Direct Covalent Immobilization Research Articles

Direct covalent immobilizaion of chiral phosphines to hollow mesoporous polystyrene nanospheres (HMPNs) for efficient heterogeneous enantioselective [2+3] cycloaddition

Low-cost and convenient strategy for the direct covalent immobilizaion of chiral phosphines into structurally superior solid supports is desirable for efficient heterogeneous enantioselective catalysis. In this paper, a universal and convenient method for the direct covalent immobilization of chiral phosphines into HMPNs is developed via Friedel-Craft alkylation to fabricate supported chiral phosphines at a low cost. Using (S)-2,2´-bis(diphenylphosphino)-1,1´-binaphthyl [(S)-BINAP] and (R,R)-diphenylbis-[(o-anisyl)-methylenephosphine] [(R,R)-DIPAMP] as examples, the as-fabricated HMPNs-supported (S)-BINAP and (R,R)-DIPAMP, namely BINAP@HMPNs and DIPAMP@HMPNs, possess a well-shaped spherical morphology with hollow interior and thin mesoporous shell, enabling reactants to easily access to catalytic sites. In enantioselective [2+3] cycloaddition, DIPAMP@HMPNs exhibits good yields (70–86 %) with excellent enantioselectivities (94–98 %ee). Furthermore, DIPAMP@HMPNs shows a good mechanical stability of spherical morphology and mesoporous structure in its reuses. Moreover, the oxidation of DIPAMP leads to the gradual deactivation of DIPAMP@HMPNs including yield and enantioselectivity, which can be restored by the reduction of tetramethyldisiloxane (TMDS)/Ti(OiPr)4.

Electrodeposited Carbonyl Functional Polymers as Suitable Supports for Preparation of the First-Generation Biosensors.

The aim of this electrochemical study was to ascertain which type of electrochemically deposited carbonyl functionalized polymer represents the most suitable electrode substrate for direct covalent immobilization of biological catalysts (enzymes). For this purpose, a triad of amperometric biosensors differing in the type of conductive polymers (poly-vanillin, poly-trans-cinnamaldehyde, and poly-4-hydroxybenzaldehyde) and in the functioning of selected enzymes (tyrosinase and alkaline phosphatase) has been compared for the biosensing of neurotransmitters (dopamine, epinephrine, norepinephrine, and serotonin) and phenyl phosphates (p-aminophenyl phosphate and hydroquinone diphosphate). The individual layers of the polymers were electrochemically deposited onto commercially available screen-printed carbon electrodes (type C110) using repetitive potential cycling in the linear voltammetric mode. Their characterization was subsequently performed by SEM imaging and attenuated total reflectance FTIR spectroscopy. Molecules of enzymes were covalently bonded to the free carbonyl groups in polymers via the Schiff base formation, in some cases even with the use of special cross-linkers. The as-prepared biosensors have been examined using cyclic voltammetry and amperometric detection. In this way, the role of the carbonyl groups embedded in the polymeric structure was defined with respect to the efficiency of binding enzymes, and consequently, via the final (electro)analytical performance.

Universal one-pot strategy for fabricating supported chiral organocatalysts via direct covalent immobilization upon hollow mesoporous polystyrene nanospheres

Heterogeneous asymmetric organocatalysis is severely limited by two problems: mult-step energy- and time-consuming covalent immobilization of expensive organocatalysts and difficulty in controlling the morphology and porous structure of supported organocatalysts. Herein, a universal strategy is presented for the direct covalent immobilization of structurally different chiral organocatalysts upon hollow mesoporous organic polystyrene nanospheres (HMOPNs) via a one-pot tandem reaction. Owing to the steric confinement and fast mass transfer originating from their hollow interior, thin shell thickness and abundant mesopores, these supported chiral organocatalysts show excellent catalytic performances in the stereo-controlled steps of the synthesis of natural products and pharmaceuticals in good to high yields (76–96%) with excellent enantioselectivities (93–99% ee). This one-pot immobiliza- tion strategy paves a universal way for direct covalent immobilization of chiral organocatalysts bearing phenyl/aryl moiety to fabricate supported chiral organocatalysts with good long-term performances, achieving the low-cost production of chiral molecules.

Plasma treatment in air at atmospheric pressure that enables reagent-free covalent immobilization of biomolecules on polytetrafluoroethylene (PTFE)

Covalent immobilization of biomolecules to surfaces is desirable in applications in biomedicine and biotechnology, such as biosensors, protein microarrays and implantable biomedical devices. Surface-embedded radicals in polymers, produced by plasma immersion ion implantation, are known to covalently immobilize biomolecules directly from buffer without additional reagents. Here we explore the prospects for creating a surface activated for direct covalent immobilization using a dielectric barrier discharge in air at atmospheric pressure, eliminating the need for vacuum and gas feed systems. We find that a surface activation process at atmospheric pressure in air can activate polytetrafluoroethylene (PTFE) in order to achieve reagent-free covalent immobilization of biomolecules. The presence of surface immobilized protein was verified by X-ray photoelectron spectroscopy (XPS), and its covalent immobilization was demonstrated by resilience to rigorous SDS washing at elevated temperature. Time course immobilization studies show that the covalent coupling capability of the activated surfaces is retained for several days. Proof-of-concept cell assays with immobilized tropoelastin demonstrate the technique’s ability to present functional cell binding molecules for the production of truly bioactive surfaces.

Understanding gold nanoparticles interactions with chitosan: Crosslinking agents as novel strategy for direct covalent immobilization of biomolecules on metallic surfaces

The development of a new method for covalent immobilization of biomolecules on the surface of bare gold nanoparticles (AuNPs) by using crosslinking agents in one step is presented. A very compatible biopolymer such as chitosan has been used as a target molecule to probe the viability of the proposed methodology. Click chemistry, based on biocompatible reactions and coupling with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide allow to analyze the covalent interactions between the metal nanoparticles and the biopolymer. Spectra deconvolution technique and Zeta potential measurements confirm the covalent interaction. The present study allows to quantify the proportion of AuNPs covalently adhered to the chitosan, which depends on the solution pH. The obtained results indicates that covalent interactions can be increased up to 25% in relation to total system interactions, which are mostly electrostatic. The proposed strategy opens up a new pathway for biomedical applications because the control of the chemical linkage can be directly performed on the nanoparticle surface without using any molecular intermediate, which may improve the encapsulation efficiency on drug delivery therapies.

Direct covalent immobilization of new nitrogen-doped carbon nanodots by electrografting for sensing applications

This paper reports a facile strategy to covalently immobilize nanosized carbon dots (CD) onto carbon conductive surfaces for sensing applications. The carbon nanodots designed with surface amine groups (N-CD) can be electrografted onto carbon electrodes and, thus, easily covalently immobilized on these conductive surfaces. They have been synthetized by a carbonization method microwave-assisted using preselected low cost and biocompatible precursors, such as d-fructose as primary carbon source and urea as N-donor reagent to obtain peripheral enriched nitrogen CD. The synthetized nanomaterial has been characterized by different techniques, that confirm the presence of size-regular amorphous structures with blue fluorescence when are irradiated with UV light. The highly stable immobilization of N-CD onto the electrode surfaces by electrografting provides hybrid electrodes with greater relative surface area and improved electron transfer properties, demonstrating to be a great promise for electrochemical sensing. Because of its good electrical conductivity, electrical properties, abundant edges sites and high catalytic activity, N-CD immobilized on carbon electrodes efficient amplify the electrochemiluminiscence (ECL) signal from the luminophore [Ru(bpy)3]2+ in a taurine sensor. A linear concentration range from 126 to 1000 μM, a sensitivity of 7.40ⅹ10−4 μM−1 and a detection limit of 37.8 μM were determined for the taurine sensor.

Plasma Ion Implantation of Silk Biomaterials Enabling Direct Covalent Immobilization of Bioactive Agents for Enhanced Cellular Responses.

Silk fibroin isolated from Bombyx mori cocoons is a promising material for a range of biomedical applications, but it has no inherent cell-interactive domains, necessitating functionalization with bioactive molecules. Here we demonstrate significantly enhanced cell interactions with silk fibroin biomaterials in the absence of biofunctionalization following surface modification using plasma immersion ion implantation (PIII). Further, PIII treated silk fibroin biomaterials supported direct covalent immobilization of proteins on the material surface in the absence of chemical cross-linkers. Surface analysis after nitrogen plasma and PIII treatment at 20 kV revealed that the silk macromolecules are significantly fragmented, and at the higher fluences of implanted ions, surface carbonization was observed to depths corresponding to that of the ion penetration. Consistent with the activity of radicals created in the treated surface layer, oxidation was observed on contact with atmospheric oxygen and the PIII treated surfaces were capable of direct covalent immobilization of bioactive macromolecules. Changes in thickness, amide and nitrile groups, refractive index, and extinction coefficient in the wavelength range 400-1000 nm as a function of ion fluence are presented. Reactions responsible for the restructuring of the silk surface under ion beam treatment that facilitate covalent binding of proteins and a significant improvement in cell interactions on the modified surface are proposed.

Structural Analysis and Protein Functionalization of Electroconductive Polypyrrole Films Modified by Plasma Immersion Ion Implantation.

Conducting polymers are good candidates for electronic biomedical devices such as biosensors, artificial nerves, and electrodes for brain tissue. Functionalizing the conducting polymer surface with bioactive molecules can limit adverse immune reactions to the foreign body and direct tissue integration. In this work, we demonstrate a simple one-step method to attach biomolecules covalently to a conductive polymer. Electrochemically synthesized polypyrrole was activated using plasma immersion ion implantation (PIII) in nitrogen. A short treatment with relatively low ion fluence (20 s) was found to enable direct covalent immobilization of protein upon incubation in a protein solution, while the protein is easily removed from untreated polypyrrole by washing in buffer. The covalent nature of the protein immobilization was demonstrated by its resistance to elution when repeatedly washed with SDS detergent. Changes in the surface properties and their evolution with time after PIII activation were studied by a combination of attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), cyclic voltammetry, and water contact angle measurements. Notable changes in the chemistry of the modified layer in polypyrrole include the appearance of nitrile groups that gradually disappear with time and oxidation of the surface that increases over time in air. The kinetics of surface energy are consistent with the generation of radicals in the modified layer that are lost predominantly through oxidation. The conductivity of the modified surface layer (64 nm in thickness) decreases for low fluence treatments and is partially restored after high fluence treatment. This simple surface modification process opens up the possibility of creating biologically active interfaces for electro-stimulating biomedical devices and electrical sensing of neurological processes.

Exploiting the Versatility of Aminated Supports Activated with Glutaraldehyde to Immobilize β-galactosidase from Aspergillus oryzae

The enzyme β-galactosidase from Aspergillus oryzae has been immobilized in aminated (MANAE)-agarose beads via glutaraldehyde chemistry using different strategies. The immobilization on MANAE-supports was first assayed at different pH values (this gave different stabilities to the immobilized enzymes) and further modified with glutaraldehyde. Dramatic drops in activity were found, even using 0.1% (v/v) glutaraldehyde. The use of a support with lower activation permitted to get a final activity of 30%, but stability was almost identical to that of the just adsorbed enzyme. Next, the immobilization on pre-activated glutaraldehyde beads was assayed at pH 5, 7 and 9. At pH 7, full, rapid immobilization and a high expressed enzyme activity were accomplished. At pH 9, some decrease in enzyme activity was observed. Direct covalent immobilization of the enzyme was very slow; even reducing the volume of enzyme/support ratio, the yield was not complete after 24 h. The stability of the biocatalyst using pre-activated supports was about 4–6 folds more stable than that of the enzyme immobilized via ion exchange at pH 5, with small differences among them. Thus, the immobilization of the enzyme at pH 7 at low ionic strength on pre-activated glutaraldehyde supports seems to be the most adequate in terms of activity, stability and immobilization rate.

A novel optical biosensor for direct and selective determination of serotonin in serum by Solid Surface-Room Temperature Phosphorescence

This paper describes a novel biosensor which combines the use of nanotechnology (non-woven nanofibre mat) with Solid Surface-Room Temperature Phosphorescence (SS-RTP) measurement for the determination of serotonin in human serum. The developed biosensor is simple and can be directly applied in serum; only requires a simple clean-up protocol. Therefore it is the first time that serotonin is analysed directly in serum with a non-enzymatic technique. This new approach is based on the covalent immobilization of serotonin directly from serum on a functional nanofibre material (Tiss®-Link) with a preactivated surface for direct covalent immobilization of primary and secondary amines, and the subsequent measurement of serotonin phosphorescent emission from the solid surface. The phosphorescent detection allows avoiding the interference from any fluorescence emission or scattering light from any molecule present in the serum sample which can be also immobilised on the nanofibre material. The determination of serotonin with this SS-RTP sensor overcomes some limitations, such as large interference from the matrix and high cost and complexity of many of the methods widely used for serotonin analysis.The potential applicability of the sensor in the clinical diagnosis was demonstrated by analysing serum samples from seven healthy volunteers. The method was validated with an external reference laboratory, obtaining a correlation coefficient of 0.997 which indicates excellent correlation between the two methods.

Depth-Resolved Structural and Compositional Characterization of Ion-Implanted Polystyrene that Enables Direct Covalent Immobilization of Biomolecules

A polystyrene film spun onto polished silicon substrates was implanted with argon ions using plasma immersion ion implantation (PIII) to activate its surface for single-step immobilization of biological molecules. The film was subsequently investigated by X-ray and neutron reflectometry, ultraviolet (UV)–visible (vis) and Fourier transform infrared (FTIR) ellipsometry, FTIR and Raman spectroscopy, as well as nuclear reaction analysis to determine the structural and compositional transformations associated with the surface activation. The ion irradiation resulted in a significant densification of the carbon structure, which was accompanied by hydrogen loss. The density and hydrogen profiles in the modified surface layers were found to agree with the expected depths of ion implantation as calculated by the Stopping and Range of Ions in Matter (SRIM) software. The data demonstrate that the reduction in film thickness is due to ion-induced densification rather than the removal of material by etching. Characte...

Direct Covalent Biomolecule Immobilization on Plasma-Nanotextured Chemically Stable Substrates.

A new method for direct covalent immobilization of protein molecules (including antibodies) on organic polymers with plasma-induced random micronanoscale topography and stable-in-time chemical functionality is presented. This is achieved using a short (1-5 min) plasma etching and simultaneous micronanotexturing process, followed by a fast thermal annealing step, which induces accelerated hydrophobic recovery while preserving important chemical functionality created by the plasma. Surface-bound biomolecules resist harsh washing with sodium dodecyl sulfate and other detergents even at elevated temperatures, losing less than 40% of the biomolecules bound even at the harshest washing conditions. X-ray photoelectron spectroscopy, secondary-ion mass spectrometry, and electron paramagnetic resonance are used to unveil the chemical modification of the plasma-treated and stabilized surfaces. The nanotextured and chemically stabilized surfaces are used as substrates for the development of immunochemical assays for the sensitive detection of C-reactive protein and salmonella lipopolysaccharides through immobilization of the respective analyte-specific antibodies onto them. Such substrates are stable for a period of 1 year with ambient storage.

Increasing binding density of yeast cells by control of surface charge with allylamine grafting to ion modified polymer surfaces

Plasma immersion ion implantation (PIII) treatment of polymers creates a biointerface capable of direct covalent immobilization of biomolecules. The immobilization of protein molecules is achieved by covalent bonds formed between embedded radicals on the treated surface and amino acid side chains and cells can be immobilized through cell-wall proteins. The attachment density of negatively charged entities on a PIII treated surface is inhibited by its negative surface charge at neutral pH. To reduce the negative charge of PIII treated surfaces in phosphate buffer (pH 7.4, 11mM), we develop an effective approach of grafting allylamine monomers onto the treated surface. The results reveal reactions between allylamine and radicals on the PIII treated surface. One of these triggers polymerization, increasing the number of amine groups grafted. As a consequence, the PIII treated polystyrene surface after allylamine exposure becomes more hydrophobic and less negatively charged in phosphate buffer. Using yeast cells as an example, we have shown a significant improvement (6–15 times) of cell density immobilized on the PIII treated surface after exposure to allylamine.

Immobilized pineapple stem bromelain activity in a wine-like medium: Effect of inhibitors

The catalytic activity of stem bromelain, covalently immobilized on chitosan beads, was characterized towards a synthetic substrate (Bz-Phe-Val-Arg-pNA), in a wine-like acidic medium containing wine inhibitors (ethanol, sulphur dioxide, grape skin and seed tannins), at their average concentration range.For all tested substances, the inhibition constant (Ki) values of immobilized bromelain were significantly higher than the corresponding values obtained in a previous work for free enzyme, thus indicating that direct covalent immobilization on chitosan beads makes protease more resistant to the inhibition effect.Immobilized protease was affected by ethanol inhibition (competitive type) only if present at abnormal concentration for real wine. Grape skin and seed tannins exerted uncompetitive inhibition to the same extent, with a Ki value close to 1 g lgallic acid eq−1. The effect of free sulphur dioxide on immobilized bromelain changed respect to free enzyme, both becoming uncompetitive inhibitor and dramatically lowering its strength.These results show that immobilized stem bromelain could have productive biotechnological applications in winemaking, even though further studies will be necessary to test its proteolytic activity towards wine proteins in real matrices.

Glycosylation of the polypropylene membrane surface via thiol–yne click chemistry for lectin adsorption

Glycosylated membrane, as one of the most important affinity membranes, permits affinity separation/purification of proteins based on carbohydrate–protein interactions. It is an important scientific issue to screen facile method for fabricating the glycosylated membrane surface with high glycosyl density. Such a surface can be fabricated by the direct covalent immobilization of carbohydrate ligands on the surfaces of microporous polypropylene membrane (MPPM). First, alkyne-functionalized membrane surface was fabricated by plasma pretreatment combined with UV-induced graft polymerization of 3-(trimethylsilyl) propargyl methacrylate. Then, the glycosylated membrane surface was directly fabricated with the thiol–yne click reaction to ensure rapid process, improved efficiency, and high glycosyl density. Chemical and physical properties of the membrane surface were characterized by ATR/FT-IR, XPS, FESEM and water contact angle measurement. Static lectin adsorption indicates that the glycosylated membrane can specifically adsorb lectin concanavalin A (Con A) other than peanut agglutinin (PNA). Break through curves from dynamic Con A adsorption show the membrane has unique properties such as strong specificity, high adsorption capacity, and reversible binding capability. We suggest that the prepared glycosylated membrane is of great potentials in affinity membrane chromatography for rapid and high-resolution separation/purification of lectins.

Covalent immobilization of a polyoxometalate in a porous polymer matrix: a heterogeneous catalyst towards sustainability

The heterogenization of a polyoxometalate (POM) catalyst by direct covalent immobilization in polymer matrices with uniform macropores and high specific surface areas was reported. Via click chemistry, organically modified POM clusters were mainly “clicked” on the functionalized channel surface of a macroporous resin. The appraisement of the catalytic performance via catalysis on tetrahydrothiophene (THT) oxidation, demonstrates that the solid catalyst is efficient and has a high selectivity. More attractively, it could be reused several times without detectable catalytic activity loss. And no POM species were detected in the filtrate, stemming from the strong covalent bonding between the POM clusters and the macroporous resin surface. Evidently, such a catalyst heterogenization strategy helps to overcome the fatal leaching problem. Therefore the POM heterogeneous material can become an ideal candidate for industrial processes and will also have great potential in practical application not only for oxidative catalysis.

Spatially controlled surface immobilization of nucleophiles via trapping of photo-generated thioaldehydes

A novel and very simple photochemical strategy that utilizes light as a facile means to provide spatio-temporal control for the direct covalent immobilization of nucleophiles is presented. The concept is based upon the efficient trapping of photogenerated thioaldehydes by amines, hydroxylamines, and thiols. Surface patterns of polymers and small molecules bearing pendant amine-, hydroxylamine- or thiol moieties were successfully generated and imaged in a time-of-flight secondary-ion mass spectrometry (ToF-SIMS) investigation.

Covalent Attachment of Biomacromolecules to Plasma-Patterned and Functionalized Carbon Nanotube-Based Devices for Electrochemical Biosensing

The interface between biomacromolecules and carbon nanotubes (CNTs) is of critical importance in developing effective techniques that provide CNTs with both biomolecular recognition and signal transduction through immobilization. However, the chemical inertness of CNT surfaces poses an obstacle to wider implementation of CNTs in bioanalytical applications. In this paper, we present a review of our recent research activities related to the covalent attachment of biomacromolecules to plasma-patterned and functionalized carbon nanotube films and their application to the fabrication of electrochemical biosensing devices. The SWCNT films were spray-deposited onto a miniaturized three-electrode system on a glass substrate and activated using highly purified atomic oxygen generated in radiofrequency plasma; this introduced oxygen-containing functional groups into the SWCNT surface without fatal loss of the original physicochemical properties of the CNTs. The carboxylated SWCNT electrodes were then selectively modified via amidation or esterification for covalent immobilization of the biomacromolecules. The plasma-treated SWCNT-based sensing electrode had an approximately six times larger effective area than the untreated SWCNT-based electrode, which significantly amplified the amperometric electrochemical signal. Finally, the efficacy of plasma-functionalized SWCNT (pf-SWCNT) as a biointerface was examined by immobilizing glucose oxidase, Legionella pneumophila ( L. pneumophila)-specific antibodies, L. pneumophila-originated DNAs, and thrombin-specific aptamers on the pf-SWCNT-based three-electrode devices. The pf-SWCNT films were found to support direct covalent immobilization of the above-listed biomacromolecules on the films and to thereby overcome the many drawbacks typically associated with simple physisorption. Thus, pf-SWCNT sensing electrodes on which biomacromolecules were covalently immobilized were found to be chemically stable and have a long lifetime.

Quantitative photochemical immobilization of biomolecules on planar and corrugated substrates: a versatile strategy for creating functional biointerfaces.

Methods for the generation of substratespresenting biomolecules in a spatially controlled manner are enabling tools for applications in biosensor systems, microarray technologies, fundamental biological studies and biointerface science. We have implemented a method to create biomolecular patterns by using light to control the direct covalent immobilization of biomolecules onto benzophenone-modified glass substrates. We have generated substrates presenting up to three different biomolecules patterned in sequence, and demonstrate biomolecular photopatterning on corrugated substrates. The chemistry of the underlying monolayer was optimized to incorporate poly(ethylene glycol) to enable adhesive cell adhesion onto patterned extracellular matrix proteins. Substrates were characterized with contact angle goniometry, AFM, and immunofluorescence microscopy. Importantly, radioimmunoassays were performed to quantify the site density of immobilized biomolecules on photopatterned substrates. Retained function of photopatterned proteins was demonstrated both by native ligand recognition and cell adhesion to photopatterned substrates, revealing that substrates generated with this method are suitable for probing specific cell receptor-ligand interactions. This molecularly general photochemical patterning method is an enabling tool for the creation of substrates presenting both biochemical and topographical variation, which is an important feature of many native biointerfaces.

Protein Tethering into Multiscale Geometries by Covalent Subtractive Printing

High-resolution patterns of proteins at multiple length scales are printed by a facile and flexible strategy using a flat elastomeric stamp and direct covalent immobilization onto mixed self-assembled monolayers of tri(ethylene glycol)- and carboxylic acid–hexa(ethylene glycol)-terminated alkanethiols on gold-coated substrates. This is a robust method for generating complex protein arrays with precise control over micrometer- and nanometer-scale pattern geometries while maintaining a protein-adsorption-resistant background.