Characterization of Three Amino-Functionalized Surfaces and Evaluation of Antibody Immobilization for the Multiplex Detection of Tumor Markers Involved in Colorectal Cancer

Abstract

Antibody microarrays are powerful and high-throughput tools for screening and identifying tumor markers from small sample volumes of only a few microliters. Optimization of surface chemistry and spotting conditions are crucial parameters to enhance antibodies' immobilization efficiency and to maintain their biological activity. Here, we report the implementation of an antibody microarray for the detection of tumor markers involved in colorectal cancer. Three-dimensional microstructured glass slides were functionalized with three different aminated molecules ((3-aminopropyl)dimethylethoxysilane (APDMES), Jeffamine, and chitosan) varying in their chain length, their amine density, and their hydrophilic/hydrophobic balance. The physicochemical properties of the resulting surfaces were characterized. Antibody immobilization efficiency through physical interaction was studied as a function of surface properties as well as a function of the immobilization conditions. The results show that surface energy, steric hindrance, and pH of spotting buffer have great effects on protein immobilization. Under optimal conditions, biological activities of four immobilized antitumor marker antibodies were evaluated in multiplex immunoassay for the detection of the corresponding tumor markers. Results indicated that the chitosan functionalized surface displayed the highest binding capacity and allowed to retain maximal biological activity of the four tested antibody/antigen systems. Thus, we successfully demonstrated the application of amino-based surface modification for antibody microarrays to efficiently detect tumor markers.

Antibody orientation on biosensor surfaces: a minireview

“…Gold, glass, copper, silicon nitride and silicon surfaces or magnetic beads represent only a small selection of surfaces used for Ab immobilization. 29,32,[42][43][44][45] Below we will review several strategies of Ab immobilization. They can be distinguished by non-covalent or covalent coupling chemistry.…”

Highly Active Ruthenium Supported on Magnetically Recyclable Chitosan‐Based Nanocatalyst for Nitroarenes Reduction

“…After ionic gelation, the peaks were deconvoluted into contributions at BE=284.6 (C−C/C−H), 286.2 (C−O and C−N), 288.1 (O−C−O and N−C=O), and 289.3 eV (O−C=O), respectively, which correspond to the structure of CS (Figure b) . The deconvoluted N 1s spectrum showed a band at BE=399.7 eV assigned to free amine groups and a weak band at BE=401.4 eV that is assigned to the protonated amino group (−NH 3 + ; Figure c) . The deconvoluted high‐resolution O 1s spectrum of Mn@CS@Ru is shown in Figure d. The peak at BE=529.9 eV is attributed to the lattice oxygen of M−O (M=Mn, Fe, Ru), and the peaks at BE=531.2, 532.3, and 533.3 eV are attributed to the O in TPP (P 3 O 10 5− ) and the −OH and O−C−O in CS .…”

Gamma Globulins Adsorption on Carbofunctional Polysiloxane Microspheres

“…On the other hand, the protein adsorption is used to bioactivate synthetic materials. The ability of surfaces to adsorb proteins is important in the case of materials used in tissue engineering [6], biosensors [7], drug carriers [8,9], adsorbents [2], protein chromatography [10] and biocatalysts [11,12]. Controlling of the affinity of proteins to surface of materials is a challenge.…”