Spatially addressable protein array: ssDNA‐directed assembly for antibody microarray

Abstract

Protein microarray offers a means for high-throughput profiling of cellular proteins to provide insights into the mechanisms of biological processes. This study describes the design and fabrication of a robust platform, spatially addressable protein array (SAPA), by exploring the specificity of ssDNA hybridization for self-assembly of semi-synthetic ssDNA-antibody conjugates which capture antigens from complex biological samples. This approach does not involve the direct immobilization of antibodies nor antigen, but instead captures the target antigens in the solution phase followed by self-directed assembly of the complex onto the surface. In an effort to optimize the platform, the effects of surface chemistry, nonspecific protein adsorption, facile preparation, and purification of ssDNA-conjugated antibody and capture of the antigen from a complex biological sample such as cell lysate were examined. This platform allowed antigen detection in cell lysate with high sensitivity (1 pM). The method described herein can be extended to the high-throughput detection of other interacting molecules in solution phase and their subsequent assembly onto any substrate.

Nanostructured Si-nanowire microarrays for enhanced-performance bio-analytics

“…Bio-analytic applications ideally require very high SNRs but obtaining the required high SNRs depends critically on three major challenges: (i) the capability to create probe sites with significantly high density, (ii) the ability to eliminate undesirable, multiphase probe-analyte diffusion-limited interfacial interactions 6 and (iii) improvement in the signal detection mechanism for signal readout. In addition to works cited previously which concentrate on creating high density probe sites, researchers have also been exploring novel detection schemes such as graphene ink 7 and quantum dots 8 for read out.…”

“…Basically, the novelty of our approach lies in two key strategies we introduce here, namely, (i) we employ a homogeneous mixing of analytes with an anti-sense DNA-conjugated antibody in the liquid phase first and then address the captured analytes to a specific location. This strategy is employed to enhance the capturing of the analyte by the antibody and to minimize antibody denaturation due to antibody-surface interaction that is normally encountered in conventional microarray technology, 6 and (ii) we provide a unique platform made by the glancing-angle-deposition and metal-assisted-chemical-etching (GLAD-MACE) technique 9 that can accommodate an extremely large number of sense-DNA that is used for addressing the captured analyte. With our GLAD-MACE method, not only can one achieve a high SNR for the DNA microarray, but more importantly, our approach of protein capturing combined with the DNA functionalized GLAD-MACE substrate provides a superior platform for protein capturing.…”

DNA-directed assembly of protein microarrays

“…Glass surfaces derivatized with polyamidoamine (PAMAM) dendrimers was first reported by Benters et al for fabricating high-density immobilization surfaces for nucleic acid and protein microarrays (76). This surface was modified to minimize non-specific binding by using carboxyl-terminated PAMAM (PAMAM-COOH) dendrimers (77). This resulted in negatively charged surfaces which substantially reduced non-specific binding (78).…”

“…This dendrimeric carboxyl surface provided highdensity immobilization of antibodies which retained high efficiency of antigen capture while maintaining low background even in the presence of a complex cell lysate mixture. PAMAM surfaces coupled with DNA-directed assembly (DDA) strategies for ASR-analyte immobilization were further shown to achieve highsensitivity antigen detection in antibody microarray applications (77,79,80).…”

“…Bailey et al (94) and our group (107) using antibody-DNA conjugates have resulted in DNA-directed self-assembly antibody microarrays where the antibody-antigen interaction was allowed to take place in a homogeneous solution before the DNA-probe-target complex is spatially separated onto a spotted DNA array for detection. Our spatially addressable protein array (SAPA) was used for the detection of fluorophore-labeled rabbit immunoglobulin G and green fluorescent protein (GFP) from both buffer solution as well as fluorophorelabeled cell lysate with a detection limit of as low as 1 pM (about 150 pg/ml) without amplification (107). The DNAencoded antibody library (DEAL) technique reported by Bailey et al uses a sandwich immunoassay approach for the detection of the human cytokines interferon-gamma, tumor necrosis factor-alpha, and interleukin-2 (IL-2).…”