Covalent Immobilization Of Biomolecules Research Articles

Reagent‐Free Covalent Immobilization of Biomolecules in a Microfluidic Organ‐On‐A‐Chip

AbstractMicrofluidic systems have become integral for lab‐on‐a‐chip and organ‐on‐a‐chip applications across numerous disciplines. These systems, typically fabricated using polydimethylsiloxane (PDMS) chips on glass substrates, lack the bioactivity required for such applications. To overcome this, biomolecules are immobilized using either oxygen (O2) plasma treatment or chemical reagents like amino silanes. However, O2 plasma treatments are unstable and cannot covalently immobilize biomolecules, while wet‐chemistry approaches are toxic, time‐consuming, and expensive. A novel microfluidic platform that combines two plasma surface treatments: Plasma‐activated coating (PAC) and atmospheric pressure plasma jet (APPJ), to enable reagent‐free covalent immobilization of biomolecules is described here. These surface treatments, unlike O2 plasma, covalently immobilized fibronectin on PDMS and glass, and significantly improved endothelial cell attachment and proliferation. By combining PAC and APPJ, a hybrid microfluidic platform with equivalent bond strength to standard O2 plasma devices, but with significantly enhanced endothelial cell growth in and artery‐on‐a‐chip model, is developed. This platform is also amenable to high‐shear applications such as coronary shear, with endothelial cells aligning with flow, as seen in human arteries. By providing reagent‐free covalent immobilization of biomolecules within a microfluidic system, this technology has the potential to radically improve organ‐on‐a‐chip development as well as lab‐on‐a‐chip systems, point‐of‐care diagnostics, and sensors.

Effect of plasma ion immersion implantation on physiochemical and biological properties of silk towards creating a versatile biomaterial platform

Silk biomaterials are widely used in tissue engineering applications due to their mechanical tuneability, processability and low immunogenic response. However, due to the lack of cell binding sites, further modification is typically required to optimize silk biomaterials towards a specific biomedical application. Plasma immersion ion implantation (PIII) is a surface modification technique that can covalently attach a wide range of bioactive molecules onto silk surfaces to trigger desirable biological responses. However, the effect of PIII treatment on the surface and bulk properties of silk biomaterials remains largely unknown. Therefore, the aim of this study is to investigate PIII-treated silk (PIII-silk) biomaterials towards their use as a platform for biomedical applications. Silk films with or without PIII treatment were examined to determine changes to the surface and bulk properties, including the chemical, mechanical, degradation properties, and the in vivo immune response. PIII treatment of silk was found to alter surface chemistry with densification of the silk surface layer and the introduction of oxygen and nitrogen functional groups. This then resulted in a significant increase in surface stiffness but also led to minor changes in tensile properties of clinically relevant silk formats and minor differences in in vitro degradation properties. Following PIII treatment, there was a significant increase in HCAEC adhesion on PIII-silk compared to untreated silk but no significant differences in fibrous encapsulation and macrophage response when implanted in vivo in a subcutaneous mouse model. Together, these results show that PIII treatment of silk minimally compromises the bulk properties and can be used to promote cellular interactions as well as a means of reagent-free, covalent immobilization of biomolecules.

Covalent immobilization of biomolecules on stent materials through mussel adhesive protein coating to form biofunctional films.

It is widely accepted that surface biofunctional modification may be an effective approach to improve biocompatibility and confer new bioactive properties on biomaterials. In this work, mussel adhesive protein (MAP) was applied as a coating on 316 L stainless steel substrates (316 L SS) and stents, and then either immobilized VEGF or CD34 antibody were added to create biofunctional films. The properties of the MAP coating were characterized by scanning electron microscope (SEM), atomic force microscope (AFM) and a water contact angle test. Universal tensile testing showed that the MAP coating has adequate adhesion strength on a 316 L stainless steel material surface. Subsequent cytotoxicity and hemolysis rate tests showed that the MAP coatings have good biocompatibility. Moreover, using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysulfosussinimide (EDC/NHS) chemistry, VEGF and CD34 antibody were immobilized on the MAP coatings. The amount and immobilized yield of VEGF on the MAP coatings were analyzed by enzyme-linked immuno-assays (ELISA). Finally, an endothelial cells culture showed that the VEGF biofunctional film can promote the viability and proliferation of endothelial cells. An in vitro CD34+ cells capturing test also verified the bioactive properties of the CD34 antibody coated stents. These results showed that the MAP coatings allowed effective biomolecule immobilization, providing a promising platform for vascular device modification.

Functionalization of Hydroxyapatite Ceramics: Raman Mapping Investigation of Silanization

Surface modification of bioceramic materials by covalent immobilization of biomolecules is a promising way to improve their bioactivity. This approach implies the use of organic anchors to introduce functional groups on the inorganic surface on which the biomolecules will be immobilized. In this process, the density and surface distribution of biomolecules, and in turn the final biological properties, are strongly influenced by those of the anchors. We propose a new approach based on Raman 2D mapping to evidence the surface distribution of organosilanes, frequently used as anchors on biomaterial surfaces on hydroxyapatite and silicated hydroxyapatite ceramics. Unmodified and silanized ceramic surfaces were characterized by means of contact angle measurements, atomic force microscopy (AFM) and Raman mapping. Contact angle measurements and AFM topographies confirmed the surface modification. Raman mapping highlighted the influence of both the ceramic’s composition and silane functionality (i.e., the number of hydrolysable groups) on the silane surface distribution. The presence of hillocks was shown, evidencing a polymerization and/or an aggregation of the molecules whatever the silane and the substrates were. The substitution of phosphate groups by silicate groups affects the covering, and the spots are more intense on SiHA than on HA.

Dendritic maleimide functionalization of core–shell (γ-Fe2O3/polymer) nanoparticles for efficient bio-immobilization

Dendritic functionalization of core–shell γ-Fe2O3/polymer MNPs for selective and covalent immobilization of biomolecules using maleimide/thiol coupling chemistry.

Surface functionalization of cyclic olefin copolymer with aryldiazonium salts: A covalent grafting method

Covalent immobilization of biomolecules on the surface of cyclic olefin copolymer (COC) is still a tough challenge. We developed a robust method for COC surface grafting through reaction with aryldiazonium. Chemical diazonium reduction generated an aryl radical and the formation of a grafted film layer on the organic surface. We also demonstrated that the chemical reduction of diazonium salt was not sufficient to form a film on the COC surface. UV illumination had to be combined with chemical reduction to graft an aryl layer onto the COC surface. We optimized organic film deposition by using different chemical reducers, different reaction times and reagent proportions. We characterized surface modifications by fluorescence microscopy and contact angle measurements, infrared spectroscopy, X-ray photoemission spectroscopy and Raman spectroscopy, and assessed the topography of the aryl film by atomic force microscopy. This original strategy allowed us to evidence various organic functions to graft biomolecules onto COC surfaces with a fast and efficient technique.

Structure and chemical composition of mixed benzylguanine‐ and methoxy‐terminated self‐assembled monolayers for immobilization of biomolecules

Covalent immobilization of biomolecules has become a subject of great interest in recent years because of the expected diversity of applications, for example, biosensors in diagnosis, lab‐on‐chip technology, and modern cell culture focused on cell adhesion, migration, and differentiation. Our approach is to produce self‐assembled monolayers (SAMs) on gold surfaces based on mixtures of a benzylguanine‐terminated thiol and a thiol, carrying ethylene glycol groups. The benzylguanine head group of such SAMs acts as a substrate for the SNAP‐tag system allowing for covalent attachment of any protein of interest fused to this tag. For this purpose, it is essential to determine the orientation and chemical composition of these layers. In this study, we use the strength of combining complementary surface analytical methods to achieve a comprehensive characterization. The chemical composition and the covalent binding of the thiols were proved by X‐ray photoelectron spectroscopy (XPS). The orientation of the SAMs together with thickness information was achieved by nondestructive depth profiles reconstructed from parallel angle‐resolved XPS data and energy‐resolved photoelectron emission spectroscopy (PES). High‐sensitivity low‐energy ion scattering and sum‐frequency‐generation spectroscopy corroborate the XPS/PES results. Copyright © 2012 John Wiley & Sons, Ltd.

Universal Biomolecule Binding Interlayers Created by Energetic Ion Bombardment

ABSTRACTThe ability to strongly attach biomolecules such as enzymes and antibodies to surfaces underpins a host of technologies that are rapidly growing in utility and importance. Such technologies include biosensors for medical and environmental applications and protein or antibody diagnostic arrays for early disease detection. Emerging new applications include continuous flow reactors for enzymatic chemical, textile or biofuels processing and implantable biomaterials that interact with their host via an interfacial layer of active biomolecules. In many of these applications it is desirable to maintain physical properties of an underlying material whilst engineering a surface suitable for attachment of proteins or peptide constructs. Nanoscale polymeric interlayers are attractive for this purpose.We have developed interlayers[1] that form the basis of a new biomolecule binding technology with significant advantages over other currently available methods. The interlayers, created by the ion implantation of polymer like surfaces, achieve covalent immobilization on immersion of the surface in protein solution. The interlayers can be created on any underlying material and ion stitched into its surface. The covalent immobilization of biomolecules from solution is achieved through the action of highly reactive free radicals in the interlayer.In this paper, we present characterisation of the structure and properties of the interlayers and describe a detailed kinetic model for the covalent attachment of protein molecules directly from solution.

Development of oligonucleotide microarray involving plasma polymerized acrylic acid

This paper presents the manufacturing of biochips by using the COOH– derived polymer coating deposited by plasma polymerization of acrylic acid. This technology is based on depositing a thin layer obtained by plasma polymerization of acrylic acid which allows a further covalent immobilization of biomolecules on glass substrates. The plasma power value was optimized to maximize the stability of plasma polymerized acrylic acid (PPAA) coatings in water, which has a very important role for such applications. In order to obtain a covalent immobilization of DNA probes on the PPAA coated surface, the activation protocol of carboxylic function was carried out with the help of N-Hydroxy Succinimide and 1-Ethyl-3-(3-DimethylAminopropyl) Carbodiimide. The efficiency of PPAA coated in microarray applications was compared with two types of commercial slides. Such surfaces have shown very interesting results in terms of relative density of attached DNA probe molecules and signal-to-background ratio measured for target DNA hybridization. Nonspecific DNA bonding measurements showed only a small amount of nonspecific physisorption between the DNA probe and the PPAA-activated surfaces. This work shows that the plasma polymerization technique can be successfully applied to produce a high-quality glass surface for the manufacturing of DNA arrays.

Surface Engineering of Polymer Sheet by Plasma Techniques and Atom Transfer Radical Polymerization for Covalent Immobilization of Biomolecules

Surface Engineering of Polymer Sheet by Plasma Techniques and Atom Transfer Radical Polymerization for Covalent Immobilization of Biomolecules

Electrogenerated indium tin oxide-coated glass surface with photosensitive interfaces: Surface analysis

We present herein a photo-immobilization technique for the localized and specific conjugation of biochip platforms with different proteinaceous bioreceptors, such as antigen or antibodies. This methodology based on a photoactivable electrogenerated polymer film, pyrrole-benzophenone, allows the covalent immobilization of biomolecules through light mediation. The surface-conductive glass platform electropolymerized with poly(pyrrole-benzophenone) thin film may then be used to affinity-coat the chip with molecular recognition probes. This glass chip electroconductive surface modification is done by the deposition of a thin layer of indium tin oxide (ITO). Thereafter, pyrrole-benzophenone monomers are electropolymerized onto the conductive metal oxide surface and then exposed to an antigen Staphylococcal Enterotoxin B (SEB)) solution and illuminated with UV light (wavelength ∼345 nm) through a mask. As a result of the photochemical reaction, a pattern thin layer of the antigen was covalently bound to the benzophenone-modified surface. Then the sample to be analyzed, along with its specific target antibody (anti-SEB antibodies), is introduced onto the glass surface and left to react with the previously photo-immobilized antigen. When the immuno-reaction is completed, the specifically attached immunoglobulin analytes are detected by using secondary antibodies conjugated with Fluorescein isothiocyanate (FITC). The fluorescence signal emanating from the biochip surface is then quantified by two methods, using a filtered intensified charge-coupled device (CCD) camera and a grating spectrometer.

Microarrays and Surface Engineering for Bioanalysis

Molecular engineering and covalent immobilization of biomolecules on material surfaces whilst retaining specificity and biological functions is a major challenge in bioarray manufacturing and analysis. Photochemical functionalization of materials enables specific bioengineering and facile manufacturing of biotech products for microanalysis with concomitant surface passivation. Surfaces engineered with polysaccharides render materials hydrophilic, biocompatible and generally applicable for probe molecule immobilization. Products and procedures described support microanalytical investigations in all '-omics' domains.

Activity of enzyme immobilized on silanized Co-Cr-Mo.

The surface of an orthopedic biomaterial was modified by the covalent immobilization of biomolecules. Derivatization of Co-Cr-Mo samples with organic and aqueous solutions of gamma-aminopropyltriethoxysilane (APS) resulted in a concentration-dependent number of reactive NH2 groups on the surface available for coupling to protein. The enzyme trypsin was used as a model biomolecule to investigate the effect of immobilization on proteolytic activity. Trypsin was coupled to the silanized samples by formation of Schiff's base linkages via glutaraldehyde. The nature of the interaction between trypsin and biomaterial was then probed by treatment with concentrated guanidine hydrochloride (GuHCl) and urea. Residual activity (following treatment with chaotropic agents) of trypsin immobilized on silanized Co-Cr-Mo was dependent both on the nature of the silane solution and on the type of chaotropic agent. Organic silanization with APS required a minimum density of approximately 49 NH2 per nm2 of nominal surface area (> 0.021 M APS) for residual activity of immobilized trypsin. For aqueous silanization, approximately 5.4 NH2/nm2 (0.51 M APS) resulted in maximal residual trypsin activity. Treatment with GuHCl removed more trypsin activity from Co-Cr-Mo samples silanized with organic solutions of APS than did treatment with urea. On the contrary, with aqueous silanization the samples possessed greater residual activity following treatment with GuHCl than following urea. Compared to simple adsorption with protein onto Co-Cr-Mo, both methods of silanization with APS resulted in superior residual immobilized enzyme activity.

Light–Dependent, Covalent Immobilization of Biomolecules on ‘Inert’ Surfaces

We describe a novel, versatile procedure for the light-dependent immobilization of ligands to 'inert' material surfaces. Covalent immobilization of ligands differing in chemical nature and complexity is accomplished under mild and non-destructive conditions. Topical interaction of ligands with organic or inorganic surfaces is mediated by photoactivable polymers with carbene generating trifluoromethyl-aryl-diazirines which serve as linker molecules. Light activation of aryl-diazirino functions at 350 nm yields highly reactive carbenes, and covalent coupling is achieved by simultaneous carbene insertion into both the ligand and inert surface. Thus, reactive functional groups are not required on either the ligand or the supporting material. These procedures are applicable whenever ligands, from molecules to cells--synthetically or genetically produced, or isolated from biological sources--need to be immobilized for improved performance.