Selective labeling of oligonucleotide monolayers by metallic nanobeads for fast optical readout of DNA-chips J. Michael Ko Èhler * , Andrea Csa Âki, Jo Èrg Reichert, R. Mo Èller, W. Straube, Wolfgang Fritzsche Institute for Physical High Technology, D-07745 Jena, Winzerlaerstr. 10, Germany Abstract Chemically modi®ed nanobeads are used for the labeling of molecular objects in ultramicroscopic techniques. In addition to this application, we found that the cooperative effect of densely immobilized ensembles of metallic nanobeads on micropatterned molecular mono®lms can be used for an optical measurement of speci®c binding events. So, a reproducible readout of hybridization on DNA chips becomes easily possible. The technique can substitute ¯uorescence methods for the readout, which are commonly used in most applications. Functionalized beads of a certain size are large enough to in¯uence light ways considerably, but small enough for speci®c chemical reactions with molecules on chip surfaces. It was found that gold particles with diameters between 15 and 60 nm are particularly suited for such a labeling of DNA-DNA-interactions on DNA-chips. Their immobilization density and sequence speci®city is very high, so that transmitted light at glass chips is reduced considerably or re¯ected light is increased, resulting in a very strong contrast. This effect can be quantitatively readout by light scanners, optical cameras or microscopes on a very small time scale. Usually, exposure times less than 10 ms are suf®cient. The chips can be stored for a long time without loss of information about the degree of hybridization. The surface chemistry for DNA immobilization and for the bead labeling as well as the readout procedure are discussed. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Biochips; Labeling; Nanobeads; Optical readout 1. Introduction The application of biochips in medical diagnostics, bio- molecular research and other promising ®elds depends strongly on reproducible labeling and readout methods easily to handle. Whereas radiolabeling methods are well suited for quantitative readout of biochips, ¯uorescence methods are largely used in cause of their better practic- ability. But, ¯uorescence labeling by molecular dyes suffers from some disadvantages like photochemical instability of chromophores or environment-depending ¯uorescence quantum yields. Nevertheless, ¯uorescence labeling be- comes more and more applied for the readout of biochips and, in particular, for DNA-chips, namely for expression studies. Labeling strategies for biochips are not restricted to small molecules like ¯uorescence dyes. In principle, particles can used for surface labeling [1]. Larger molecules, clusters and nanosized particles have a chemical behavior similar to small molecules and can so be used as speci®c chemical labels. Nanobeads are well introduced in the labeling of cellular and molecular objects for ultramicroscopy [2]. They are also of interest for the optical readout of miniaturized sensor devices [3]. In addition, beads can be modi®ed by oligonucleotide surface ®lms and used for chemical inter- action of beads [4]. Clustering of surface-modi®ed nano- beads by selective hybridization of complementary single strand DNA in solution was detected by the shift of absorp- tion wavelength in optical spectroscopy [5]. A sequence- speci®c bonding of DNA-modi®ed nanoparticles was demonstrated at micropatterned oligonucleotide mono®lms [6]. Here, the binding of nanometer-sized DNA-modi®ed gold nanoparticles is studied as a model for possible appli- cations of surface-modi®ed metal nanoparticles as labels in biochip techniques. Therefore, the density of oligonucleo- tide molecules at the surface of the beads as well as at the chip surface was investigated. The density of bounded nanoparticles is studied in dependence on bead concentra- tion and on the ratio of complementary to non-complemen- tary surface-immobilized DNA. Finally, the read-out of Sensors and Actuators B 76 2001) 166±172 * Corresponding author. 0925-4005/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII:S0925-400501)00619-0