Immobilization Of Peptides Research Articles

Polyglycerol based coatings to reduce non-specific protein adsorption in sample vials and on SPR sensors

Coatings based on dendritic polyglycerol (dPG) were investigated for their use to control nonspecific protein adsorption in an assay targeted to analyze concentrations of a specific protein. We demonstrate that coating of the sample vial with dPG can significantly increase the recovery of an antibody after incubation. First, we determine the concentration dependent loss of an antibody due to nonspecific adsorption to glass via quartz crystal microbalance (QCM). Complementary to the QCM measurements, we applied the same antibody as analyte in an surface plasmon resonance (SPR) assay to determine the loss of analyte due to nonspecific adsorption to the sample vial. For this purpose, we used two different coatings based on dPG. For the first coating, which served as a matrix for the SPR sensor, carboxyl groups were incorporated into dPG as well as a dithiolane moiety enabling covalent immobilization to the gold sensor surface. This SPR-matrix exhibited excellent protein resistant properties and allowed the immobilization of amyloid peptides via amide bond formation. The second coating which was intended to prevent nonspecific adsorption to glass vials comprised a silyl moiety that allowed covalent grafting to glass. For demonstrating the impact of the vial coating on the accuracy of an SPR assay, we immobilized amyloid beta (Aβ) 1-40 and used an anti-Aβ 1-40 antibody as analyte. Alternate injection of analyte into the flow cell of the SPR device from uncoated and coated vials, respectively gave us the relative signal loss (1−RUuncoated/RUcoated) caused by the nonspecific adsorption. We found that the relative signal loss increases with decreasing analyte concentration. The SPR data correlate well with concentration dependent non-specific adsorption experiments of the analyte to glass surfaces performed with QCM. Our measurements show that rendering both the sample vial and the sensor surface is crucial for accurate results in protein assays.

Development of Pleurocidin-poly(vinyl alcohol) electrospun antimicrobial nanofibers to retain antimicrobial activity in food system application

Pleurocidin is a novel antimicrobial peptide with advantages of broad microbial inhibition spectrum and thermal/pH/salt-tolerance. However, direct application of Pleurocidin into food systems caused its activity loss. This study aims to preserve the bioactivity of Pleurocidin in real foodstuffs by incorporating it in poly(vinyl alcohol) electrospun nanofiber. A pattern of sustained release of Pleurocidin from fiber, following initial burst release was successfully achieved, which could be tuned by varying environmental temperature. In vitro study clearly illustrated that Pleurocidin-incorporated fiber could efficiently inhibit foodborne pathogens. More importantly, released Pleurocidin showed higher inhibition efficiency than free Pleurocidin against Escherichia coli O157:H7 in a real food system – apple cider, mainly due to the protection from negative influence of environmental components by polymeric fiber matrix. The maintenance of Pleurocidin activity suggests an extensively promising application of electrospinning technology in antimicrobial peptide immobilization towards improved food safety.

Kinetics of immobilisation and release of tryptophan, riboflavin and peptides from whey protein microbeads

This study investigated the kinetics of immobilisation and release of riboflavin, amino acids and peptides from whey microbeads. Blank whey microbeads were placed in solutions of the compounds. As the volume of microbeads added to the solution was increased, the uptake of the compounds increased, to a maximum of 95% for the pentapeptide and 56%, 57% and 45% for the dipeptide, riboflavin and tryptophan respectively, however, the rate of uptake remained constant. The rate of uptake increased with increasing molecule hydrophobicity. The opposite was observed in the release studies, the more hydrophobic compounds had lower release rate constants (kr). When whey microbeads are used as sorbents, they show excellent potential to immobilise small hydrophobic molecules and minimise subsequent diffusion, even in high moisture environments.

Biochemical surface modifications to titanium implants using the tresyl chlorideactivated method.

Tresyl chloride (2,2,2-trifluoroethanesulfonyl chloride)-activated method is an easy and simple method for immobilizing biological molecules onto a titanium surface. Tresyl chloride was directly dropped onto a titanium surface without any solvent. Tresylated titanium disks were immersed in a protein or peptide solution to give protein- or peptide-immobilized titanium. Frontier molecular orbital calculations suggested that tresylation of the OH compound improved its reactivity towards the amine groups of protein compounds. Quartz-crystal microbalance-dissipation measurements suggested that ionic interaction between the oxygen of the terminal hydroxyl groups of titanium and the nitrogen of fibronectin was important for fibronectin immobilization. Initial cell attachment was enhanced by fibronectin or collagen immobilization. Fibronectin and fibronectin-derived peptide immobilization improved the expression of specific genes related to bone formation. The immobilization of cytokine onto titanium enhanced bone formation following its implantation into rat femur defects. The tresyl chloride-activated method is useful for immobilizing biological molecules onto titanium surfaces.

Development of a catheter functionalized by a polydopamine peptide coating with antimicrobial and antibiofilm properties

Catheter-associated urinary tract infections (CAUTIs) are the most common hospital-acquired infections worldwide, aggravating the problem of antimicrobial resistance and patient morbidity. There is a need for a potent and robust antimicrobial coating for catheters to prevent these infections. An ideal coating agent should possess high antimicrobial efficacy and be easily and economically conjugated to the catheter surface. In this study, we report a simple yet effective immobilization strategy to tether a potent synthetic antimicrobial peptide, CWR11, onto catheter-relevant surfaces. Polydopamine (PD) was deposited as a thin adherent film onto a polydimethylsiloxane (PDMS) surface to facilitate attachment of CWR11 onto the PD-functionalized polymer. Surface characterization of the CWR11-tethered surfaces confirmed the successful immobilization of peptides onto the PD-coated PDMS. The CWR11-immobilized PDMS slides displayed excellent antimicrobial (significant inhibition of 5×104 colony-forming units of CAUTI-relevant microbes) and antibiofilm (∼92% enhanced antibacterial adherence) properties. To assess its clinical relevance, the PD-based immobilization platform was translated onto commercial silicone-coated Foley catheters. The CWR11-impregnated catheter displayed potent bactericidal properties against both Gram-positive and Gram-negative bacteria, and retained its antimicrobial functionality for at least 21days, showing negligible cytotoxicity against human erythrocyte and uroepithelial cells. The outcome of this study demonstrates the proof-of-concept potential of a polydopamine–CWR11-functionalized catheter to combat CAUTIs.

Effect of hydrophobic mismatch on domain formation and peptide sorting in the multicomponent lipid bilayers in the presence of immobilized peptides.

In the plasma membranes, many transmembrane (TM) proteins/peptides are anchored to the underlying cytoskeleton and/or the extracellular matrix. The lateral diffusion and the tilt of these proteins/peptides may be greatly restricted by the anchoring. Here, using the coarse-grained molecular dynamics simulation, we investigated the domain formation and peptide sorting in the ternary lipid bilayers in the presence of the immobilized peptide-grid and peptide-cluster. We mainly focused on examining the combining effect of the peptide immobilization and hydrophobic mismatch on the domain formation and peptide sorting in the lipid bilayers. Compared to the lipid bilayers inserted with free TM peptides, our results showed that, because of the tilt restriction imposed on the peptides, the hydrophobic mismatch effect more significantly influences the domain size, the dynamics of domain formation, and the peptide sorting in our systems. Our results provide some theoretical insights into understanding the formation of nanosized lipid rafts, the protein sorting in the lipid rafts and the interaction between the cytoskeleton, the extracellular matrix, and the plasma membranes.

An electrochemically switched smart surface for peptide immobilization and conformation control.

We report an electrochemically switched smart surface for controlled peptide immobilization and conformation control. This dynamic surface is based on self-assembled monolayers (SAMs) containing surface-bound trimethoxybenzene moieties, which can undergo electrochemically modulated surface activation to be stepwisely converted to two catechol derivatives. This new smart surface can be used to realize stepwise immobilization of a peptide, and more importantly, to control peptide conformation on a surface. We demonstrate herein that with one electrochemical activation step, a linear peptide containing an RGD sequence can be attached onto the SAMs. With the subsequence activation step, the attached linear RGD peptide can be converted into cyclic conformation. The SAMs bounded with linear and cyclic RGD exhibit different adhesion behaviors to fibroblasts cells. The reaction procedure can be well-monitored by cyclic voltammetry (CV), electrochemical surface enhanced Raman microscopy (EC-SERS), and X-ray photoelectron spectroscopy (XPS). It is believed this robust smart surface can find wide applications in surface immobilization of bioactive moieties.

ChemInform Abstract: Controlled Covalent Surface Immobilization of Proteins and Peptides Using Plasma Methods

AbstractReview: 67 refs.

Effectiveness of antimicrobial peptide immobilization for preventing perioperative cornea implant-associated bacterial infection.

Titanium (Ti) is a promising candidate biomaterial for an artificial corneal skirt. Antimicrobial peptide (AMP) immobilization may improve the bactericidal effect of the Ti substrate. In this study, we tested the bactericidal efficacy of a functionalized Ti surface in a rabbit keratitis model. A corneal stromal pocket was created by a femtosecond laser. The Ti films were then inserted into the pocket, and Staphylococcus aureus or Pseudomonas aeruginosa was inoculated into the pocket above the implant films. The corneas with Ti-AMP implants were compared with the corneas implanted with unprotected Ti by slit lamp observation and anterior segment optical coherence tomography (AS-OCT). Inflammatory responses were evaluated by bacterium counting, hematoxylin-eosin staining, and immunostaining. There was a lower incidence and a lesser extent of infection on rabbit corneas with Ti-AMP implants than on those with unprotected Ti implants. The bactericidal effect of AMP against S. aureus was comparable to that of postoperative prophylactic antibiotic treatment; hence, SESB2V AMP bound to the Ti implant provided functional activity in vivo, but its efficacy was greater against S. aureus than against P. aeruginosa. This work suggests that SESB2V AMP can be successfully functionalized in a rabbit keratitis model to prevent perioperative corneal infection.

Diamond nanowires modified with poly[3-(pyrrolyl)carboxylic acid] for the immobilization of histidine-tagged peptides.

Coating boron-doped diamond nanowires (BDD NWs) with a conducting polymer, poly[3-(pyrrolyl)carboxylic acid], has been reported. Polymer coating was achieved through electropolymerization of 3-(pyrrolyl)carboxylic acid at the electrode interface by amperometrically biasing the BDD NWs interface until a predefined charge has passed. The poly[3-(pyrrolyl)carboxylic acid] modified BDD NWs (PPA-BDD NWs) were characterized by scanning electron microscopy (SEM) and cyclic voltammetry (CV). Using a deposition charge of 11 mC cm(-2) resulted in a thin polymer film deposition. The availability of the carboxylic groups of the polymer coated BDD NWs electrode was demonstrated through copper ion (Cu(2+)) chelation. The resulting complex was successfully used for the site-specific immobilization of histidine-tagged peptides. The binding process was followed by electrochemical impedance spectroscopy (EIS). The Cu(2+)-chelated PPA-BDD NWs interface showed peptide loading capability comparable to those of commercially available interfaces and can be easily regenerated several times using ethylenediaminetetraacetic acid (EDTA).

Immobilization of RGD peptide onto the surface of apatite-wollastonite ceramic for enhanced osteoblast adhesion and bone regeneration

The arginine-glycine-aspartic (RGD) acid peptide was grafted to the surface of apatite-wollastonite (AW) ceramic in an effort to improve its cell adhesion, proliferation and osteoinduction. RGD peptide was covalently immobilized onto the surface of AW ceramic via the synthetic cross linker AAPTS-E and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The modified surfaces were characterized by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). The chemical analysis indicated that RGD peptide had been immobilized onto the AW surface successfully. The growth of osteoblast-like cells (MG63) showed that modifying the AW surface with RGD peptide enhanced the cell adhesion and proliferation. And the histological evaluation of RGD-AW showed that the bone regeneration and remodeling process were significantly enhanced compared to the original AW ceramics after 2, 4 and 8 weeks implantation in rabbit’s femoral condyles.

Peptide microarray with ligands at high density based on symmetrical carrier landscape phage for detection of cellulase.

Peptide microarrays evolved recently as a routine analytical implementation in various research areas due to their unique characteristics. However, the immobilization of peptides with high density in each spot during the fabricating process remains a problem, which will affect the performance of the resultant microarray greatly. To respond to this challenge, a novel peptide immobilization method using symmetrical phage carrier was developed in this work. The cellulytic enzyme endoglucanase I (EG I) was used as a model for selection of its specific peptide ligands from the f8/8 landscape library. Three phage monoclones were selected and identified by the specificity array, of which one phage monoclone displaying the fusion peptide EGSDPRMV (phage EGSDPRMV) could bind EG I specifically with highest affinity. Subsequently, the phage EGSDPRMV was used directly to construct peptide microarray. For comparison, major coat protein pVIII fused EG I specific peptide EGSDPRMV (pVIII-fused EGSDPRMV) which was isolated from phage EGSDPRMV was also immobilized by traditional method to fabricate peptide microarray. The fluorescent signal of the phage EGSDPRMV-mediated peptide microarray was more reproducible and about four times higher than the value for pVIII-fused EGSDPRMV-based microarray, suggesting the high efficiency of the proposed phage EGSDPRMV-mediated peptide immobilization method. Further, the phage EGSDPRMV based microarray not only simplified the procedure of microarray construction but also exhibited significantly enhanced sensitivity due to the symmetrical carrier landscape phage, which dramatically increased the density and sterical regularity of immobilized peptides in each spot. Thus, the proposed strategy has the advantages that the immobilizing peptide ligands were not disturbed by their composition and the immobilized peptides were highly regular with free amino-terminal.

"Click" immobilization of a VEGF-mimetic peptide on decellularized endothelial extracellular matrix to enhance angiogenesis.

We show that coating of decellularized extracellular matrix (DC-ECM) on substrate surfaces is an efficient way to generate a platform mimicking the native ECM environment. Moreover, the DC-ECM can be modified with a peptide (QK) mimicking vascular endothelial growth factor without apparently compromising its integrity. The modification was achieved through metabolic incorporation of a “clickable” handle to DC-ECM followed by rapid attachment of the QK peptide with an azido tag using copper-catalyzed click reaction. The attachment of the QK peptide on to DC-ECM in this way further enhanced the angiogenic responses (formation of branched tubular networks) of endothelial cells.

Voltammetric Behavior of Peptide-Modified Porous Silicon after Metal Complexation

Hybrid nanomaterials based on organic layer covalently grafted on porous silicon (PSi) nanostructure appear as promising systems for innovative applications such as detecting of traces amounts and/or removing metal cations in water effluents. In this work, we focused on the functionalization of the PSi nanostructure by the peptide GlyCysGlyCys, which forms stable complexes with metal ions. This property is exploited to achieve toxic metal recognition in water using electrochemical methods. Peptide immobilization was achieved using multi-step reactions; GlyCysGlyCys was anchored on a previously prepared carboxyl-terminated PSi surface, using EDC/NHS coupling agents. This scheme is compatible with the mild conditions required for preserving the probe activity of the peptide. At each step of the functionalization, the surface was monitored by Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). Electrochemical behavior of such modified electrode was carried out after Nickel accumulation on the surface, by means of cyclic voltammetry. The recorded cyclic voltammograms showed a quasi-irreversible process corresponding to the Ni2+/Ni0 couple.

Covalent immobilization of hLf1-11 peptide on a titanium surface reduces bacterial adhesion and biofilm formation

Bacterial infection represents a major cause of implant failure in dentistry. A common approach to overcoming this issue and treating peri-implant infection consists in the use of antibiotics. However, the rise of multidrug-resistant bacteria poses serious concerns to this strategy. A promising alternative is the use of antimicrobial peptides due to their broad-spectrum activity against bacteria and reduced bacterial resistance responses. The aim of the present study was to determine the in vitro antibacterial activity of the human lactoferrin-derived peptide hLf1-11 anchored to titanium surfaces. To this end, titanium samples were functionalized with the hLf1-11 peptide either by silanization methods or physical adsorption. X-ray photoelectron spectroscopy analyses confirmed the successful covalent attachment of the hLf1-11 peptide onto titanium surfaces. Lactate dehydrogenase assay determined that hLf1-11 peptide did not affect fibroblast viability. An outstanding reduction in the adhesion and early stages of biofilm formation of Streptococcus sanguinis and Lactobacillus salivarius was observed on the biofunctionalized surfaces compared to control non-treated samples. Furthermore, samples coated with the hLf1-11 peptide inhibited the early stages of bacterial growth. Thus, this strategy holds great potential to develop antimicrobial biomaterials for dental applications.

Whey microbeads as a matrix for the encapsulation and immobilisation of riboflavin and peptides

Whey microbeads manufactured using a cold-set gelation process, have been used to encapsulate bioactives. In this study whey microbeads were used to encapsulate riboflavin using 2 methods. Riboflavin was added to the microbead forming solution however diffusional losses of riboflavin occurred during the subsequent bead preparation. To overcome riboflavin loss, a second approach to ‘load’ whey microbeads by soaking in riboflavin was assessed. Significantly (p⩽0.05) higher concentrations of riboflavin were obtained in ‘loaded’ microbeads (361mg/L) compared to riboflavin added to the microbead forming solution (48mg/L). Riboflavin uptake by the microbeads was shown to be via a partition process. As partitioning is often driven by hydrophobic interactions the uptake of amino acids and peptides of varying hydrophobicities by the microbeads was examined. The % encapsulation increased with increasing molecule hydrophobicity with a maximum of 89% encapsulation. Whey microbeads are well suited to act as sorbents for encapsulation.

Different Interfacial Behaviors of Peptides Chemically Immobilized on Surfaces with Different Linker Lengths and via Different Termini

Molecular structures such as conformation and orientation are crucial in determining the activity of peptides immobilized to solid supports. In this study, sum frequency generation (SFG) vibrational spectroscopy was applied to investigate such structures of peptides immobilized on self-assembled monolayers (SAMs). Here cysteine-modified antimicrobial peptide cecropin P1 (CP1) was chemically immobilized onto SAM with a maleimide terminal group. Two important characteristics, length of the poly(ethylene glycol) (PEG) segment in the SAM and location of the cysteine residue in the peptide, were examined using SFG spectroscopy to determine the effect of each on surface immobilization as well as peptide secondary structure and its orientation in the immobilized state. Results have shown that while each length of PEG chain studied promotes chemical immobilization of the target peptide and prevents nonspecific adsorption, CP1 immobilized on long-chain (PEG2k) maleimide SAMs shows random coil structure in water, whereas CP1 demonstrates α-helical structure when immobilized on short-chain (with four ethylene glycol units - (EG4)) maleimide SAMs. Placement of the cysteine residue at the C-terminus promotes the formation of α-helical structure of CP1 with a single orientation when tethered to EG4 maleimide SAM surfaces. In contrast, immobilization via the N-terminal cysteine of CP1 results in a random coil or lying-down helical structure. The bacteria capturing/killing capability was tested, showing that the surface-immobilized CP1 molecules via C- and N- terminal cysteine exhibit only slight difference, even though they have different secondary structures and orientations.

Immobilisation of cell‐binding peptides on poly‐ε‐caprolactone (PCL) films: A comparative XPS study of two chemical surface functionalisation methods

Successful interaction between cells and biomaterial surfaces is crucial for biomedical devices, and incorporation of peptides such as RGD (Arg‐Gly‐Asp) at the polymer interface can substantially promote cell adhesion and proliferation. X‐ray photoelectron spectroscopy (XPS) has been used to characterise poly‐ε‐caprolactone (PCL) films modified by aminolysis and the introduction of RGD peptides via carbodiimide (CDI) and thiol‐halogen ‘click’ chemistry. The nitrogen signal acts as an elemental indicator for successful attachment, and changes in the chemical environment are reflected in the carbon and oxygen spectra. Chlorine and sulfur provide additional chemical indicators of reaction progress in the thiol method, with the selective nature of the Cl–S reaction reflected in the complete loss of Cl signal and appearance of S, avoiding potential amine‐peptide side‐reactions. Comparison of the XPS elemental concentrations indicated an estimate of 2–3% peptide functionalisation on the PCL surface for both methods, correlating with the improvement in Schwann cell response observed after peptide immobilisation. The enhanced selectivity of peptide attachment to the polymer surface demonstrated with XPS for the novel method based on thiol chemistry shows its potential for development as a biomimetic scaffold for peripheral nerve injury. Copyright © 2014 John Wiley & Sons, Ltd.

Nanoscale engineering of low-fouling surfaces through polydopamine immobilisation of zwitterionic peptides

We report a versatile approach for the design of substrate-independent low-fouling surfaces via mussel-inspired immobilisation of zwitterionic peptides. Using mussel-inspired polydopamine (PDA) coatings, zwitterionic glutamic acid- and lysine-based peptides were immobilised on various substrates, including noble metals, metal oxides, polymers, and semiconductors. The variation of surface chemistry and surface wettability upon surface treatment was monitored with X-ray photoelectron spectroscopy (XPS) and water contact angle measurements. Following peptide immobilisation, the surfaces became more hydrophilic due to the strong surface hydration compared with PDA-coated surfaces. The peptide-functionalised surfaces showed resistance to human blood serum adsorption and also effectively prevented the adhesion of gram-negative and gram-positive bacteria (i.e., Escherichia coli and Staphylococcus epidermidis) and mammalian cells (i.e., NIH 3T3 mouse embryonic fibroblast cells). The versatility of mussel-inspired chemistry combined with the unique biological nature and tunability of peptides allows for the design of low-fouling surfaces, making this a promising coating technique for various applications.

Site specific immobilization of a potent antimicrobial peptide onto silicone catheters: evaluation against urinary tract infection pathogens.

Bacterial colonization of urinary catheters is a common problem leading to Catheter Associated Urinary Tract Infections (CAUTIs) in patients, which result in high treatment costs and associated complications. Due to the advantages of antimicrobial peptides (AMPs) compared to most other antimicrobial molecules, an increasing number of AMP-coated surfaces is being developed but their efficacy is hindered by suboptimal coating methods and loss of peptide activity upon surface tethering. This study aims to address this issue by employing a methodic approach that combines a simple selective chemical immobilization platform developed on a silicone catheter with the choice of a potent AMP, Lasioglossin-III (Lasio-III), to allow site specific immobilization of Lasio-III at an effective surface concentration. The Lasio-III peptide was chemically modified at the N-terminal with a cysteine residue to facilitate cysteine-directed immobilization of the peptide onto a commercial silicone catheter surface via a combination of an allyl glycidyl ether (AGE) brush and polyethylene glycol (PEG) based chemical coupling. The amount of immobilized peptide was determined to be 6.59 ± 0.89 μg cm-2 by Sulfo-SDTB assay. The AMP-coated catheter showed good antimicrobial activity against both Gram positive and negative bacteria. The antimicrobial properties of the AMP-coated catheter were sustained for at least 4 days post-incubation in a physiologically relevant environment and artificial urine and prevented the biofilm growth of E. coli and E. faecalis. Adenosine tri-phosphate leakage and propidium iodide fluorescence studies further confirmed the membranolytic mode of action of the immobilized peptide. To the best of our knowledge, this is the first proof-of-concept study that reports the efficacy of AMP immobilization by sulfhydryl coupling on a real catheter surface.