Assembly of bionanostructures onto beta-cyclodextrin molecular printboards for antibody recognition and lymphocyte cell counting

The assembly of complex bionanostructures onto beta-cyclodextrin (betaCD) monolayers has been investigated with the aims of antibody recognition and cell adhesion. The formation of these assemblies relies on host-guest, protein-ligand, and protein-protein interactions. The buildup of a structure consisting of a divalent bis(adamantyl)-biotin linker, streptavidin (SAv), biotinylated protein A (bt-PA), and an Fc fragment of a human immunoglobin G (IgG-Fc) was studied with surface plasmon resonance (SPR) spectroscopy. Patterns of this bionanostructure were obtained via microcontact printing of the divalent linker at the molecular printboard, followed by the subsequent attachment of the proteins. Fluorescence microscopy showed that the buildup of these bionanostructures on the betaCD monolayers is highly specific. On the basis of these results, bionanostructures were made in which whole antibodies (ABs) were used instead of the IgG-Fc. These ABs were bound to the SAv layer via biotinylated protein G (bt-PG) or via a biotinylated AB. These constructions yielded specifically bound ABs with a less than maximal density, as shown by SPR spectroscopy and atomic force microscopy (AFM). Finally, the immobilization of ABs to the molecular printboard was used to create platforms for lymphocyte cell count purposes. Monoclonal ABs (MABs) were attached to the SAv layer using bt-PG, an engineered biotin functionality, or through nonspecific adsorption. The binding specificity of the immobilized cells was the highest on the buildup made from bt-PG, which is attributed to an optimized orientation of the antibodies. An approximately linear relationship between the numbers of seeded cells and counted cells was demonstrated, rendering the platform potentially suitable for lymphocyte cell counting.     

Recruitment of receptors at supported lipid bilayers promoted by the multivalent binding of ligand-modified unilamellar vesicles

The development of model systems that mimic biological interactions and allow the control of both receptor and ligand densities, is essential for a better understanding of biomolecular processes, such as the recruitment of receptors at interfaces, at the molecular level. Here we report a model system based on supported lipid bilayers (SLBs) for the investigation of the clustering of receptors at their interface. Biotinylated SLBs, used as cell membrane mimics, were functionalized with streptavidin (SAv), used here as receptor. Subsequently, biotinylated small (SUVs) and giant (GUVs) unilamellar vesicles were bound to the SAv-functionalized SLBs by multivalent interactions and found to induce the recruitment of both SAv on the SLB surface and the biotin moieties in the vesicles. The recruitment of receptors was investigated with quartz crystal microbalance with dissipation monitoring (QCM-D), which allowed the identification of the biotin and SAv densities necessary to obtain receptor recruitment. At approx. 0.6% of biotin in the vesicles, a transition between dense and low vesicle packing was observed, which coincided with the transitions between recruitment in the vesicles vs. recruitment in the SLB and between full and partial use of the biotin moieties in the vesicle. Direct optical visualization of the clustering at the interface of individual GUVs with the SLB platform was achieved with fluorescence microscopy, showing recruitment of SAv at the contact area as well as the deformation of the vesicles upon binding. Different vesicle binding regimes were observed for lower and higher biotin densities in the vesicles and at the SLBs. A more quantitative analysis of the molecular parameters implied in the interaction, indicated that approx. 10% of the vesicle area constitutes the contact area. Moreover, the SUV binding and recruitment appeared to be fast on the analysis time scale, whereas the binding of GUVs is slower due to the larger SLB area over which SAv recruitment needs to occur. The mechanisms revealed in this study may provide insight in biological processes in which recruitment occurs.

The development of model systems that mimic biological interactions and allow the control of both receptor and ligand densities, is essential for a molecular understanding of biomolecular processes, such as the recruitment of receptors at interfaces.     

Non-specific and specific interactions on functionalized polymer surface studied by FT-SPR

Fourier transform surface plasmon resonance (FT-SPR) was utilized to study specific and non-specific interactions between proteins and a biotinylated polymer film by monitoring adsorptions of streptavidin (SAv) and bovine serum albumin (BSA) on the polymer films. The biotinylated polymer, poly(lactide-co-2,2-dihydroxymethyl-propylene carbonate-graft-biotin) [P(LA-co-DHC/biotin)], was prepared by ring-opening copolymerization of lactide and a OH-bearing cyclic carbonate monomer, followed by biotinylation of the OH groups. The copolymer was coated onto the FT-SPR chip and vacuum-dried, hydrated at 70°C, and treated with a blocking agent respectively to achieve different surface status. The FT-SPR results showed that the vacuum-dried film had the most BSA adsorption; hydration treatment led to migration of the biotin moieties from inner film to surface and thus resulted in less BSA adsorption; blocking layer on the polymer surface saturated the active sites for physical and chemical adsorptions on the surface and thus weakened the BSA adsorption. Adsorption of SAv displayed similar polymer-surface-status dependence, i.e., more adsorption on vacuum-dried surface, less adsorption on hydrated surface and the least adsorption on blocked surface. Compared with BSA, SAv showed more enhanced adsorptions on P(LA-co-DHC/biotin) surface because of the specific interaction of biotin moieties in the polymer with SAv molecules, especially on the blocked surface. The above semi-quantified results further indicate that the FT-SPR system is suitable for investigating interactions between polymer surface and bio-molecules.     

The effective concentration of unbound ink anchors at the molecular printboard

Self-assembled monolayers terminating in beta-cyclodextrin cavities can be used to bind ink molecules and so provide a molecular printboard for nanopatterning applications. Multivalent or multisite binding strengthens the attachment of large inks and provides more robust patterns. In the present work we use computer simulations to probe the behavior of functionalized dendrimer inks at the printboard. We performed a series of long 10 ns fully atomistic molecular dynamics (MD) simulations to measure the effective local concentration of unbound ink anchor groups at the printboard for a variety of binding modes and also for the partial unbinding prerequisite for ink diffusion on the printboard. These simulations allow us to describe the conformational space occupied by partially bound inks and estimate the likelihood of an additional binding interaction. Furthermore, by simulating the shift from a divalent to monovalent binding mode we show that the released anchor quickly moves to the periphery of the dendrimer binding hemisphere but then reapproaches the printboard and remains in the vicinity of alternative binding sites. Secondary electrostatic interactions between the protonated dendrimer core and hydroxyl groups at the entrance to the beta-cyclodextrin cavities give "flattened" dendrimer binding orientations and may aid dendrimer diffusion on the printboard, allowing the dendrimer to "walk" along the printboard by switching between different partially bound states and minimizing complete unbinding to bulk solution, crucial for the application of the printboard in, for example, medical diagnostics.     

Electrochemical rectification by redox-labeled bioconjugates: molecular building blocks for the construction of biodiodes

In the present work, we describe the properties of a bifunctional redox-labeled bioconjugate at electrode surfaces mediating the electron transfer across the electrode-electrolyte interface. We show that the assembly of ferrocene-labeled streptavidin on biotinylated electrodes results in a reproducible unidirectional current flow in the presence of electron donors in solution. Such rectifying films were built up by spontaneous binding of tetrameric streptavidin molecules to biotin centers immobilized on the electrode surface. Due to the high affinity of biotin to streptavidin, such bifunctional films completely bind any biotinylated compounds. The charge transport between donors in solution and the Au electrode is mediated by the ferrocene moieties, allowing us to develop a molecular rectifier. Our experimental results suggest that such redox-labeled proteins with a high binding capacity constitute a promising alternative to organic compounds used in molecular electronics.     

Free energy balance predicates dendrimer binding multivalency at molecular printboards

Self-assembled monolayers (SAMs) terminating in beta-cyclodextrin (beta-CD) cavities can be used to bind ink molecules and so provide a molecular printboard for nanopatterning applications. Multivalent, or multisite, binding strengthens the attachment of large inks to the printboard, yielding more robust patterns. We performed fully atomistic molecular dynamics (MD) simulations in bulk explicit solvent to probe the conformational space available to dendrimer and dendrite ink molecules, in both free and bound environments. We show that accurate treatment of both pH effects and binding conformations gives calculated binding modes in line with known binding multivalencies. We identify and quantify the steric frustration causing small, low-generation dendrimer inks to bind to the printboard using just a subset of the available anchor groups. Furthermore, we show that the enhanced binding energy of multisite attachment offsets the steric strain, the feasibility of a given binding mode thus determined by the relative magnitudes of the unfavorable steric strain and favorable multisite binding free energies. We use our experimentally validated model of dendrimer binding to predict the binding mode of novel fluorophoric dendrites and find divalent binding, consistent with confocal microscopy imaging of pattern formation at molecular printboards.     

Writing with molecules on molecular printboards

Nanotechnology aspires to create functional materials with characteristic dimensions of the order 1-100 nm. One requirement to make nanotechnology work is to precisely position molecules and nanoparticles on surfaces, so that they may be addressed and manipulated for bottom-up construction of nanoscale devices. Here we review the concept of a "molecular printboard". A molecular printboard is a monolayer of host molecules on a solid substrate on which guest molecules can be attached with control over position, binding strength, and binding dynamics. To this end, cyclodextrins were immobilized in monomolecular layers on gold, on silicon wafers and on glass. Guest molecules (for example, adamantane and ferrocene derivatives) bind to these host surfaces through supramolecular, hydrophobic inclusion interaction. Multivalent interactions are exploited to tune the binding strength and dynamics of the interaction of guest molecules with the printboard. Molecules can be positioned onto the printboard using supramolecular microcontact printing and supramolecular dip-pen nanolithography due to the specific interaction between the 'ink' and the substrate. In this way, nanoscale patterns can be written and erased on the printboard. Currently, the molecular printboard is exploited for nanofabrication, for example in combination with electroless deposition of metals and by means of supramolecular layer-by-layer deposition.     

Towards a fluorescent molecular switch for nucleic acid biosensing

A novel fluorescent molecular switch for the detection of nucleic acid hybridization has been explored in relation to the development of a structure that would be amenable for operation when immobilized for solid-phase analyses. The structure was prepared by self-assembly, and used Neutravidin as the central multivalent docking molecule, a newly synthesized biotinylated long-chain linker for intercalating dye that was modified with thiazole orange (TO) at one end, and a biotinylated probe oligonucleotide. Self-assembly of the biotinylated components on adjacent Neutravidin binding sites allowed for physical placement of an oligonucleotide probe molecule next to tethered TO. The TO located at the end of the flexible linker chain was available to intercalate, and could report if a duplex structure was formed by a probe–target interaction by means of fluorescence intensity. Subsequently, regeneration of the single-stranded probe was possible without loss of the intercalator to solution. The switch constructs were assembled in solution and subsequently immobilized onto biotin functionalized optical fibers to complete the sensor design. Solution-phase fluorescence lifetime data showed a biexponential behavior for switch constructs, suggesting intercalation as well as a significant secondary binding mode for the immobilized TO. It was found that the secondary binding mechanism for the dye to DNA could be decreased, thus shifting the dye to intercalative binding modes, by adjusting the solution conditions to a pH below the pI of Neutravidin, and by increasing the ionic strength of the buffer. Preliminary work demonstrated that it was possible to achieve up to a fivefold increase in fluorescence intensity on hybridization to the target.     

Supramolecular architectures of streptavidin on biotinylated self-assembled monolayers. Tracking biomolecular reorganization after bioconjugation

In this work we have studied the supramolecular bioconjugation of streptavidin (SAv) on biotinylated self-assembled monolayers. By using the quartz crystal microbalance technique with dissipation we were able to follow in real time the biomolecular reorganization within the film. The overall process could be described as an early stage involving a significant increase in surface coverage followed by another stage where the SAv layer slowly reached the asymptotic coverage. Finally, a reorganization process takes place in the bioconjugated film. These results on the kinetics of biomolecular reorganization can be described in terms of the Lifshitz-Slyozov law. These are the first experimental results demonstrating the complexity and the different time scales involved on the bioconjugation of SAv at solid-liquid interfaces. We consider that these findings could have strong implications on the molecular design of biosensing platforms.     

Post-capillary reaction detection in capillary electrophoresis based on the streptavidin-biotin interaction. Optimization and application to single cell analysis.

A class-selective post-capillary reaction detection method for capillary electrophoresis is described in which a streptavidin-fluorescein isothiocyanate (streptavidin-FITC) conjugate is used to detect biotin moieties. The selective binding of biotin moieties to the streptavidin-FITC conjugate causes an enhancement of fluorescence proportional to the concentration of biotin present. After capillary electrophoresis the separated analytes react with streptavidin-FITC in a coaxial reactor and are then detected either by a benchtop spectrofluorometer (2.5 microM detection limit) or by an epi-fluorescence microscope (1 x 10(-7) M detection limit). The method is used to examine biotinylated species in a crude mammalian cell lysate which was found to contain 83+/-3 fmol in 3600 cell volumes. In addition, it is used to examine the uptake of biotin by individual sea urchin oocytes. The results indicate that, in the oocytes, biocytin is the prevalent form of biotin and its concentration varies widely between cells (mean=2+/-2 microM).     

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.     

Controlling biotinylation of microgels and modeling streptavidin uptake

Compared are two approaches for the biotinylation of poly(N-isopropylacrylamide-co-vinylacetic acid) microgels, 300-nm diameter, water swollen particles with a corona of carboxyl groups. The biotinylated microgels are a platform for bioactive water-based ink. Streptavidin binding was measured as a function of biotin density, and the results were interpreted with a new model that predicts the minimum local density of biotins required to capture a streptavidin. An amino-polyethylene glycol derivative of biotin gave higher biotin contents than a biotin hydrazide. However, the streptavidin content versus biotin content results for both biotin derivatives fell on the same master curve with maximum biotin coverage of 0.11?mg of bound streptavidin per milligram of biotinylated microgel. Exclusion experiments showed that streptavidin was too big to penetrate the cross-linked microgel structure; thus, the conjugated streptavidin was restricted to the microgel surface. The colloidal stability of the microgels was preserved, and the biotinylated products showed good hydrolytic stability.     

Quantitative Assessment of Tip Effects in Single-Molecule High-Speed Atomic Force Microscopy Using DNA Origami Substrates

High-speed atomic force microscopy (HS-AFM) is widely employed in the investigation of dynamic biomolecular processes at a single-molecule level. However, it remains an open and somewhat controversial question, how these processes are affected by the rapidly scanned AFM tip. While tip effects are commonly believed to be of minor importance in strongly binding systems, weaker interactions may significantly be disturbed. Herein, we quantitatively assess the role of tip effects in a strongly binding system using a DNA origami-based single-molecule assay. Despite its femtomolar dissociation constant, we find that HS-AFM imaging can disrupt monodentate binding of streptavidin (SAv) to biotin (Bt) even under gentle scanning conditions. To a lesser extent, this is also observed for the much stronger bidentate SAv–Bt complex. The presented DNA origami-based assay can be universally employed to quantify tip effects in strongly and weakly binding systems and to optimize the experimental settings for their reliable HS-AFM imaging.     

Quantitative Assessment of Tip Effects in Single‐Molecule High‐Speed Atomic Force Microscopy Using DNA Origami Substrates

High‐speed atomic force microscopy (HS‐AFM) is widely employed in the investigation of dynamic biomolecular processes at a single‐molecule level. However, it remains an open and somewhat controversial question, how these processes are affected by the rapidly scanned AFM tip. While tip effects are commonly believed to be of minor importance in strongly binding systems, weaker interactions may significantly be disturbed. Herein, we quantitatively assess the role of tip effects in a strongly binding system using a DNA origami‐based single‐molecule assay. Despite its femtomolar dissociation constant, we find that HS‐AFM imaging can disrupt monodentate binding of streptavidin (SAv) to biotin (Bt) even under gentle scanning conditions. To a lesser extent, this is also observed for the much stronger bidentate SAv–Bt complex. The presented DNA origami‐based assay can be universally employed to quantify tip effects in strongly and weakly binding systems and to optimize the experimental settings for their reliable HS‐AFM imaging.     

In silico engineering of tailored ink-binding ability at molecular printboards.

Molecular recognition between guest ink molecules and beta-cyclodextrin (beta-CD) cavities at self-assembled monolayers provides a molecular printboard for nanopatterning applications. We recently used molecular dynamics (MD) simulations to describe the specificity of ink-printboard binding and here extend the simulations to include charged cyclodextrin hosts, necessary to broaden the chemistry of molecular printboards and bind charged inks such as the ferrocenium cation. Shifting to high pH, or alternatively grafting a charged sidearm onto beta-CD, created three distinct types of anionic beta-CD cavity and we used electronic structure calculations and MD simulations to measure host-guest charge transfer and binding strengths. We find that steric recognition of uncharged organic molecules is retained at the charged printboards, and that improved guest-host electrostatic contacts can strengthen binding of larger inks while penalising small inks, enhancing the level of discrimination. A prudent choice of complementary host-guest shape and charge states thus provides a means of tuning both ink binding strength and specificity at molecular printboards.     

Cerium complexes of cyclodextrin dimers as efficient catalysts for luminol chemiluminescence reactions

The chemiluminescence of a luminol-H(2)O(2) system is found to be remarkably enhanced by the Ce(IV) complexes of EDTA-bridged cyclodextrin dimers. The dimers were proved to work much more efficiently than the corresponding monomer. The cavity shape of cyclodextrin moieties and their cooperation displayed an important role in amplifying the chemiluminescence. Further modification of either the cyclodextrin rims or the EDTA linker altered significantly the catalytic abilities of the cyclodextrin dimers, and the examination of the effect of substituents on the chemiluminescence outputs suggested that the proximity between the cyclodextrin cavity and the metallic center might account for the amelioration of the chemiluminescence output.     

Quantitative FT-IRRAS spectroscopic studies of the interaction of avidin with biotin on functionalized quartz surfaces

The interaction of avidin with biotin was studied on functionalized quartz surfaces terminated with 3-aminopropyltrimethoxysilane (3-APTMS), 2,2'-(ethylenedioxy)bis(ethylenediamine) (DADOO), and fourth-generation amine-terminated polyamidoamine (G4-NH2 PAMAM) dendrimers with the use of Fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS). In particular, the molecular recognition ability of these surfaces was quantified through FT-IRRAS in combination with the use of an alkyne dicobalt hexacarbonyl probe coupled with avidin. The degree of nonspecific adsorption of avidin was determined by exposure of the amine-terminated and/or biotinylated surfaces to solutions of biotin-saturated avidin. The results indicate that the biotinylated 3-APTMS layer exhibits a very low specific binding capacity for avidin (on the order of 0.15 pmol of avidin/cm2) and substantial nonspecific adsorption. Both the binding capacity and the specificity were greatly improved when the 3-APTMS layer on quartz was modified through serial chemisorption of glutaraldehyde (GA), DADOO, and/or G4-NH2 PAMAM dendrimer layers. Among these layers, the biotinylated G4-NH2 PAMAM dendrimer layer exhibited the highest capacity for avidin binding (2.02 pmol of avidin/cm2) with a specificity of approximately 90%. This effect can be attributed to the efficient packing/ordering of the binding dendrimer layer, leading to a more dense and better organized layer of biotin headgroups on the subsequent biotinylated surface.     

Thermoresponsive dendronized copolymers for protein recognitions based on biotin-avidin interaction

Thermoresponsive biotinylated dendronized copolymers carrying dendritic oligoethylene glycol(OEG) pendants were prepared via free radical polymerization,and their protein recognitions based on biotin-avidin interaction investigated.Both first(PG1)and second generation(PG2)dendronized copolymers were designed to examine possible thickness effects on the interaction between biotin and avidin.Inherited from the outstanding thermoresponsive properties from OEG dendrons,these biotinylated cylindrical copolymers show characteristic thermoresponsive behavior which provides an envelope to capture avidin through switching temperatures above or below their phase transition temperatures(T_cps).Thus,the recognition of polymer-supported biotin with avidin was investigated with UV/vis spectroscopy and dynamic laser light scattering.In contrast to the case for PG1,the increased thickness for copolymer PG2 hinders partially and inhibits the recognition of biotin moieties with avidin either below or above its T_cp.This demonstrates the significant architecture effects from dendronized polymers on the biotin moieties to shift onto periphery of the collapsed aggregates,which should be a prerequisite for protein recognition.These kinds of novel thermoresponsive copolymers may pave a way for the interesting biological applications in areas such as reversible activity control of enzyme or proteins,and for controlled delivery of drugs or genes.     

Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells

Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. The structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). The binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. The interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. The apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines. The interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture.     

Characterization of an Inclusion Complex of 5,10,15,20-Tetrakis(4-sulfonatophenyl)-porphinato Iron and an O-Methylated β-Cyclodextrin Dimer Having a Pyridine Linker and Its Related Complexes in Aqueous Solution

The structure of a carbon monoxide adduct (CO-hemoCD) of a 1:1 complex of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinato iron(II) (Fe(II)TPPS) and an O-methylated β-cyclodextrin dimer having a pyridine linker (1) has been determined by means of NMR spectroscopy and molecular mechanics (MM) calculation. The results indicate the structure as that the sulfonatophenyl groups at the 5- and 15-positions of Fe(II)TPPS are incorporated into two cyclodextrin cavities of 1 to form a 1:1 inclusion complex (hemoCD), whose Fe(II) center is coordinated by a carbon monoxide (CO) molecule. CO-hemoCD possesses a C _2v symmetrical nature that is supported by MM calculation. The energy minimized structure of CO-hemoCD suggests that the CO–Fe(II) part is significantly covered by two cyclodextrin moieties resulting in a cage effect in CO binding phenomenon. Other spectroscopic results of relating complexes also support the structure of hemoCD deduced from the results concerning CO-hemoCD.