Functionalized nanocomposite coating of a glass surface for oligonucleotide immobilization

A new type of coating for manufacturing DNA chips was constructed on the basis of an organicinorganic nanocomposite based on the polyvinylbutyral-tetraethoxysilane copolymer. The organosilicon composite was functionalized by introduction of ethanolamine vinyl ether copolymers, which contain amino groups and anchor vinyloxide units capable of reacting with silanol groups of the nanocomposite. The resulting coatings form a film on glass slides with a high surface density of amino groups (up to 700 groups/nm²) suitable for three-dimensional immobilization of oligonucleotides. The use of bifunctional reagents (e.g., phenylene diisothiocyanate) for the attachment of oligonucleotides bearing amino linkers to the amino-containing surface provides an immobilization density of 0.5–1.6 pmol/mm². Immobilization with a higher density (10–12 pmol/mm²) was achieved for attachment to amino-containing glass slides upon the use of oligonucleotides containing a selectively activated terminal phosphate group. The activation of oligonucleotides was carried out with the triphenylphosphine-dithiodipyridine pair in the presence of dimethylaminopyridine N-oxide. The resulting DNA chips were shown to be useful in principle for DNA detection.     

A new heterobifunctional reagent for immobilization of biomolecules on glass surface

Synthesis of a new heterobifunctional reagent, [N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine] (NTMTA) is described for the immobilization of a variety of biomolecules on glass surface. Its triethoxysilyl group reacts with glass surface and trifluoroethanesulfonate ester structure reacts selectively with aminoalkyl/mercaptoalkyl function in biomolecules. The immobilization can be achieved by two ways involving two steps. The first route involves the reaction of NTMTA with glass beads followed by attachment of aminoalkyl- or mercaptoalkylated biomolecules. The second one involves the reaction of biomolecules, viz., oligonucleotides, proteins, etc., with NTMTA via their aminoalkyl or mercaptoalkyl functions to form a biomolecule conjugate, which is then reacted with glass beads (unmodified) to complete immobilization process. This has been demonstrated by successful immobilization of 5'-mercaptoalkyl- or aminoalkylated oligonucleotides and some commonly used enzymes on glass beads using NTMTA reagent.     

Functionalized kaolin as support for endoglucanase immobilization

Bioprocess and Biosystems Engineering - Endoglucanases are an enzyme of cellulases complex that has a great potential for many technological applications. One of the issues of its use concerns the...     

One-end immobilization of individual DNA molecules on a functional hydrophobic glass surface

We demonstrate an effective method for DNA immobilization on a hydrophobic glass surface. The new DNA immobilizing technique is extremely simple compared with conventional techniques that require heterobifunctional crosslinking reagent between DNA and substrate surface that are both modified chemically. In the first process, a coverslip was treated with dichlorodimethylsilane resulting in hydrophobic surface. lambda DNA molecules were ligated with 3'-terminus disulfide-modified 14 mer oligonucleotides at one cohesive end. After reduction of the disulfide to sulfhydryl (thiol) groups the resulting thiol-modified lambda DNA molecules were reacted on silanized coverslip. Fluorescent observation showed that the thiol-modified lambda DNA molecules were anchored specifically to the hydrophobic surface at one terminus, although non-specific binding of the DNA molecules was suppressed. It was observed that the one-end-attached DNA molecule was bound firmly to the surface and stretched reversibly in one direction when a d.c. electric field was applied.     

A chemically modified glass surface that facilitates transglutaminase-mediated protein immobilization

An amino-modified glass surface for enzymatic protein immobilization by microbial transglutaminase (MTG) was developed. Diamine substrates with secondary amino groups in the linker moiety, like triethylenetetramine (TETA), exhibited at most a 2-fold higher reactivity in the MTG-catalyzed reaction compared to those with the alkyl linker. A 96-well glass plate was subsequently modified with selected diamine substrates. Validation of the modified surface by enzymatic immobilization of enhanced green fluorescent protein tagged with a glutamine donor-substrate peptide (LLQG) of MTG revealed that the protein loading onto the TETA-modified glass surface was approximately 15-fold higher than that on the unmodified one.     

Dual immobilization of biomolecule on the glass surface using cysteine as a bifunctional linker

A novel method for highly efficient enzyme immobilization on the glass surface, by incorporating cysteine as a linker has been demonstrated. The internal glass surface of test tube was pretreated with (3-mercaptopropyl) trimethoxysilane sol–gel and cysteine capped silver nanoparticles, to generate a cysteine layer. This, cysteine rich surface is then used to covalently immobilize alkaline phosphatase on both groups (amino and carboxyl) of cysteine through carbodiimide and glutaraldehyde treatment. The cysteine capped silver nanoparticles were synthesized with an average nanoparticle size of 61 nm as determined by particle size analyzer, while cysteine capping of nanoparticles was confirmed by Fourier transform infra-red spectroscopy. Enhanced enzymatic activity of about 73% was obtained using the dual immobilization technique, while 40% enzyme activity was recovered with carboxyl group and 51% with amino group only. The re-usability of the enzyme immobilized test tube was found to be 8 times and the enzyme retained 85% of its initial activity. With such high immobilization efficiency, cysteine provides a new approach for enhanced immobilization and its integration into different industrial processes and biosensor technology.     

Use of porous glass fiber as a support for biocatalyst immobilization

Summary Porous glass fiber has a very high surface area and good mechanical properties that make it an excellent support for biocatalyst immobilization. By packing aligned glass fibers in a tubular reactor such that the fibers are all parallel to the axis of the tube, the resulting pressure drop is considerably smaller than for a similar bed of packed beads. The utility of this support was demonstrated by immobilizing -glucoamylase by silane-glutaraldehyde coupling, and measuring its activity toward converting maltose to glucose. Using optimized immobilization conditions, an enzyme loading of 1.5 mg protein perm ² surface area was obtained, with an activity of 370 units/g glass at 50°C. The half-life of the immobilized glucoamylase was more than twice as long as that of the free enzyme.     

Trehalase immobilization on aminopropyl glass for analytical use

Trehalase is the enzyme which hydrolyzes the disaccharide trehalose into two alpha-D-glucose molecules. In this article, we present the immobilization of trehalase on aminopropyl glass particles. The enzyme was extracted from Escherichia coli Mph2, a strain harboring the pTRE11 plasmid, which contains the trehalase gene. The partially purified enzyme had a specific activity of 356 U/mg and could be used for quantifying trehalose in the presence of sucrose, maltose, lactose, starch, and glycogen. Partially purified trehalase was immobilized by covalent coupling with retention of its catalytic activity. The support chosen for the majority of the experiments reported was aminopropyl glass, although spherisorb-5NH(2) and chitin were also tested. The immobilized enzyme was assayed continuously for 40 h, at pH 6.0 and 30 degrees C, and no release of enzyme molecules was detected during this procedure. The best condition found for storing the enzyme-support complex was at 4 degrees C in the presence of 25 mM sodium maleate, containing 7 mM beta-mercaptoethanol, 1 mM ethylenediaminetetraacetic acid (EDTA), and 50% glycerol. The enzyme under these conditions was stable, retaining approximately 100% of its initial activity for at least 28 days. The immobilized enzyme can be employed to detect trehalose molecules in micromolar concentration. The optimum pH value found was 4.5 and the K(m) app. 4.9 x 10(-3) M trehalose at pH 4.6 and 30 degrees C, with V(max) of 5.88 mumol glucose . min.(-1), as calculated by a Lineweaver-Burk plot. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 33-39, 1997.     

Functionalized polymeric liposomes. Efficient immobilization of alpha chymotrypsin

A polymerizable phospholipid has been synthesized having a latent aldehyde moiety in the head group (1). Photopolymerized vesicles which have been prepared from this phosphatidylcholine have been successfully conjugated to alpha chymotrypsin. A high degree of loading was achieved with significant retention of enzymatic function.     

Functionalized, biocompatible coating for superparamagnetic nanoparticles by controlled polymerization of a thioglycosidic monomer

It is demonstrated that water-soluble, glucosylated poly(pentafluorostyrene) derivatives revealed favorable coating material properties for magnetic iron oxide nanoparticles. To prepare the coating material in high reproducibility and purity as well as in sufficient amounts, a new route of synthesis is established. The preparation and characterization of the glucosylated, tetrafluorostyryl monomer, by thiol-para-fluorine "click" reaction, and its polymerization, via nitroxide-mediated radical process, is presented in detail. In addition, the coating material and the resulting particle properties are investigated by means of XPS, DLS, TGA, TEM, and cryo-TEM as well as flow cytometry. The glycopolymer acts as an appropriate stabilizing agent for the superparamagnetic nanoparticles by the formation of an approximately 10 nm thick shell, as shown by the XPS analysis. Furthermore, the application of FITC-labeled glycopolymer yielded fluorescent, superparamagnetic nanoparticles, which can be used for monitoring cell-carbohydrate interactions, because these particles show no cytotoxicity toward 3T3 fibroblasts.     

Effective diffusivity and tortuosity in a porous glass immobilization matrix

The effective diffusivity of glucose in porous glass beads was determined using a transient method. Predictions for the intraparticle and surface concentrations were made by an analytical solution of the mass balance. The value of the diffusivity was expected to be lower than the value of the corresponding diffusion coefficient in water, but the opposite was observed. This effect results from intraparticle fluid flow, leading to high values of the apparent effective glucose diffusivity. To measure diffusion only and to prevent any internal convection during the diffusion experiment, the pores of the porous glass beads were filled with Ca-alginate gel. For these glass beads (internal porosity, , equal to 0.56), we found an effective glucose diffusivity of 2.2×10^–10 m²/s at 30°C. Using the relationship to effective intraparticle diffusivity (D_eff)=effective diffusivity in 1% Ca-alginate beads (D_gel) / (with the tortuosity factor) this gives =1.7. For known and measuring by the method described, the D_eff can be calculated for other porous materials or diffusing substances. Knowledge of the exact value of the effective diffusivity is a necessity in bioreactor modelling and was demonstrated by prediction of the residence time distribution profiles in a packed-bed bioreactor containing immobilized yeast cells.     

Derivatization of controlled pore glass beads for solid phase oligonucleotide synthesis

An improved and simplified procedure for the attachment of nucleosides onto long chain alkylamine controlled pore glass beads (LCAA-CPG) is presented. This procedure uses 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide (DEC) to couple nucleoside 3'-succinates directly to the LCAA-CPG. The preparation of nucleoside 3'-succinate anhydrides, p-nitrophenyl, or pentachlorophenyl esters and the use of highly toxic dicyclohexylcarbodiimide (DCC) is no longer required. Procedures involving acidic activation of the LCAA-CPG before derivatization and a pre-synthesis capping are also described, which prevent the formation of oligonucleotides linked by 3'-phosphates to the LCAA-CPG. Evidence is presented indicating that this type of linkage is responsible for the apparently greater than 100% coupling yields observed for the first coupling cycle.     

Attachment of oligonucleotide probes to poly carbodiimide-coated glass for microarray applications

Oligonucleotide-based DNA microarrays are becoming increasingly useful tools for the analysis of gene expression and single nucleotide polymorphisms (SNPs). Here, we present a method that permits the manufacture of microarrays from non-modified oligonucleotides on a poly carbodiimide-coated glass surface by UV-irradiation. The use of UV-irradiation facilitates an increase in the level of signal intensity, but it does not affect signal discrimination by the oligonucleotides immobilized on the surface. The signal intensity obtained for an array fabricated using non-modified oligonucleotides with UV-irradiation is ~7-fold greater than that without UV-irradiation. The detection of SNPs was tested to ascertain whether this technique could discriminate specific hybridization signals without causing significant UV-irradiation-induced damage to the immobilized oligonucleotides. We found that this immobilization method provides greater hybridization signals and a better match/mismatch ratio of SNPs than do the established aminosilane techniques. Application of this technology to manufacturing DNA microarrays for sequence analysis is discussed.     

A microcontact printing approach to the immobilization of oligonucleotide brushes

Solution hybridized oligonucleotides were immobilized onto surfaces via micro-contact printing. Besides micro-patterning of the substrate, sequential dehybridization and rehybridization were monitored via laser scanning microscopy, which assess the surface tethering of the oligonucleotides into a brush.     

Direct immobilization of DNA oligomers onto the amine-functionalized glass surface for DNA microarray fabrication through the activation-free reaction of oxanine

Oxanine having an O-acylisourea structure was explored to see if its reactivity with amino group is useful in DNA microarray fabrication. By the chemical synthesis, a nucleotide unit of oxanine (Oxa-N) was incorporated into the 5′-end of probe DNA with or without the -(CH₂)_n- spacers (n = 3 and 12) and found to immobilize the probe DNA covalently onto the NH₂-functionalized glass slide by one-pot reaction, producing the high efficiency of the target hybridization. The methylene spacer, particularly the longer one, generated higher efficiency of the target recognition although there was little effect on the amount of the immobilized DNA oligomers. The post-spotting treatment was also carried out under the mild conditions (at 25 or 42°C) and the efficiencies of the immobilization and the target recognition were evaluated similarly, and analogous trends were obtained. It has also been determined under the mild conditions that the humidity and time of the post-spotting treatment, pH of the spotting solution and the synergistic effects with UV-irradiation largely contribute to the desired immobilization and resulting target recognition. Immobilization of DNA oligomer by use of Oxa-N on the NH₂-functionalized surface without any activation step would be employed as one of the advanced methods for generating DNA-conjugated solid surface.     

A novel immobilization strategy using oligonucleotide as linker for small molecule microarrays construction

A novel immobilization method based on oligonucleotide as linker has been developed for small molecule microarrays (SMMs) construction. The oligonucleotide tail was employed as a linker in solid-phase synthesis. Small molecules could be easily conjugated at the 5′ end of the oligonucleotide, previously modified with a functional group. Being a reactive species, the oligonucleotide was activated by UV irradiation, for the attachment of the conjugate to the slide surface. The method was successfully applied to structurally distinct small molecules, including biotin, antibiotic and drug. This immobilization strategy showed high efficiency, 1.1 fmol of small molecules in the spotting solution per spot gave a detectable signal (mean S/N = 10.9). The results suggest that it is very promising for exploring interaction between small molecules and proteins, and high throughput detecting the chemical compounds.     

Chemoselective surface immobilization of proteins through a cleavable peptide

Surface immobilization of biomolecules is a fundamental step in several experimental techniques such as surface plasmon resonance analysis and microarrays. Oxime ligation allows reaching chemoselective protein immobilization with the retention of native-like conformation by proteins. Beside the need for chemoselective ligation of molecules to surface/particle, equally important is the controlled release of the immobilized molecules, even after a specific binding event. For this purpose, we have designed and assessed in an SPR experiment a peptide linker able to (i) anchor a given protein (enzymes, receptors, or antibodies) to a surface in a precise orientation and (ii) release the immobilized protein after selective enzymatic cleavage. These results open up the possibility to anchor to a surface a protein probe leaving bioactive sites free for interaction with substrates, ligands, antigens, or drugs and successively remove the probe-ligand complex by enzymatic cleavage. This peptide linker can be considered both an improvement of SPR analysis for macromolecular interaction and a novel strategy for drug delivery and biomaterial developments.     

Dendrimeric coating of glass slides for sensitive DNA microarrays analysis

Successful use and reliability of microarray technology is highly dependent on several factors, including surface chemistry parameters and accessibility of cDNA targets to the DNA probes fixed onto the surface. Here, we show that functionalisation of glass slides with homemade dendrimers allow production of more sensitive and reliable DNA microarrays. The dendrimers are nanometric structures of size-controlled diameter with aldehyde function at their periphery. Covalent attachment of these spherical reactive chemical structures on amino-silanised glass slides generates a reactive ~100 Å layer onto which amino-modified DNA probes are covalently bound. This new grafting chemistry leads to the formation of uniform and homogenous spots. More over, probe concentration before spotting could be reduced from 0.2 to 0.02 mg/ml with PCR products and from 20 to 5 µM with 70mer oligonucleotides without affecting signal intensities after hybridisation with Cy3- and Cy5-labelled targets. More interestingly, while the binding capacity of captured probes on dendrimer-activated glass surface (named dendrislides) is roughly similar to other functionalised glass slides from commercial sources, detection sensitivity was 2-fold higher than with other available DNA microarrays. This detection limit was estimated to 0.1 pM of cDNA targets. Altogether, these features make dendrimer-activated slides ideal for manufacturing cost-effective DNA arrays applicable for gene expression and detection of mutations.     

Novel peptide surface for reversible immobilization of concanavalin A

Concanavalin A (Con A) was spontaneously adsorbed on polymyxin B surface. This peptide-lectin interaction was strong, K(D)=1.9 x 10(-10), based predominantly on creation of hydrophobic bonds, and was completely reversible. Concanavalin A on polymyxin B (PmB) retained higher binding capacity for yeast mannan, compared with covalently immobilized lectin. Kinetics of mannan-concanavalin A interaction were significantly different in dependence on type of concanavalin A immobilization.