Orthogonal Immobilization Research Articles

Bioinstructive Coatings for Hematopoietic Stem Cell Expansion Based on Chemical Vapor Deposition Copolymerization.

We report the chemical vapor deposition (CVD) of dual-functional polymer films for the specific and orthogonal immobilization of two biomolecules (notch ligand delta-like 1 (DLL1) and an RGD-peptide) that govern the fate of hematopoietic stem and progenitor cells. The composition of the CVD polymer and thus the biomolecule ratio can be tailored to investigate and optimize the influence of the relative surface concentrations of biomolecules on stem cell behavior. Prior to cell experiments, all surfaces were characterized by infrared reflection adsorption spectroscopy, time-of-flight secondary ion mass spectrometry, and X-ray photoelectron spectroscopy to confirm the presence of both biomolecules. In a proof-of-principle stem cell culture study, we show that all polymer surfaces are cytocompatible and that the proliferation of the hematopoietic stem and progenitor cells is predominantly influenced by the surface concentration of immobilized DLL1.

Preparation of Functional Alternating Polymer Brushes and Their Orthogonal Surface Modification through Microcontact Printing.

This paper reports microcontact printing (μCP) to immobilize an alkoxyamine initiator (regulator) on glass and silicon substrates and subsequent surface-initiated alternating nitroxide-mediated copolymerization (siNMP) of hexafluoroisopropyl acrylate (HFIPA) and 7-octenylvinyl ether (OVE). The resulting patterned polymer brushes are analyzed by using atomic force microscopy (AFM). In addition, site-specific post-functionalization of the alternating polymer brushes by applying two orthogonal surface reactions is achieved with thiols and amines through μCP. The versatility of this post-polymerization modification approach is demonstrated by site-selective immobilization of small organic molecules, fluorophores, and ligands providing a binary bioactive surface. The successful side-by-side orthogonal immobilization is verified by using X-ray photoelectron spectroscopy (XPS) and fluorescence microscopy.

Electrochemically Inducible Surfaces for Patterning Two Distinct Molecules

Herein, we report on a new multicomponent patterning method based on electrochemically inducible self‐assembled monolayers on gold. Two different masked functional groups on monolayers are activated to give amine and acetylene moieties through electrochemical activations at negative potential and positive potential, respectively. The resulting amine and acetylene groups are further used as chemical handles for incorporation of ligand molecules via well‐known chemoselective conjugation reactions such as amine‐specific conjugation chemistry and click reaction. The chemical conversions of masked functional groups to amine and acetylene groups were characterized by cyclic voltammetry. We demonstrated the orthogonal immobilization of two fluorescent dyes on the patterned surface along the patterned features. Our strategy can provide a useful platform technology for the preparation of multicomponent ligand‐patterned substrates with various advantages such as chemical flexibility, mild reaction conditions, and high yields of two orthogonal chemical reactions of amine‐specific conjugation and click reaction.

Immobilization strategy for enhancing sensitivity of immunosensors: L-Asparagine–AuNPs as a promising alternative of EDC–NHS activated citrate–AuNPs for antibody immobilization

This paper addresses the question – Is EDC–NHS activated gold nanoparticles modified electrode surface the best available option for antibody immobilization for immunosensor fabrication? Is there any other alternative covalent immobilization strategy for orthogonal orientation of antibody, ensuring enhanced sensitivity of immunosensors? Does EDC–NHS activation of carboxyl functionalized nanoparticles surface really leads to orthogonal or directed immobilization of antibody? Gold nanoparticles synthesized using L-Asparagine as reducing and stabilization agent were employed for orthogonal immobilization of antibody for immunosensor fabrication. Anti-CA125 antibody was used as a model system for immunosensor fabrication. A comparative evaluation of immunosensors fabricated using L-Asparagine stabilized gold nanoparticles and citrate stabilized gold nanoparticles via different immobilization strategies/chemistries was done. The three strategies involved immobilization of Anti-CA125 antibody – (1) after EDC–NHS activation of citrate stabilized gold nanoparticles, (2) directly onto citrate stabilized gold nanoparticles and (3) directly onto L-Asparagine stabilized gold nanoparticles modified electrode surfaces. Comparative evaluation of Impedimetric response characteristics showed 2.5 times increase in sensitivity (349.36Ω/(IU/mL)/cm2) in case of third strategy as compared to first (147.53Ω/(IU/mL)/cm2) and twice that of second strategy (166.24Ω/(IU/mL)/cm2). Additionally, an extended dynamic range of 0–750IU/mL was observed while for others it was up to 500IU/mL. Amino acid coated gold nanoparticles ensured orthogonal immobilization, lesser randomization, with 88% of active antibody available for antigen binding as opposed to other two strategies with less than 30% active antibody.

Bioactive surface functionalization

Reactive chemical vapor deposition (CVD) polymerization provides a substrate-independent platform for effective functionalization of virtually any solid substrates, flat, or curved, even with complex geometries. This article reviews bioactive surface functionalization strategies based on CVD polymerization and highlights commonly used surface chemistries. These reactions include alkyne–azide “click” chemistry, reactions of active esters with amine, aldehydes/ketones with hydrazides and alkoxyamines, thiols with alkenes and alkynes and surface-initiated atom transfer radical polymerization. The resulting biofunctional surface coatings can facilitate orthogonal immobilization of more than one type of ligand on a substrate. CVD polymer coatings with nanoscale thicknesses are widely applicable in biomedical applications and can be easily integrated into microand nanodevice fabrication. VC 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40315.

Orthogonal surface functionalization through bioactive vapor‐based polymer coatings

ABSTRACTReactive chemical vapor deposition (CVD) polymerization provides a substrate‐independent platform for effective functionalization of virtually any solid substrates, flat, or curved, even with complex geometries. This article reviews bioactive surface functionalization strategies based on CVD polymerization and highlights commonly used surface chemistries. These reactions include alkyne–azide “click” chemistry, reactions of active esters with amine, aldehydes/ketones with hydrazides and alkoxyamines, thiols with alkenes and alkynes and surface‐initiated atom transfer radical polymerization. The resulting biofunctional surface coatings can facilitate orthogonal immobilization of more than one type of ligand on a substrate. CVD polymer coatings with nanoscale thicknesses are widely applicable in biomedical applications and can be easily integrated into micro‐ and nanodevice fabrication. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40315.

Sequential Nucleophilic Substitutions Permit Orthogonal Click Functionalization of Multicomponent PEG Brushes

Multicomponent poly(ethylene glycol) (PEG) brushes (i.e., ≥ 2 adjacent PEG brushes) can be used to engineer culture substrates with microscale, nonfouling regions decorated with covalently immobilized ligands that mediate biospecific interactions. However, synthesizing such brushes with orthogonal immobilization chemistries to permit differential biofunctionalization is nontrivial and often requires synthesis of PEG-co-polymers. To simplify synthesis and enhance the versatility of such substrates, we developed a protocol for generating orthogonal click-functionalized multicomponent PEG brushes using sequential nucleophilic substitutions by sodium azide, ethanolamine, and propargylamine. The novel application of propargylamine-mediated substitution functionalizes PEG brushes with acetylene groups, and for the first time, ethanolamine-mediated substitution is shown to be sufficient for passivating the "living" polymer chain ends between brush synthesis steps. Thus, our multicomponent PEG brushes present dual orthogonal chemistries (i.e., azido and acetylene groups) for ligand immobilization via versatile copper-free click reactions, which are useful for in situ surface modifications during cell culture.

Differential anodic oxidation of single organic linkage molecules enabling orthogonal bio-immobilization

We report a novel method for preparing electrode surfaces that present thiol-containing or thiol-reactive groups using a single organic silane linkage terminated with 1,3-dithiane via differential anodic oxidation of 1,3-dithiane. We found that the surface groups resulting from the anodic oxidation reaction differed according to the concentration of H2O in the electrolyte solution during the oxidation reaction. The differential anodic oxidation was used successfully for the orthogonal immobilization of two different proteins. We demonstrated also the preparation of an immunoassay platform via orthogonal immobilization, which was then used to detect multiple target antigens based on a sandwich immunoassay.

Oriented Protein Immobilization using Covalent and Noncovalent Chemistry on a Thiol-Reactive Self-Reporting Surface

We report the fabrication of a patterned protein array using three orthogonal methods of immobilization that are detected exploiting a fluorogenic surface. Upon reaction of thiols, the fluorogenic tether reports the bond formation by an instantaneous rise in (blue) fluorescence intensity providing a means to visualize the immobilization even of nonfluorescent biomolecules. First, the covalent, oriented immobilization of a visible fluorescent protein (TFP) modified to display a single cysteine residue was detected. Colocalization of the fluorescence of the immobilized TFP and the fluorogenic group provided a direct tool to distinguish covalent bond formation from physisorption of proteins. Subsequent orthogonal immobilization of thiol-functionalized biomolecules could be conveniently detected by fluorescence microscopy using the fluorogenic surface. A thiol-modified nitrilotriacetate ligand was immobilized for binding of hexahistidine-tagged red-fluorescing TagRFP, while an appropriately modified biotin was immobilized for binding of Cy5-labeled streptavidin.

Orthogonal, metal-free surface modification by strain-promoted azide–alkyne and nitrile oxide–alkene/alkyne cycloadditions

In this article we present a fast and efficient methodology for biochemical surface patterning under extremely mild conditions. Micropatterned azide/benzaldoxime-surfaces were prepared by microcontact printing of a heterobifunctional cyclooctyne oxime linker on azide-terminated self-assembled monolayers (SAMs). Strain-promoted azide–alkyne cycloaddition (SPAAC) in combination with microcontact printing allows fast and effective surface patterning. The resulting bifunctional azide/oxime substrates could successfully be used for metal-free, orthogonal immobilization of various biomolecules by 1,3-dipolar cycloadditions at room temperature. Azide-decorated areas were modified by reaction with a cyclooctyne-conjugate using SPAAC, while benzaldoxime-decorated areas were activated by in situ oxidation to the reactive nitrile oxides and subsequent nitrile oxide cycloaddition with alkene- and alkyne-functionalized bioconjugates. In addition, orthogonal double immobilization was achieved by consecutive and independent SPAAC and nitrile oxide cycloadditions.

Towards Multipotent Coatings: Chemical Vapor Deposition and Biofunctionalization of Carbonyl‐Substituted Copolymers

AbstractFuture advances in designing bioactive materials, such as antithrombotic coatings for cardiovascular stents, will require widely applicable and robust methods of surface modification. In this paper, we report on the development of multifunctional polymer coatings made by chemical vapor deposition (CVD) copolymerization. Polymer coatings of various [2.2]paracyclophane derivatives were co‐deposited in controlled ratios and their chemical composition verified by FT‐IR and X‐ray photoelectron spectroscopy. Furthermore, preliminary biocompatibility of these coatings was assessed using human umbilical vein endothelial cells and 3T3 murine fibroblasts. The parallel immobilization of two different antithrombotic biomolecules onto a CVD‐based copolymer is also demonstrated by orthogonal immobilization strategies.magnified image