Optimizing antibody immobilization strategies for the construction of protein microarrays.

Silicon microchips with immobilized antibodies were used to develop microfluidic enzyme immunoassays using chemiluminescence detection and horseradish peroxidase (HRP) as the enzyme label. Polyclonal anti-atrazine antibodies were coupled to the silicon microchip surface with an overall dimension of 13.1 x 3.2 mm, comprising 42 porous flow channels of 235-microm depth and 25-microm width. Different immobilization protocols based on covalent or noncovalent modification of the silica surface with 3-aminopropyltriethoxysilane (APTES) or 3-glycidoxypropyltrimethoxysilane (GOPS), linear polyethylenimine (LPEI, MW 750,000), or branched polyethylenimine (BPEI, MW 25,000), followed by adsorption or covalent attachment of the antibody, were evaluated to reach the best reusability, stability, and sensitivity of the microfluidic enzyme immunoassay (microFEIA). Adsorption of antibodies on a LPEI-modified silica surface and covalent attachment to physically adsorbed BPEI lead to unstable antibody coatings. Covalent coupling of antibodies via glutaraldehyde (GA) to three different functionalized silica surfaces (APTES-GA, LPEI-GA, and GOPS-BPEI-GA) resulted in antibody coatings that could be completely regenerated using 0.4 M glycine/HCl, pH 2.2. The buffer composition was shown to have a dramatic effect on the assay stability, where the commonly used phosphate buffer saline was proved to be the least suitable choice. The best long-term stability was obtained for the LPEI-GA surface with no loss of antibody activity during one month. The detection limits in the microFEIA for the three different immuno surfaces were 45, 3.8, and 0.80 ng/L (209, 17.7, and 3.7 pM) for APTES-GA, LPEI-GA, and GOPS-BPEI-GA, respectively.

Microfluidic Enzyme Immunoassay Using Silicon Microchip with Immobilized Antibodies and Chemiluminescence Detection

3‐Aminopropyltriethoxysilane-Functionalized Bioanalytical Platforms for Biosensors and Diagnostics Sandeep Kumar Vashist,*,†,‡ Edmond Lam, Sabahudin Hrapovic, Keith B. Male, and John H. T. Luong †HSG-IMIT Institut für Mikround Informationstechnik, Georges-Koehler-Allee 103, 79110 Freiburg, Germany ‡Laboratory for MEMS Applications, Department of Microsystems Engineering IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany National Research Council Canada, Montreal, Quebec H4P 2R2, Canada Innovative Chromatography Group, Irish Separation Science Cluster (ISSC), Department of Chemistry and Analytical, Biological Chemistry Research Facility (ABCRF), University College Cork, Cork, Ireland

/pdf/immobilization-of-antibodies-and-enzymes-on-3-3um0ob31sw.pdf

Immobilization of antibodies and enzymes on 3-aminopropyltriethoxysilane-functionalized bioanalytical platforms for biosensors and diagnostics.

Optimization and characterization of biomolecule immobilization on silicon substrates using (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde linker

Chitosan, a biopolymer found in the exoskeletons of shellfish, has been shown to be antibacterial, biodegradable, osteoconductive, and has the ability to promote organized bone formation. These properties make chitosan an ideal material for use as a bioactive coating on medical implant materials. In this study, coatings made from 86.4% de-acetylated chitosan were bound to implant-quality titanium. The chitosan films were bound through a three-step process that involved the deposition of 3-aminopropyltriethoxysilane (APTES) in toluene, followed by a reaction between the amine end of APTES with gluteraldehyde, and finally, a reaction between the aldehyde end of gluteraldehyde and chitosan. Two different metal treatments were examined to determine if major differences in the ability to bind chitosan could be seen. X-ray photoelectron spectroscopy (XPS) was used to examine the surface of the titanium metal and to study the individual reaction steps. The changes to the titanium surface were consistent with the anticipated reaction steps, with significant changes in the amounts of nitrogen, silicon, and titanium that were present. It was demonstrated that more APTES was bound to the piranha-treated titanium surface as compared to the passivated titanium surface, based on the amounts of titanium, carbon, nitrogen, and silicon that were present. The metal treatments did not affect the chemistry of the chitosan films. Using toluene to bond APTES on titanium surfaces, rather than aqueous solutions, prevented the formation of unwanted polysiloxanes and increased the amount of silane on the surface for forming bonds to the chitosan films. Qualitatively, the films were more strongly attached to the titanium surfaces after using toluene, which could withstand the ultrahigh vacuum environment of XPS, as compared to the aqueous solutions, which were removed from the titanium surface when exposed to the ultrahigh vacuum environment of XPS.

XPS study on the use of 3-aminopropyltriethoxysilane to bond chitosan to a titanium surface.

A convenient and efficient method for the site-directed incorporation of aldehydes generated on the oligosaccharide moieties at the C-terminal of immunoglobulin (IgG) using NaIO4oxidation reaction is explored as a means of ensuring controlled assembly of IgG antibodies onto aminopropyltriethoxylsilane (APTES) derivatized silicon wafer surfaces. The orientation and antigen binding capacity (AgBC) of site-directly assembled IgG antibodies on derivatized surfaces were investigated using atomic force microscopy (AFM) and enzyme immunoassay (EIA), respectively. A major difference in preferential orientation is observed when the incubation of derivatized surfaces with oxidized IgG molecules is compared in two different kinds of buffer solutions. We obtained the stable and homogeneous IgG layer without loss of the AgBC on the APTES derivatized surface using the controlled incubation condition.

Controlled Site-Directed Assembly of Antibodies by Their Oligosaccharide Moieties onto APTES Derivatized Surfaces.

Site-directed immobilization of immunoglobulin G on 3-aminopropyltriethoxylsilane modified silicon wafer surfaces

An antibody can be specifically oxidized with periodate (NaIO4) on the carbohydrate side chains at its C-terminal. Rabbit anti-hepatitis B surface antigen (anti-HBsAg) IgG antibodies were bound to the silicon wafer surface by covalent bonds between aldehydes generated on the carbohydrate side chains of the antibodies and the reactive amine groups of 3-aminopropyltriethoxylsilane(APTES)-modified silicon wafer surfaces. A control experiment was also performed by direct attachment of antibodies to glutaraldehyde-treated silicon surfaces. Two different coupling antibody strategies were investigated in this paper. Atomic force microscopy was used to observe the orientation of the site-directed and random attachment of rabbit anti-HBsAg IgG antibodies and the conservation of their antigen-binding capacity (AgBC) was assessed using an enzyme immunoassay (EIA).

Xu Bin

Papers

Controlled Site-Directed Assembly of Antibodies by Their Oligosaccharide Moieties onto APTES Derivatized Surfaces.

Site-directed immobilization of immunoglobulin G on 3-aminopropyltriethoxylsilane modified silicon wafer surfaces

Orientation of Antibodies on a 3-Aminopropyltriethoxylsilane-Modified Silicon Wafer Surface