Specific Capture of Peptide‐Receptive Major Histocompatibility Complex Class I Molecules by Antibody Micropatterns Allows for a Novel Peptide‐Binding Assay in Live Cells

Binding assays with fluorescently labeled ligands and recombinant receptor proteins are commonly performed in 2D arrays. But many cell surface receptors only function in their native membrane environment and/or in a specific conformation, such as they appear on the surface of live cells. Thus, receptors on live cells should be used for ligand binding assays. Here, it is shown that antibodies preprinted on a glass surface can be used to specifically array a peptide receptor of the immune system, i.e., the major histocompatibility complex class I molecule H‐2K^b, into a defined pattern on the surface of live cells. Monoclonal antibodies make it feasible to capture a distinct subpopulation of H‐2K^b and hold it at the cell surface. This patterned receptor enables a novel peptide‐binding assay, in which the specific binding of a fluorescently labeled index peptide is visualized by microscopy. Measurements of ligand binding to captured cell surface receptors in defined confirmations apply to many problems in cell biology and thus represent a promising tool in the field of biosensors.     

Binding stoichiometry of DNA adducts with antibody studied by capillary electrophoresis and laser-induced fluorescence

Four oligonucleotides (fluorescently labeled and unlabeled 16- and 90-mer), each containing a single adduct of benzo[a]pyrene diol epoxide (BPDE), were synthesized and used to study the binding stoichiometry between the DNA adduct and its antibody. The free oligonucleotide and its complexes with mouse monoclonal antibody were separated using capillary electrophoresis and detected with laser-induced fluorescence (LIF). Two complexes, representing the 1:1 and 1:2 stoichiometry between the antibody and the DNA adduct, were clearly demonstrated. The stoichiometry depended upon the relative concentrations of the antibody and the DNA adducts. A new approach examining the binding of the antibody with a mixture of a tetramethylrhodamine (TMR)-labeled and unlabeled BPDE-16-mer revealed insights on ligand redistribution and exchange between the labeled and unlabeled BPDE-16-mer oligonucleotides in the complexes. The observation of this unique behavior has not been possible previously with other binding studies. A mixture of the antibody with the TMR-labeled BPDE- 16-mer and an unlabeled BPDE-90-mer further revealed the formation of three fluorescent complexes: antibody with one TMR-BPDE-16-mer molecule, antibody with two TMR-BPDE- 16-mer molecules, and antibody with one TMR-BPDE-16-mer and one BPDE-90-mer. The three complexes clearly demonstrated binding stoichiometry and ligand redistribution/exchange.     

A quantitative assessment of heterogeneity for surface-immobilized proteins

Many biotechnological applications use protein receptors immobilized on solid supports. Although, in solution, these receptors display homogeneous binding affinities and association/dissociation kinetics for their complementary ligand, they often display heterogeneous binding characteristics after immobilization. In this study, a fluorescence-based fiber-optic biosensor was used to quantify the heterogeneity associated with the binding of a soluble analyte, fluorescently labeled trinitrobenzene, to surface-immobilized monoclonal anti-TNT antibodies. The antibodies were immobilized on silica fiber-optic probes via five different immobilization strategies. We used the Sips isotherm to assesses and compare the heterogeneity in the antibody binding affinity and kinetic rate parameters for these different immobilization schemes. In addition, we globally analyzed kinetic data with a two-compartment transport-kinetic model to analyze the heterogeneity in the analyte-antibody kinetics. These analyses provide a quantitative tool by which to evaluate the relative homogeneity of different antibody preparations. Our results demonstrate that the more homogeneous protein preparations exhibit more uniform affinities and kinetic constants.     

Studies of protein--DNA interactions by capillary electrophoresis/laser-induced fluorescence polarization

Protein-DNA interactions were studied on the basis of capillary electrophoretic separation of bound from free fluorescent probe followed by on-line detection with laser-induced fluorescence polarization. Changes in electrophoretic mobility and fluorescence anisotropy upon complex formation were monitored for the determination of binding affinity and stoichiometry. The method was applied to study the interactions of single-stranded DNA binding protein (SSB) with synthetic oligonucleotides and single-stranded DNA. Increases in fluorescence anisotropy and decreases in electrophoretic mobility upon their binding to SSB were observed for the fluorescently labeled 11-mer and 37-mer oligonucleotide probes. Fluorescence anisotropy and electrophoretic mobility were used to determine the binding constants of the SSB with the 11-mer (5 x 10(6) M(-1)) and the 37-mer (23 x 10(6) M(-1)). Alternatively, a fluorescently labeled SSB was used as a probe, and the formation of multiple protein-DNA complexes that differ in stoichiometry was observed. The results demonstrate the applicability of the method to study complex interactions between protein and DNA.     

Thin gold film-assisted fluorescence spectroscopy for biomolecule sensing

We present a configuration for fluorescence spectroscopy that exploits the optical properties of semitransparent gold films and widely available instrumentation. This method enables monitoring of biomolecule interactions with small molecules tethered on substrates in multicomponent environments. The neurotransmitter serotonin (5-hydroxytryptamine) was covalently attached to self-assembled monolayers on thin gold films at low density to facilitate antibody recognition. Protein-binding studies were performed in a fluorescently labeled immunoassay format. We find that the use of this method enables evaluation of nonspecific binding and relative quantification of specific binding between competing binding partners. This fluorescence spectroscopy technique has the potential to assess biosensor or medical device responses in complex biological matrices.     

Analysis of cell binding and internalization of multivalent PEG-based gene delivery vehicles

RGD peptides have been incorporated into several gene delivery vehicles to enhance specific interactions of nonviral vehicles with the cell surface. However, there are contradictory results regarding the effect of linear RGD peptides on specific cell surface binding of polyethylene glycol (PEG)-conjugated gene delivery vehicles. This study sought to understand how coupling RGD peptides to PEG vehicles affects cell binding and internalization using a novel four arm PEG backbone. Coupling multiple RGD peptides to the PEG backbone increased the affinity of the vehicle for the cell surface, and that the PEG backbone did not reduce the affinity of RGD peptides for integrin receptors in both kinetic and equilibrium studies. Kinetic studies suggest that cellular internalization of PEG-based vehicles is not regulated by the RGD peptides on the vehicle, but rather by nonspecific interactions with heparan sulfate proteoglycans either alone or in combination with integrins. These results suggest that while increasing the number of RGD peptides per vehicle increases cell binding, but it does not contribute to increased internalization or transfection efficiency.     

Rationally Designed Peptide Interface for Potential Modulated Cell Adhesion and Migration

Conformational changes of peptides are critically important in the control of their biological activities. Here, a quaternary ammonium group‐terminated RGD‐containing peptide (RGD‐NMe₃) is designed, which may undergo reversible conformational switch upon different electrochemical potentials. Potential responsive peptide interfaces are constructed on gold substrates with RGD‐NMe₃ in a tetra (ethylene glycol) background. It is demonstrated that by applying positive and negative potentials, the RGD peptide can be reversibly switched between linear and cyclic conformation, which can be used in reversible controlling of cell adhesion/migration on the interface. Furthermore, by combining microfluidics, adhesion of the cells in specific areas on the surface and subsequent directional migration of the cells can be controlled. It is believed that this straightforward potential modulation mechanism for peptide conformation control may find a wide use in design responsive peptide interfaces.     

Method for distinguishing specific from nonspecific protein-ligand complexes in nanoelectrospray ionization mass spectrometry

A new methodology for distinguishing between specific and nonspecific protein-ligand complexes in nanoelectrospray ionization mass spectrometry (nanoES-MS) is described. The method involves the addition of an appropriate reference protein (P(ref)), which does not bind specifically to any of the solution components, to the nanoES solution containing the protein(s) and ligand(s) of interest. The occurrence of nonspecific protein-ligand binding is monitored by the appearance of nonspecific (P(ref) + ligand) complexes in the nanoES mass spectrum. Furthermore, the fraction of P(ref) undergoing nonspecific ligand binding provides a quantitative measure of the contribution of nonspecific binding to the measured intensities of protein and specific protein-ligand complexes. As a result, errors introduced into protein-ligand association constants, K(assoc), as determined with nanoES-MS, by nonspecific ligand binding can be corrected. The principal assumptions on which this methodology is based, namely, that the fraction of proteins and protein complexes that engage in nonspecific ligand binding during the nanoES process is determined by the number of free ligand molecules in the offspring droplets leading to gaseous ions and is independent of the size and structure of the protein or protein complex, are shown to be generally valid. The application of the method for the determination of K(assoc) for two protein-carbohydrate complexes, under conditions where nonspecific ligand binding is prevalent, is demonstrated.     

Determination of binding constants on microarrays with confocal fluorescence detection

Confocal laser scanning microscopy was employed for the determination of binding constants of receptor-ligand interactions in a microarray format. Protocols for a localized immobilization of amine containing substances on glass via GOPTS (3-glycidyloxypropyl)trimethoxysilane) were optimized with respect to the detection of ligand binding by fluorescence. Compatibility with miniaturization by nanopipetting devices was ensured during all steps. The interaction of the tripeptide L-Lys-D-Ala-D-Ala with vancomycin immobilized on glass served as a model. To minimize consumption of ligand, binding constants were determined by stepwise titration of binding sites. The binding constant of the unlabeled ligand was determined by competitive titration with a fluorescently labeled analogue. The determined binding constants agreed well with those determined by other techniques, previously. Labeled ligand bound stronger than the unlabeled one. This difference was dye-dependent. Still, binding was specific for the tripeptide moiety confirming that ligand and fluorescent analogue competed for the same binding sites these results validate the determination of binding constants by competitive titration. The protocols established for confocal fluorescence detection are applicable to axially resolved detection modalities and screening for unlabeled ligands by competitive titration in general.     

Cell chip with nano-scale peptide layer to detect dopamine secretion from neuronal cells

A cell chip with a nano-scaled thin film of cysteine modified synthetic oligopeptide C(RGD)4 was fabricated to detect dopamine secretion from neuronal cells. Thin C(RGD)4 peptide layer was fabricated on chip surface for increasing the binding affinity of cells to gold electrode surface, which is essential for the electrochemical detection of dopamine released from PC12 cells. The structural formation of the peptide thin film was confirmed by both atomic force microscopy (AFM) and scanning electron microscopy (SEM). Redox characteristics of chemical dopamine were firstly characterized by voltammetric tool to compare the dopamine released from PC12 cells. Cells grown on the chip were then subjected to cyclic voltammetric (CV) analysis after 48 hours of incubation. The intensities of reduction peaks were found to be increased with increasing the concentrations of PC12 cells. In addition, the electrochemical redox signal increased more in the cells treated with glucose and potassium compared to the control group. Hence, the developed cell chip can be used to determine the effects of drugs on living cells electrochemically.     

Three-dimensional mesoporous gold film to enhance the sensitivity of electrochemical detection

Cell-cell and cell-extracellular matrix (ECM) adhesion are fundamental and important in the development of a cell-based chip. In this study, a novel, simple, rapid, and one-step technique was developed for the fabrication of a uniform three-dimensional mesoporous gold thin film (MPGF) onto a gold (Au) coated glass plate based on an electrochemical deposition method. Scanning electron microscopy images demonstrated that the resulting MPGF electrode had uniformly distributed pores with diameters of about 20 nm. The cyclic voltammetric behavior of [Fe(CN)(6)](4-/3-) coupled onto MPGF and Au electrodes demonstrated that the MPGF electrode had a higher electrocatalytic sensitivity and reversibility than the bare Au electrode. The Arg-Gly-Asp (RGD) sequence containing the peptide was immobilized on the MPGF and bare Au substrates. HeLa cancer cells were then cultured on the RGD peptide layer. The successful immobilization of the peptide and cells was confirmed by atomic force microscopy. The cell proliferation and viability were evaluated by cyclic voltammetry and Trypan blue dyeing assay. These results indicated that the RGD/MPGF modified electrodes showed an electrochemical sensitivity in the detection of cancer cells which is approximately three times higher, especially at low cell density, than RGD/Au electrodes. This much improved sensitivity of the MPGF modified electrode demonstrates the potential for the fabrication of a highly sensitive and low-cost cell-based chip for rapid cancer detection.     

Nanolithographic control of the spatial organization of cellular adhesion receptors at the single-molecule level

The ability to control the placement of individual molecules promises to enable a wide range of applications and is a key challenge in nanoscience and nanotechnology. Many biological interactions, in particular, are sensitive to the precise geometric arrangement of proteins. We have developed a technique which combines molecular-scale nanolithography with site-selective biochemistry to create biomimetic arrays of individual protein binding sites. The binding sites can be arranged in heterogeneous patterns of virtually any possible geometry with a nearly unlimited number of degrees of freedom. We have used these arrays to explore how the geometric organization of the extracellular matrix (ECM) binding ligand RGD (Arg-Gly-Asp) affects cell adhesion and spreading. Systematic variation of spacing, density, and cluster size of individual integrin binding sites was used to elicit different cell behavior. Cell spreading assays on arrays of different geometric arrangements revealed a dramatic increase in spreading efficiency when at least four liganded sites were spaced within 60 nm or less, with no dependence on global density. This points to the existence of a minimal matrix adhesion unit for fibronectin defined in space and stoichiometry. Developing an understanding of the ECM geometries that activate specific cellular functional complexes is a critical step toward controlling cell behavior. Potential practical applications range from new therapeutic treatments to the rational design of tissue scaffolds that can optimize healing without scarring. More broadly, spatial control at the single-molecule level can elucidate factors controlling individual molecular interactions and can enable synthesis of new systems based on molecular-scale architectures.     

Air-stable G protein-coupled receptor microarrays and ligand binding characteristics

This paper described novel strategies to achieve air-stable G protein-coupled receptor (GPCR) microarrays and the uses of the microarrays for ligand profiling. Specifically, GPCR cell membrane fragments were suspended in a buffered solution containing bovine serum albumin (BSA) and disaccharide sucrose or trehalose and used for fabricating GPCR microarrays. During the array fabrication and postfabrication processes, BSA molecules were found to effectively form packed layer(s) surrounding the GPCR membranes immobilized onto the predetermined printing area, thereby stabilizing the membrane microspots. The use of disaccharides was shown to protect the integrity and functionality of GPCR microarrays from the typical deterioration of the membranes when fabricated and stored under dry conditions. To utilize the ability of fluorescence technology for multichannel detection as well as to maximize the capability of GPCR microarrays for multiplexed binding assays, several fluorescently labeled ligands were synthesized and optimized for multiplexing binding assays. A schematic microarray of five GPCRs had been used as a model for characterizing the association and dissociation rate constants of labeled ligands binding to their respective receptors in the microarrays. Interestingly, distinct receptor-ligand interactions exhibited different dependence on the type of pH reagent as well as the species and concentration of cations used in a binding assay buffered solution. The potential mechanisms and implications for the uses of air-stable GPCR microarrays were discussed.     

Fluorescence polarization based displacement assay for the determination of small molecules with aptamers

The conversion of an aptamer-target binding event into a detectable signal is an important step in the development of aptamer-based sensors. In this work, we show that the displacement of a fluorescently labeled oligo from the aptamer by the target can be detected by fluorescence polarization (FP). We used Ochratoxin A (OTA), a small organic molecule (MW = 403) as a case study. A detection limit of 5 nM OTA was achieved. The method presented here provides an advantage over fluorophore-quenching systems and other steady-state fluorescence approaches in that no modification of the aptamer or the target is required. Additionally, the signal is produced by the displacement event itself, so no further aggregation or conformational events have to be considered. This analytical method is particularly useful for small targets, as for large targets a direct measurement of the FP change of a labeled aptamer upon binding can be used to determine the concentration of the target. The results presented here demonstrate that aptamers and inexpensive labeled oligos can be used for rapid, sensitive, and specific determination of small molecules by means of FP.     

Analysis of the (Trimethylsilyl)propionic Acid-β(12-28) Peptide Binding Equilibrium with NMR Spectroscopy

The binding of a small molecule, (trimethylsilyl)propionic acid (TSP), to a 17-residue peptide, β(12-28), is examined using (1)H NMR spectroscopy. β(12-28) (VHHQKLVFFAEDVGSNK) is a central fragment of the 40-42-residue Alzheimer's-associated Aβ peptide. This peptide has been previously shown to form soluble aggregates in low-pH aqueous solution. The TSP resonance is broadened appreciably in solutions containing relatively high concentrations (～2 mM) of the peptide. The changes in TSP line width measured by titration of a peptide solution with TSP indicate a 1:1 binding stoichiometry. If the concentrations of both the peptide and TSP are reduced by 1 order of magnitude, the resonances of both species are sharp, suggesting that TSP binds predominately to the aggregated peptide. Nuclear Overhauser effect experiments indicate that the TSP interacts predominately with the side chains of the aliphatic peptide residues Leu(17) and Val(18). Pulsed-field gradient NMR measurements of TSP and peptide diffusion coefficients provide a more quantitative picture of the TSP-peptide binding equilibrium. The measured diffusion coefficients were used to calculate the fractions of the free and bound TSP. These results substantiate the conclusion that the stoichiometry of the TSP-peptide binding equilibrium is essentially 1:1 and further indicate anticooperative behavior in solutions containing an excess of TSP resulting in a dissociation of the peptide aggregates.     

Immobilization of RGD peptide on HA coating through a chemical bonding approach

In this work, Arg-Gly-Asp (RGD) sequence containing peptide was immobilized on hydroxyapatite (HA) coatings through a chemical bonding approach in two steps, surface modification with 3-aminopropyltriethoxysilane (APTES) and RGD immobilization. The results indicate that RGD has been successfully immobilized on HA coatings. Comparing with physical adsorption coatings, the chemically bonded RGD on the coatings shows much better anti-wash-out ability. Since RGD is able to recognize cell-membrane integrins on biointerfaces, the present method will be an effective way to favor interaction of cells with HA coatings.     

Two-photon excitation fluorescence cross-correlation assay for ligand-receptor binding: cell membrane nanopatches containing the human micro-opioid receptor

Current ligand-receptor binding assays for G-protein coupled receptors cannot directly measure the system's dissociation constant, Kd, without purification of the receptor protein. Accurately measured Kd's are essential in the development of a molecular level understanding of ligand-receptor interactions critical in rational drug design. Here we report the introduction of two-photon excitation fluorescence cross-correlation spectroscopy (TPE-FCCS) to the direct analysis of ligand-receptor interactions of the human micro opioid receptor (hMOR) for both agonists and antagonists. We have developed the use of fluorescently distinct, dye-labeled hMOR-containing cell membrane nanopatches ( approximately 100-nm radius) and ligands, respectively, for this assay. We show that the output from TPE-FCCS data sets can be converted to the conventional Hill format, which provides Kd and the number of active receptors per nanopatch. When ligands are labeled with quantum dots, this assay can detect binding with ligand concentrations in the subnanomolar regime. Interestingly, conjugation to a bulky quantum dot did not adversely affect the binding propensity of the hMOR pentapeptide ligand, Leu-enkephalin.     

Dextran‐based coating system for the immobilization of cell adhesion promoting molecules on titanium surfaces

Immobilization of adhesive peptides interacting with cellular integrin receptors onto metallic implant surfaces represents a promising approach to improve osseointegration of implants into the surrounding tissue. In the present study, a functional dextran‐based coating system consisting of an amino titanate adhesion promoter with dendritic structure and a carboxymethyl dextran was established to bind an RGD‐containing adhesive peptide via a selective coupling methodology onto titanium surfaces. The three‐step reaction procedure was characterized by X‐ray photoelectron spectroscopy. In cell adhesion experiments it could be demonstrated that dextran coatings containing immobilized RGD promote attachment and spreading of fibroblast and pre‐osteoblastic cells compared to native as well as CMD‐coated titanium surfaces without RGD. The direct attachment of the RGD sequence to the metal surface via the amino titanate adhesion promoter did not increase pre‐osteoblastic cell spreading, whereas coupling of RGD to the polymeric carboxy­methyl dextran layer slightly enhanced spreading of the cells.     

Investigations on the in vitro bioactivity of swift heavy oxygen ion irradiated hydroxyapatite

Poly(propylene fumarate) (PPF) is an ultraviolet-curable and biodegradable polymer with potential applications for bone regeneration. In this study, we designed and fabricated three-dimensional (3D) porous scaffolds based on a PPF polymer network using micro-stereolithography (MSTL). The 3D scaffold was well fabricated with a highly interconnected porous structure and porosity of 65%. These results provide a new scaffold fabrication method for tissue engineering. Surface modification is a commonly used and effective method for improving the surface characteristics of biomaterials without altering their bulk properties that avoids the expense and long time associated with the development of new biomaterials. Therefore, we examined surface modification of 3D scaffolds by applying accelerated biomimetic apatite and arginine-glycine-aspartic acid (RGD) peptide coating to promote cell behavior. The apatite coating uniformly covered the scaffold surface after immersion for 24 h in 5-fold simulated body fluid (5SBF) and then the RGD peptide was applied. Finally, the coated 3D scaffolds were seeded with MC3T3-E1 pre-osteoblasts and their biologic properties were evaluated using an MTS assay and histologic staining. We found that 3D PPF/diethyl fumarate (DEF) scaffolds fabricated with MSTL and biomimetic apatite coating can be potentially used in bone tissue engineering.     

Preferential binding of a novel polyhistidine peptide dendrimer ligand on quantum dots probed by capillary electrophoresis

Fluorescence detection coupled to capillary electrophoresis (CE-FL) effectively separates molecules in solution and at the same time allows monitoring of the fluorescence spectrum of each individual species. The integration of separation and fluorescence detection results in a powerful method superior to the ensemble in-cuvette fluorescence measurement, in probing the binding interaction between ligands and quantum dots (QDs) in complex solutions. Fo?rster resonance energy transfer (FRET) between fluorescent ligands and QDs could be readily detected by CE-FL, which together with the migration times of the fluorescent peaks provides an indication of the binding interaction between ligands and QDs. In the present study, the binding interaction between a multivalent ligand, polyhistidine peptide denderimer (PHPD), and CdSe-ZnS QDs was probed by CE-FL using the monovalent hexahistidine peptide as a control. Cy5 labeled PHPD assembles on glutathione capped QDs, showing a higher FRET signal than that of the assembly between Cy5 labeled hexahistidine peptide and QDs. Capillary electrophoresis further revealed that PHPD outcompetes other QD binding small molecules, peptides, and proteins in cell lysate. Our study demonstrates the power of CE-FL in analyzing the binding interaction between ligands and QDs in a complex binding solution. It also shows that clustering surface binding motifs yields multivalent ligands that can preferentially assemble with nanoparticles.