helicity was maintained at 58% at 65 degrees C, increasing to the original value upon cooling to 25 degrees C. Beyond 0.15 mM AOT, the helicity sharply decreased until 3 mM AOT. A particular AOT concentration required to induce the maximal protective effect ([AOT]REQ) was examined at different HSA concentrations. [AOT]REQ shifted to higher values with an increase of the protein concentration. From the protein concentration dependences of [AOT]REQ, the maximal protection was estimated to require 8.0 and 5.0 AOT ions per a molecule of HSA and BSA, respectively. The AOT concentration, where the protective effect was observed, was too low to form its micelle-like aggregate. Then the protein structures might be stabilized by a cross-linking of surfactant monomers bound to specific sites. These specific sites might exist between a group of nonpolar residues and a positively charged residue located on several sets of amphiphilic helical rods in the proteins. Such a unique function of the double-tailed ionic surfactant is first presented by its characteristic nature as an amphiphilic material.     

Surface plasmon microscopy of biotin-streptavidin binding reactions on UV-photopatterned alkanethiol self-assembled monolayers

We report surface plasmon imaging of streptavidin binding to photopatterned biotinylated alkanethiol self-assembled monolayers (SAMs) on gold. Micrometer-scale patterns of a mixed biotin- and hydroxyl-terminated monolayer were formed in an inert, hydroxy-terminated alkanethiol monolayer using a UV-photopatterning procedure. Using surface plasmon microscopy, contrast is readily observed between the mixed biotin- and hydroxy-terminated SAM region after specific binding of streptavidin has occurred and the pure hydroxy-terminated region where nonspecific binding of streptavidin is negligible. Surface plasmon microscopy was also able to monitor in situ and in real time the binding of streptavidin to the patterned SAMs. The ability of surface plasmon microscopy to detect and spatially resolve 2-dimensional monolayer binding events may prove useful in diagnostic applications involving the parallel interrogation at surface biomolecular arrays.     

Specific binding of beta-endorphin to the isolated renal basolateral membranes in vitro

Binding of human beta-endorphin (beta-EP) to rat renal basolateral membranes was characterized using [125I]Tyr27-beta-EP ([125I]beta-EP) as a primary ligand. Ten millimolar of ethylenediaminetetra acetic acid (EDTA) completely inhibited the degradation of [125I]beta-EP in the incubation mixture at 4 degrees C, thus making it possible to quantitatively examine the [125I]beta-EP binding. The specific binding of [125I]beta-EP to the basolateral membranes was reversible and saturable, and a nonlinear least-squares regression analysis of a saturation isotherm revealed two different classes of specific binding sites. One class had an apparent dissociation constant (Kd) of 0.68 nM and a lower number of binding sites (33 fmol/mg protein), whereas the other class had a lower affinity (apparent Kd of 210 nM) and a higher number of binding sites (7.3 pmol/mg protein). Inhibition of the [125I]beta-EP binding by naloxone (10 microM) was approximately only 20%, and that by D-Ala2-D-Leu5-enkephalin (10 microM) was null, suggesting the major role of a non-opioid binding component in specific [125I]beta-EP binding to basolateral membranes. Moreover, a 50% inhibition by 10 microM of dynorphin(1-13) suggests that a certain region of the primary structure of beta-EP, excluding at least the NH2-terminal enkephalin sequence, is of particular importance for the [125I]beta-EP binding. These lines of evidence suggest the existence of two different classes of specific binding sites for beta-EP on the renal basolateral membranes, and the high-and low-affinity bindings may be attributed to opioid and non-opioid receptors, respectively, as judged by known characteristics of opioid and non-opioid receptors in other peripheral tissues.     

Steric crowding effects on target detection in an affinity biosensor

This work quantifies the impact of steric crowding on whole antibody (Ab) receptor immobilization and target Ab detection and also demonstrates how the versatile biotin/streptavidin receptor immobilization system must be tuned to optimize target detection in designing biosensors. Results are demonstrated on a label-free optical biosensor fabricated from n-type macroporous porous silicon (PSi) with approximately 88-107 nm diameter pores. We employ a sandwich assay scheme comprising a linking chemistry (biotin/streptavidin) to attach biotinylated anti-rabbit IgG (receptor) to detect rabbit IgG (target). A "bottom-up" approach was taken to investigate each layer of the sandwich assay to optimize target binding. Steric crowding was observed to hinder subsequent layer binding for each layer in the sandwich (biotin, streptavidin, and receptor). Our results give definitive evidence that the onset of steric crowding within the biotin layer occurs at a surface coverage of 57%, which is much higher compared to that from published work on well-ordered self-assembled biotin monolayers on planar gold surfaces. This difference is attributed to the topographical heterogeneity of the PSi substrate. Streptavidin (SA) binding to surface-linked biotin was altered by preblocking the streptavidin binding sites with biotin. Through consistent trends in data, preblocking SA was shown to reduce steric crowding within the SA layer, which translated into increased receptor immobilization. The final detection range of rabbit IgG was 0.07-3 mg mL(-1) (0.4-17 ng mm(-2)), and binding specificity was demonstrated by employing an anti-chicken IgG control receptor. This study underlines the importance of considering binding avidity and surface topography in optimizing chip-based biosensors.     

Interfacial recognition reactions as seen by fluorescence-, surface plasmon-and atomic force microscopies

We report on various microscopic investigations of the specific recognition and binding reaction between a biotinylated lipid layer and streptavidin. First, we present fluorescence microscopic evidence for the preferential adsorption of the protein to only the fluid matrix of a monolayer at the water-air interface if the latter is compressed to the phase transition region where crystalline domains coexist with expanded phase. Surface plasmon microscopy shows that this selectivity is preserved also if the monofilm is first transferred to a solid support (but still in contract with the aqueous phase) and then exposed to a streptavidin-containing solution. Finally, atomic force microscopic pictures taken at the monolayer-electrolyte interface are presented that confirm this preferential binding.     

Thermally changing lattice distance of lamella in the hydrated solid of octadecyltrimethylammonium chloride

A temperature scanning small-angle X-ray scattering measurement was carried out for the hydrated solids of octadecyltrimethylammonium chloride (OTAC). A gradual change of the lattice spacing of lamella-like structure from 40 nm at 5 degrees C to 20 nm at 18 degrees C was observed in the melting process of the hydrated solid that was incubated at 4 degrees C for a period of 24 h in the aqueous solution, while little change of the lattice spacing of about 20 nm was observed in the same process of the hydrated solid that was incubated at 4 degrees C for a period about 10 min. This indicates structural changes of the hydrated solid during the incubation at 4 degrees C and in the melting process. Corresponding to the nanostructure changes, broad endothermic peaks were observed at temperatures from 13 to 22 degrees C for the former hydrated solid and at temperatures from 15 to 21 degrees C for the latter hydrated solid in difference scanning calorimetry measurements. The structure change at temperatures below 13 degrees C is considered to be athermal from the fact that no endothermic peak is observed there. Large dielectric dispersions at frequencies at about 10 kHz were observed for the suspensions of hydrated solids but not for the solutions of dissolved solids. It was found that the electric conductance of the hydrated solid suspensions was much lower than that of the solutions of dissolved solids. The observed electric properties indicate that an amount of the free chloride ion is very small and that the chloride ions binding to the ammonium groups are movable in the hydrated solids by responding to an applied electric field. The electric conductance of suspension of the hydrated solid being incubated at 4 degrees C for 10 min was 4 times as large as that of a suspension of the hydrated solid being incubated at the same temperature for 24 h. This indicates that the structural change of the OTAC hydrated solid at 4 degrees C is related to the chloride ion binding to the hydrated solid. The experimental results described above suggest that the lamella in the hydrated solid of OTAC is undulated and that the wavelength of undulation increases with the incubation at a temperature much lower than the melting temperature.     

Characterization of thyroxine-albumin binding using high-performance affinity chromatography. I. Interactions at the warfarin and indole sites of albumin.

A high-performance affinity column containing immobilized human serum albumin (HSA) was used to study the binding of thyroxine at the warfarin and indole sites of HSA. Frontal analysis, using R-warfarin and L-tryptophan as probes for these sites, demonstrated that the immobilized HSA had binding behavior equivalent to that observed for HSA in solution. By injecting R-warfarin or L-tryptophan in the presence of excess thyroxine, it was found that thyroxine was binding directly to both types of site. The warfarin and indole sites had relatively strong binding for thyroxine, with association constants at 37 degrees C of 1.4 x 10(5) and 5.7 x 10(5) M-1, respectively. The value of delta G for these sites ranged from -7 to -8 kcal/mol and had a significant entropy component. The techniques used in this study are not limited to thyroxine-HSA interactions, but should also be valuable in examining the site-specific binding of other drugs and hormones to HSA.     

Specific interaction of desthiobiotin lipids and water-soluble biotin compounds with streptavidin

As shown for biotin lipids (Ref. 1), the formation of perfect 2-D crystalline streptavidin domains can also be observed in the plane of desthiobiotin lipid monolayers. The binding constant of streptavidin with desthiobiotin (K_a = 5·10¹³ mol⁻¹) is lower than that with biotin (K_a = 10¹⁵ mol⁻¹) (Ref. 2). By adding free biotin into the subphase a competitive replacement and a detaching of the streptavidin domains from the desthiobiotin lipid monolayer takes place. Streptavidin domains built at receptor lipid monolayers are still functional. As could be shown, there are two biotin binding sites at each protein molecule that are fully accessible to biotin (Ref. 1). This can be proven by the interaction with biotinylated ferritin and fluoresceinated biotin. Further coupling of an anti-FITC-antibody can proceed and a second protein layer can be formed. Using a bifunctional biotin linker a second crystalline streptavidin layer underneath the first one can be obtained.     

Ordering transitions triggered by specific binding of vesicles to protein-decorated interfaces of thermotropic liquid crystals

We report that specific binding of ligand-functionalized (biotinylated) phospholipid vesicles (diameter = 120 ± 19 nm) to a monolayer of proteins (streptavidin or anti-biotin antibody) adsorbed at an interface between an aqueous phase and an immiscible film of a thermotropic liquid crystal (LC) [nematic 4'-pentyl-4-cyanobiphenyl (5CB)] triggers a continuous orientational ordering transition (continuous change in the tilt) in the LC. Results presented in this paper indicate that, following the capture of the vesicles at the LC interface via the specific binding interaction, phospholipids are transferred from the vesicles onto the LC interface to form a monolayer, reorganizing and partially displacing proteins from the LC interface. The dynamics of this process are accelerated substantially by the specific binding event relative to a protein-decorated interface of a LC that does not bind the ligands presented by the vesicles. The observation of the continuous change in the ordering of the LC, when combined with other results presented in this paper, is significant, as it is consistent with the presence of suboptical domains of proteins and phospholipids on the LC interface. An additional significant hypothesis that emerges from the work reported in this paper is that the ordering transition of the LC is strongly influenced by the bound state of the protein adsorbed on the LC interface, as evidenced by the influence on the LC of (i) "crowding" of the protein within a monolayer formed at the LC interface and (ii) aging of the proteins on the LC interface. Overall, these results demonstrate that ordering transitions in LCs can be used to provide fundamental insights into the competitive adsorption of proteins and lipids at oil-water interfaces and that LC ordering transitions have the potential to be useful for reporting specific binding events involving vesicles and proteins.