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. 2010 Feb 8;20(3):409-421.
doi: 10.1002/adfm.200901293.

Multifunctional Dendrimer-templated Antibody Presentation on Biosensor Surfaces for Improved Biomarker Detection

Hye Jung Han  1 , Rangaramanujam M Kannan  1 , Sunxi Wang  2 , Guangzhao Mao  2 , Juan Pedro Kusanovic  3 , Roberto Romero  4
Affiliations

Multifunctional Dendrimer-templated Antibody Presentation on Biosensor Surfaces for Improved Biomarker Detection

Hye Jung Han et al. Adv Funct Mater. .

Abstract

Dendrimers, with their well-defined globular shape and a high density of functional groups, are ideal nanoscale materials for templating sensor surfaces. This work exploits dendrimers as a versatile platform for capturing biomarkers with improved sensitivity and specificity. Synthesis, characterization, fabrication, and functional validation of the dendrimer-based assay platform are described. Bifunctional hydroxyl/thiol functionalized G4-polyamidoamine (PAMAM) dendrimer is synthesized and immobilized on to the polyethylene-glycol (PEG)-functionalized assay plate by coupling PEG-maleimide and dendrimer thiol groups. Simultaneously, part of the dendrimer thiol groups are converted to hydrazide functionalities. The resulting dendrimer-modified surface is coupled to the capture antibody in the Fc region of the oxidized antibody. This preserves the orientation flexibility of the antigen binding region (Fv) of the antibody. To validate the approach, the fabricated plates are further used as a solid phase for developing a sandwich type ELISA to detect IL-6 and IL-1β, important biomarkers for early stages of chorioamnionitis. The dendrimer-modified plate provides assays with significantly enhanced sensitivity, lower nonspecific adsorption, and a detection limit of 0.13 pg ml-1 for IL-6 luminol detection and 1.15 pg ml-1 for IL-1β TMB detection, which are significantly better than those for the traditional ELISA. The assays were validated in human serum samples from normal (non-pregnant) woman and pregnant women with pyelonephritis. The specificity and the improved sensitivity of the dendrimer-based capture strategy could have significant implications for the detection of a wide range of cytokines and biomarkers since the capture strategy could be applied to multiplex microbead assays, conductometric immunosensors and field effect biosensors.

Keywords: IL-1β; Interleukin-6; antibody conjugation; biosensors; cytokine; dendrimers.

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Figures

Figure 1
Figure 1
Emission spectra of thiol/hydroxyl G4-PAMAM 4 and PyMPO immobilized ELISA plate in MeOH for different dendrimer concentration [Ex: 410 nm].
Figure 2
Figure 2
Fluorescence intensity of antibody-dendrimer conjugate reaction mixture. [Ex: 410 nm in PBS pH 7.4] (A) Free dendrimer 6 (276 fM) before immunoprecipitation (IP). (B) Reaction mixture of antibody-dendrimer conjugation before IP. (C) Free dendrimer 6 after IP. (D) Reaction mixture of antibody-dendrimer conjugation after IP. (E) Eluted bound antibody-dendrimer conjugate, dendrimer 6 and antibody mixture. (F) Eluted nonspecifically adsorbed free dendrimer 6. (G) Free antibody (50 μg ml-1, 333 nM).
Figure 3
Figure 3
(a) AFM height image of 2k PEG-OH and 3.4k PEG-Mal modified ELISA plate, conducted in air with the tapping mode. (b) AFM height image of dendrimer 4 attached on PEG modified substrate, conducted in air with the tapping mode. (c) A typical AFM height image of antibody-dendrimer conjugate attached on PEG modified ELISA plate. The image was conducted in tapping mode in liquid cell filled with PBS pH 7.4.
Figure 4
Figure 4
Standard curves for IL-6 ELISA measured with the dendrimer plate, IL-6 kit and plate prepared by physical adsorption of anti-human IL-6 antibody.
Scheme 1
Scheme 1
Synthesis of hydroxyl/LC-PDP conjugated G4-PAMAM dendrimer 4.
Scheme 2
Scheme 2
Schematic representation of ELISA plate modification with PEG and immobilization of hydroxyl/LC-PDP-G4 PAMAM 4 onto PEG layer.
Scheme 3
Scheme 3
Schematic representation of antibody immobilization on to the dendrimer.
See this image and copyright information in PMC

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