Immobilization Of Peptides Research Articles

Immobilization of Peptides with Distinct Biological Activities onto Stem Cell Culture Substrates Using Orthogonal Chemistries

We have used the orthogonal carbodiimide condensation and copper-catalyzed azide-alkyne "click" cycloaddition (CuAAC) reactions to prepare self-assembled monolayers that present distinct peptides to stem cells in a bioinert background. The approach involved first forming mixed SAMs with three components: (i) an azide-terminated hexaethylene glycol alkanethiolate (HS-EG6-N3), (ii) a carboxylate-terminated hexaethylene glycol alkanethiolate (HS-EG6-COOH), and (iii) a triethylene glycol alkanethiolate (HS-EG3). An acetylene-bearing peptide and an amine-terminated peptide were then immobilized to these substrates using a "click" CuAAC reaction and a carbodiimide condensation reaction, respectively. Polarization-modulated infrared reflectance-absorbance spectroscopic analysis demonstrated formation of well-ordered, close-packed self-assembled monolayers (SAMs), chemoselective conjugation of amine-terminated peptides to surface carboxylate groups, and subsequent conjugation of acetylene-terminated peptides to the azide groups on SAMs. Varying the mole fraction of HS-EG6-N3, HS-EG6-COOH, and HS-EG3 during SAM formation allowed for control over the densities of each peptide on the substrate. Substrates presenting varying surface densities of RGESP (a nonfunctional peptide), RGDSP (a cell adhesion peptide), or TYRSRKY (a heparin/heparan sulfate-binding peptide) were then used to characterize the relationship between peptide surface density and human mesenchymal stem cell (hMSC) adhesion. Results demonstrate that RGESP does not influence RGDSP-mediated adhesion of hMSCs, which indicates that a second peptide with distinct bioactivity can be immobilized alongside RGDSP to characterize the influence of two peptides on hMSC behavior. Our results also demonstrate that RGDSP and TYRSRKY act synergistically to promote hMSC adhesion in the absence of serum. Interestingly, heparin sequestered by TYRSRKY inhibits cell adhesion on substrates presenting RGDSP = 0.1% and > or = 0.1% TYRSRKY or RGDSP = 1% and > or = 0.5% TYRSRKY. Taken together, these results indicate that two peptides can be controllably presented to stem cells on the same otherwise bioinert SAM substrate, and that multiple, distinct extracellular moieties act in concert to regulate hMSC adhesion.

Preparation, characterization, and binding profile of molecularly imprinted hydrogels for the peptide hepcidin

AbstractMolecularly imprinted hydrogels for the capture of the peptide hormone hepcidin were prepared by water‐in‐oil (w/o) suspension polymerization under mild conditions. Spherical and relatively uniformly sized gel beads were routinely obtained after optimization of the synthetic methodology. The polymers were analyzed by Fourier transform infrared spectroscopy, optical microscopy, and scanning electron microscopy. Although the imprinted materials exhibited higher affinity towards the epitope template (hepcidin N‐terminus) than their corresponding blank polymers, the full‐length target peptide was found strongly bound to all the hydrogels tested. However, by using whole fluorescent hepcidin as the print species, the imprinting effect was more pronounced. Moreover, bovine serum albumin did not bind to the poly N‐isopropylacrylamide (PNIPAm)‐based polymers. Thus, polymeric “sponges” for biomacromolecules with size‐exclusion effect were developed, useful for peptide concentration, immobilization and/or purification from serum samples. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 1721–1731, 2010

An XPS study on the covalent immobilization of adhesion peptides on a glass surface

Covalent attachment of adhesive peptides to biomaterials surfaces can result in the formation of a bioactive and biomimetic surface. We have demonstrated that titanium surfaces grafted with adhesion peptides, reproducing sequences of fibronectin and vitronectin, can increase osteoblast adhesion compared to non-treated surfaces. We now extend our investigation to peptide immobilization on glass for studying human osteoblast adhesion and spreading. Silanization was used to anchor adhesion peptides to the glass surface through a selective or a non-selective immobilization. Investigated samples were analysed by XPS spectroscopy. Comparison between the results obtained using two different peptides and applying selective and non-selective immobilization will be discussed.

Native chemical ligation in the synthesis of internally modified oligonucleotide–peptide conjugates

Peptide-oligonucleotide conjugates have frequently been synthesized to improve cellular delivery of antisense or antigene compounds, to allow the immobilization of peptide and protein conjugates on DNA arrays, or to decorate nucleic acid architectures with peptide functions. In such applications, the site of conjugation is of little importance, and peptides have predominantly been appended to one of the terminal ends of the oligonucleotide by using an oxime-, thioether-, or disulfide-linkage or native chemical ligation. We, herein, demonstrate the first coupling of peptides to sequence internal sites. This attachment mode provides better control of the spatial arrangement of peptides presented by self-assembled nucleic acid scaffolds. Internal modification requires special phosphoramidite building blocks that can be used in automated DNA synthesis. For this purpose, Fmoc/StBu-protected cysteine was attached via an aminopropargyl linker to the C5-position of uridine. The rigid triple bond conferred a high reactivity in native chemical ligation reactions of 5-6mer peptide thioesters with up to 15 nucleotides long oligonucleotides. The desired peptide-oligonucleotide conjugates were obtained in high yields after purification. UV melt experiments revealed that the peptide modification does not hamper nucleic acid hybridization. This finding marked an important step in our research program devoted to studies of multivalent presentation of peptides via modular assembly of nucleic acid complexes.

Immobilization and Incorporation of Antigenic Peptide P17-1 from HIV-1 P17 Protein in Nanostructured Films

The immobilization of antigenic peptides in nanostructured films is promising for the development of highly specific immunosensors. In this work, we analyze the peptide p17-1 (LSGGELDRWEKIRLRPGG), derived from the HIV-1 p17 protein, immobilized into Layer-by-Layer (LbL) films and incorporated into Langmuir monolayers of phospholipids. The LbL film was assembled using different polyelectrolytes but only poly(allylamine) hydrochloride (PAH) was efficient for the peptide immobilization. The intensity in the UV-Vis. spectra of PAH/p17-1 films increased exponentially with the number of layers, which may indicate that the peptide can be reorganized in each bilayer adsorbed. Fluorescence and circular dichroism (CD) spectra indicated that the interaction with the film did not induce an alpha helix conformation in p17-1, analogously to what occurs in an aqueous solution and in contrast to the organized peptide in a methanol solution. The maximum emission for p17-1 fluorescence occurred at 340 nm in methanol, compatible with tryptophan residue buried in the solvent, while for p17-1 in an LbL film the maximum appeared at 355 nm. This red shift is consistent with the tryptophan being exposed to the environment. The CD spectra confirmed these results showing the random structure for p17-1 in the LbL films and an α-helix structure in methanol solution. The lack of structure is the probable reason for the low sensitivity toward anti-p17 observed in amperometric sensors made with PAH/p17-1 LbL films. With regard to the Langmuir monolayers, p17-1 was found to affect the surface pressure isotherms of dipalmitoyl phosphatidyl glycerol (DPPG), even at a concentration as low as 0.5 mol%. This cooperative interaction of p17-1 and DPPG may perhaps be exploited in designing new architectures for producing immunosensors based on antigenic peptides.

LPS interactions with immobilized and soluble antimicrobial peptides

A promising approach in sepsis therapy is the use of peptides truncated from serum- and membrane-proteins with binding domains for LPS: antimicrobial peptides (AMPs). AMPs can be useful in combination with conventional antibiotics to increase killing and neutralize LPS. Although many AMPs show a high specificity towards bacterial membranes, they can also exhibit toxicity, i.e. non-specific membrane lysis, of mammalian cells such as erythrocytes and therefore, unsuitable as systemic drugs. A way to overcome this problem may be an extracorporeal therapy with immobilized peptides. This study will compare neutralization of LPS using different AMPs in solution and when immobilized on to solid phases. The peptides ability to neutralize LPS-induced cytokine release in whole blood will also be tested. The peptides are truncated derivates from the known AMPs LL-37, SC4, BPI, S3Δ and CEME. Two different methods were used to immobilize peptides, biomolecular interaction analysis, and Pierce SulfoLink Coupling Gel. To investigate LPS binding in solution the LAL test was used. After whole blood incubation with LPS and AMPs ELISA was used to measure TNFα, IL-1β and IL-6 production. The results suggest that immobilization of antimicrobial peptides does not inhibit their capacity to neutralize LPS, although there are differences between the peptides tested. Thus, peptides derived from LL-37 and CEME were more efficient both in LPS binding and neutralizing LPS-induced cytokine production.

Prevention of bacterial biofilms by covalent immobilization of peptides onto plasma polymer functionalized substrates

In this study, robust antibacterial coatings were created on stainless steel through the covalent grafting of antibacterial peptides onto an organic–polymeric interlayer deposited by RF-glow discharge plasma. X-Ray photoelectron spectroscopy was used to characterize and optimize the two steps of the coating process. The biocidal activity of these surfaces was demonstrated against both Gram+ and Gram− bacteria using ISO tests. 3 to 6 log10 reductions of both Gram+ and Gram− bacterial strains were obtained compared to uncoated stainless steel and depending on the particular antibacterial peptide immobilized. Importantly the antibacterial surfaces were resistant to several cleaning conditions. The latter is significant as the stability of such antibacterial surfaces in close to real life conditions is a major concern and leaching, de-lamination, rearrangement and ageing of the coating can lead to insufficient long term biofilm resistance of the surface.

Degree of immobilization of synthetic RGDS(PO3H2)PA peptides on titanium surfaces

The purpose of this study was to characterize the chemical interaction between titanium surfaces and the peptide RGDS(PO(3)H(2))PA (P-RGD) synthesized from RGD peptide (RGD) and o-phospho-L-serine (P-Ser), and to determine the degree of peptide immobilization on the titanium surface. X-ray photoelectron spectroscopy showed that the adsorption amount of RGD was significantly smaller than those of P-Ser and P-RGD (p<0.05). Furthermore, although it appeared that P-RGD bonded to the surface, ultrasonic rinsing with water caused it to dissociate, releasing RGD and leaving only S(PO(3)H(2))PA bonded to the surface. These findings show that although it remains difficult to obtain a stable P-RGD layer, the phosphate functional group greatly improves immobilization of the molecule on titanium surfaces.

PbSe nanocrystal growth as nanocubes and nanorods on peptide nanotubes via different directed-assembly pathways

Pb-binding TAR-1 peptides (Ile-Ser-Leu-Leu-His-Ser-Thr) were covalently conjugated on a bolaamphiphile peptide nanotube substrate and the precursors of PbSe were incubated at room temperature. This resulted in the growth of highly crystalline PbSe nanocubes on this biomimetic cylindrical substrate. The growth mechanism to generate nanocubes occurs via the directed self-assembly of nanoparticles and then nanoparticle fusion. The peptide conformation and the cylindrical peptide nanotube substrate play important roles in the mesoscopic crystallization of PbSe nanocubes. Changing the buffer for the peptide immobilization process from 2-(N-morpholino)ethanesulfonic acid to phosphate induces a transformation in the nanocrystal shape from nanocube to nanorods. The conformational change of the TAR-1 peptide on the nanotubes due to the change in the buffer seems to be responsible for aggregating intermediate nanoparticles in different directions for the directed fusion and mesoscopic crystallization of PbSe into the different shapes.

Parylene-A coated microplate for covalent immobilization of proteins and peptides

Various types of microplates made of polystyrene have been widely used for immunoassays. A new microplate suitable for the covalent immobilization of proteins and peptides was developed by thermal deposition of amino-modified parylene (parylene-A) on the microplate. The primary amine groups of the parylene-A was exploited for the covalent coupling of proteins and peptides. The optical transmittance at the wavelength of 400–500 nm was estimated to be suitable for the application to immunoassays. The immobilization efficiency of the parylene-A coated microplate was demonstrated to be far improved in comparison to the conventional microplate by using horseradish peroxidase (HRP), anti-HRP antibody and a peptide with 9-residues as model biomolecules.

Poly(N-isopropylacrylamide) gel beads prepared by waterin- oil suspension polymerization for the adsorption of fluorescently tagged hepcidin

AbstractPoly(N-isopropylacrylamide) (PNIPAm) gel beads were prepared by water in oil (w/o) suspension polymerization using aqueous acetonitrile to dissolve the monomer phase, toluene or cyclohexane as the continuous phase and ethyl cellulose (EC) as the stabilizer. The beads were analyzed by FT-IR and their morphology was investigated by optical and electron microscopy. The advantages of this novel synthetic method for PNIPAm preparation include efficient polymerization and mild experimental conditions, as well as the formation of spherical and relatively uniformly sized particles. The microgels were then evaluated for their efficiency to absorb fluorescently labelled hepcidin. The beads were found to rapidly capture the target peptide from neutral aqueous solutions. Notably, bovine serum albumin (BSA) did not bind to the polymer beads at the cross-linking ratio used; therefore, size-exclusion properties may be controlled by finely tuning monomer to cross-linker ratio and/or monomer concentration during polymer design. The route could potentially be useful for diagnostic, drug delivery or peptide immobilization, as well as for the molecular imprinting of peptides and proteins.

Facile construction of fluorescent peptide microarrays: One-step fluorescent derivatization of sub-microscale peptide aldehydes for selective terminal immobilization

In this note, we demonstrate the utility of bifunctional fluorescent linkers to facilitate the construction of peptide microarrays with either an N- or a C-terminal alkylamine for directionally preferred peptide immobilization. Significantly, these small tags facilitate high-performance liquid chromatography (HPLC) profiling while limiting interference with antigen–antibody interactions after peptide immobilization. In a model peptide–antibody binding assay, a sequence-dependent orientation effect of antibody binding to a series of peptide ligands was demonstrated. This approach provides a strategy that can be applied to a variety of peptide microarray-based detection systems.

Development of peptide immobilization method using PS-tagged cushion protein

Development of peptide immobilization method using PS-tagged cushion protein

Photochemical Immobilization of Proteins and Peptides on Benzophenone-Terminated Boron-Doped Diamond Surfaces

The successful covalent linking of green fluorescence protein and streptavidin to patterned benzophenone-modified boron-doped diamond (BDD) electrodes is demonstrated. Photoreactive benzophenone moieties were covalently grafted to oxidized diamond surfaces via an esterification reaction. Patterned BDD surfaces were obtained using a UV/ozone lithographic approach either on hydrogen-terminated BDD or on poly(ethylene)-glycol-modified BDD surfaces. UV light (lambda = 365 nm) irradiation of the patterned BDD surfaces in the presence of green fluorescence protein (GFP) or streptavidin resulted in the covalent immobilization of the proteins. The presence of poly(ethylene) glycol chains reduces significantly the nonspecific adsorption of the proteins. The success of the photoimmobilization of streptavidin was evidenced through biomolecular interaction with avidin. The preservation of the biological activity was furthermore underlined by photoimmobilization of peptides directly onto benzophenone modified BDD using a photomask.

The effect of matrix stiffness on mesenchymal stem cell differentiation in a 3D thixotropic gel

Recent studies have demonstrated the effect of matrix stiffness on the phenotype and differentiation pathway of mesenchymal stem cells (MSCs). MSCs differentiated into neural, myogenic or osteogenic phenotypes depending on whether they were cultured on two-dimensional (2D) substrates of elastic moduli in the lower (0.1–1 kPa), intermediate (8–17 kPa) or higher ranges (34 kPa). In this study, MSCs were cultured in thixotropic gels of varying rheological properties, and similar results were found for the three-dimensional (3D) culture as for the previous findings in 2D culture. For the 3D cell cultures in thixotropic gels, the liquefaction stress ( τ y), the minimum shear stress required to liquefy the gel, was used to characterize the matrix stiffness. The highest expressions of neural (ENO2), myogenic (MYOG) and osteogenic (Runx2, OC) transcription factors were obtained for gels with τ y of 7, 25 and 75 Pa, respectively. Immobilization of the cell-adhesion peptide, RGD, promoted both proliferation and differentiation of MSCs, especially for the case of the stiffer gels (>75 Pa). This study demonstrated the usefulness of thixotropic gels for 3D cell culture studies, as well as the use of τ y as an effective measure of matrix stiffness that could be correlated to MSC differentiation.

Tat peptide mediated cellular uptake of SiO 2 submicron particles

Internalization of nano- and microparticles into live cells correlates closely with their potential applications, functions, cytotoxicity and intracellular drug delivery. Particularly, delivery of a large variety of cargoes such as proteins, peptides, nucleic acids and small particles into cells could be enhanced by some ligands such as Tat peptide. In this work, the ability of Tat mediated cellular uptake was assessed. The Tat peptide was covalently immobilized to fluorescein tagged SiO 2 particles (FITC–SiO 2–NH 2 particles) with a diameter of 200 nm. BCA protein assay determined that the grafting amount of the Tat peptide could be controlled within a range of 0–3.5 μg/mg SiO 2 particles by the Tat feeding amount. Surface immobilization of the Tat peptide did not bring apparent changes on the surface morphology and charge property of the SiO 2–NH 2 particles. By contrast, the surface charge of both the FITC–SiO 2–NH 2 particles and the FITC–SiO 2–Tat particles was reversed from slight positive in Dulbecco's Modified Eagles Medium (DMEM) to slight negative in DMEM/fetal bovine serum, conveying adsorption of plasma proteins on the particles. Flow cytometry measurement showed that the FITC–SiO 2–Tat particles were internalized by HepG2 cells with a significant faster rate and a higher number of particles than that of the FITC–SiO 2–NH 2 particles. Moreover, internalization of the Tat peptide decorated particles was less influenced by the low temperature at 4 °C. The Tat decoration affected the subcellular distribution of the particles as well, resulting in localization of the particles in the cell nucleus. No obvious cytotoxicity was detected for both the FITC–SiO 2–NH 2 particles and the FITC–SiO 2–Tat particles.

Surface immobilization of fibronectin-derived PHSRN peptide on functionalized polymer films – Effects on fibroblast spreading

The Pro-His-Ser-Arg-Asn (PHSRN) sequence in fibronectin is a second cell-binding site that synergistically affects Arg-Gly-Asp (RGD). The PHSRN peptide also induces cell invasion and accelerates wound healing. We report on the surface immobilization of PHSRN by spontaneous adsorption on polysiloxane thin films which have different surface free energy characteristics. Low-surface energy (hydrophobic) polysiloxane and the corresponding high-surface energy (hydrophilic) surfaces obtained by UV–ozone treatments were used as adsorbing substrates. The peptide adsorption process was investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy. Both adsorption kinetics and peptide rearrangement dynamics at the solid interface were significantly different on the surface-modified films compared to the untreated ones. Fibroblast cells cultures at short times and in a simplified environment, i.e., a medium-free solution, were prepared to distinguish interaction events at the interface between cell membrane and surface-immobilized peptide for the two cases. It turned out that the cell-adhesive effect of immobilized PHSRN was different for hydrophobic compared to hydrophilic ones. Early signatures of cell spreading were only observed on the hydrophilic substrates. These effects are explained in terms of different spatial arrangements of PHSRN molecules immobilized on the two types of surfaces.

Synthesis of specific nanoparticles for targeting tumor angiogenesis using electron-beam irradiation

Angiogenesis plays a critical role in both growth and metastasis of tumors. Vascular endothelial growth factor (VEGF) is an endogenous mediator of tumor angiogenesis. Blocking associations of the VEGF with its corresponding receptors (KDR) have become critical for anti-tumor therapy. A cyclo-peptide (CBO-P11), derived from VEGF, able to inhibit the interaction between the growth factor and its receptor, was synthesized in our laboratory to provide a target for angiogenesis. We have prepared biocompatible poly(vinylidene fluoride) (PVDF) nanoparticles in order to obtain long blood circulating systems. Electron-beam (EB) irradiation was used to activate the PVDF nanoparticles. From electron paramagnetic resonance (EPR) measurements, we studied the radical stability in order to optimize the radio-grafting of acrylic acid (AA). Further functionalization of PVDF-g-PAA nanoparticles with the cyclo-peptide via a spacer arm was also possible by performing coupling reactions. High resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) and MALDI mass spectrometry allowed us to follow each chemical step of this peptide immobilization. We designed a new nanodevice suggesting a great potential for targeting angiogenesis. 7727-21-1

Micropatterned Fiber Scaffolds for Spatially Controlled Cell Adhesion

Because the local microstructure plays a pivotal role for many biological functions, a wide range of methods have been developed to design precisely engineered substrates for both fundamental biological studies and biotechnological applications. However, these techniques have been by-and-large limited to flat surfaces. Herein, we use electrohydrodynamic co-spinning to prepare biodegradable three-dimensional fiber scaffolds with precisely engineered, micrometre-scale patterns, wherein each fiber is comprised of two distinguishable compartments. When bicompartmental fiber scaffolds are modified via spatially controlled peptide immobilization, highly selective cell guidance at spatial resolutions (<10 µm), so far exclusively reserved for flat substrates, is achieved. Microstructured fiber scaffolds may have utility for a range of biotechnological applications including tissue engineering or cell-based assays.

Immobilization of enzyme on detonation nanodiamond for highly efficient proteolysis

Immobilization of enzyme on detonation nanodiamond (dND, 3–10 nm) and its application for efficient proteolysis have been demonstrated. By evaluation of the Michaelis constant ( K m) and maximum velocity ( V max) of immobilized enzyme, its activity was not impaired significantly by immobilization. And enzyme immobilized on dNDs exhibited much better thermal and chemical stabilities than its free counterpart and maintained high activity even after 10 times reuse. The efficient proteolysis by trypsin immobilized on dNDs (dND-trypsin) is demonstrated with the digestion of myoglobin (or other model protein) in a short time (5 min). Large numbers of identified peptides obtained by dNDs-trypsin enable a higher degree of sequence coverage and more positive identification of proteins than those obtained by in-solution digestion and the commercial immobilized trypsin beads, respectively. Moreover, immobilization of peptide- N-glycosidase F (PNGase F) on dNDs was realized and resulted in faster sequential glycosidase digestion of glycopeptides in less than 10 min.