Superparamagnetic bead interactions with functionalized surfaces characterized by an immunomicroarray Peter Durand Skottrup 1 , Mikkel Fougt Hansen, Jacob Moresco Lange, Maria Deryabina, Winnie Edith Svendsen, Mogens Havsteen Jakobsen, Martin Dufva * Department of Micro- and Nanotechnology, Technical University of Denmark, DTU Nanotech, Building 345 East, DK-2800 Kongens Lyngby, Denmark article info Article history: Received 25 November 2009 Received in revised form 8 April 2010 Accepted 20 April 2010 Available online 24 April 2010 Keywords: Magnetic bead Microarray Silica Streptavidin Biotin abstract Magneto-resistive sensors capable of detecting superparamagnetic micro-/nano-sized beads are promis- ing alternatives to standard diagnostic assays based on absorbance or fluorescence and streptavidin-func- tionalized beads are widely used as an integral part of these sensors. Here we have developed an immunomicroarray for systematic studies of the binding properties of 10 different micro-/nano-sized streptavidin-functionalized beads to a biotin substrate immobilized on SiO 2 with or without surface mod- ification. SiO 2 surface cleaning, immobilized substrate concentration and surface blocking conditions were optimized. Polyethylene glycol-based surfaces with different end groups on the anchor molecule, 2,4,6-trichloro-1,3,5-triazine (TsT), were synthesized and compared with the standard (3-aminopro- pyl)triethoxysilane (APTS)/glutaraldehyde chemistry. APTS/glutaraldehyde, directly linked TsT and bare H 2 O 2 -activated SiO 2 performed better than polyethylene glycol-modified surfaces. Two beads, Masterbe- ads and M-280 beads, were found to give superior results compared with other bead types. Antibody/ antigen interactions, illustrated by C-reactive protein, were best performed with Masterbeads. The results provide important information concerning the surface binding properties of streptavidin-functionalized beads and the immunomicroarray can be used when optimizing the performance of bead-based biosensors. Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction Micro- and nano-sized superparamagnetic beads are widely used in biochemical laboratories for the capture and purification of biological targets. Beads can be modified with antibodies, nu- cleic acid probes or streptavidin and thereby capture desired ana- lytes in solution. They are used for many different kinds of applications, such as the capture and isolation of cells from com- plex suspensions, the isolation of proteins and nucleotides by immunoprecipitation, to more advanced applications such as the isolation of phospho- and glycopeptides for mass spectrometry analysis [1]. Magnetic beads are also popular in the very active re- search area concerning the ability to perform pathogen detection on site, often referred to as ‘‘point of care” (POC) analysis. POC analysis should be performed in a precise and rapid manner at the site where it is needed, e.g. in hospitals, at the doctor’s office on the battlefield and in agricultural production, preferably using hand-held systems [2,3]. Superparamagnetic micro- and nano- sized beads can be used in combination with magneto-resistive sensors based on, for example, the giant magnetoresistance (GMR) effect, the magnetic tunnelling effect or the planar Hall ef- fect (PHE) for the detection of biomarkers, toxins and pathogens [4,5]. For example, biological warfare pathogens have been de- tected using a GMR micro-bead sensor that measured nucleotide hybridizations [6,7]. Magneto-resistive sensors can potentially be made very compact and sensitive and could prove useful as alter- natives or supplements to standard diagnostic laboratory-based assays, such as enzyme-linked immunosorbent assay (ELISA) and real-time PCR, as well as for POC detection of pathogens or toxins [4,5]. In sandwich-type assays surface immobilized ligands capture the analyte, followed by bead binding to the analyte. The trapping of beads on the surface can then be measured by the magnetoresis- tive sensor (for a review see, [4]). Unlike fluorescence detection, in which small molecules (fluorochromes) are tethered to the target or tracers and subsequently detected by optical means, magneto- resistive sensors are based on the detection of relatively large beads, which often have diameters in the range 200–3000 nm. At the molecular scale beads can therefore be considered as indepen- dent surfaces and thus two surface-bound molecules must interact in bead-based assays. It has been suggested that immobilization of 1742-7061/$ - see front matter Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2010.04.019 * Corresponding author. Tel.: +45 4525 6324; fax: +45 4588 7762. E-mail address: martin.dufva@nanotech.dtu.dk (M. Dufva). 1 Present address: Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Jagtvej 162, DK-2100 Copenhagen, Denmark. Acta Biomaterialia 6 (2010) 3936–3946 Contents lists available at ScienceDirect Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat