Nanometer-thin, Soft Polymer Cushions: A Novel Coating on Chips for the Analysis of Genes and Proteins

Now that the sequences of the human genome are known, increasing emphasis is being placed on gene expression analysis (genomics) and on the study of the structure, function and expression of proteins (proteomics). Typical biological samples (such as cell extracts, tissue samples or blood) contain a large number of different DNAs, RNAs and especially proteins often in very low concentrations. The analysis of these complex samples requires novel multiplexed sensing platforms with high sensitivity and selectivity. Fluorescent microarrays with thousands of detection spots are currently being used and their sensitivity and selectivity is rapidly increasing thanks to the use of engineered surfaces [1].

A novel DNA biosensor chip surface: a successful collaboration between ETHZ and Zeptosens

A novel, dedicated biosensor chip surface for DNA/RNA microarray analysis has been developed in a close collaboration between ETHZ BioInterfaceGroup (Laboratory for Surface Science and Technology) and the sensor company Zeptosens. It consists of a single layer of densely packed poly(ethylene glycol) (“PEG”) chains attached to surface and form a soft cushion to immobilize recognition molecules. PEG is one of the few materials that strongly resist the adsorption of proteins. We have applied a novel type of copolymer, poly(L-lysine)-graft-poly(ethylene glycol) (“PLL-g-PEG”) to form such PEG brushes at charged tantalum oxide surfaces through the spontaneous adsorption and formation of a nanometer-thin layer [2] (Fig. 1). Since only one layer of molecules is required, very little polymer material is needed, 1 g being ideally sufficient to coat 1000 m² of surface. The underlying tantalum oxide film serve as waveguiding layer, allowing the detection and quantification of biospecific interface reactions through the measurement of fluorescence that is excited in the evanescent field.

Figure 1. Poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) adsorbs to negatively charged metal oxide surfaces. The interactiveness of the surface (from interactive to highly repulsive) can be tailored through the surface density and length of the PEG chains [2].

Adjusting the spacing between the PEG chains of the brush allows for the control of the strength of the physical interaction between the DNA and the surface, with intermediate spacings providing sufficient adhesion strength and optimum efficiency in the hybridization (recognition) step (Figure 2a) [3]. Oligonucleotides or cDNAs are spotted on a chip to produce microarrays that allow differential gene expression analysis of dozens or hundreds of genes. Figure 2b shows a fluorescence image from fluorescently labeled cDNA captured on a SensiChipTM microarray with such a tailored surface architecture, measured after hybridization using the SensiChip Reader of Zeptosens AG (an instrument for using the evanescent field generated above a planar waveguide to excite fluorescence in a highly sensitive and surface-selective manner [1]). The use of the PLL-g-PEG polymer coating leads to spots with an excellent signal-to-background ratio and with the preferred columnar fluorescence spot morphology (Figure 2c).

Figure 2. a) DNA as guest molecules inside an open PEG brush host surface: intermediate PEG chain (red) surface density allows for optimum ability of the DNA (in light blue) to recognize their complementary DNA for analysis of genetic information; b) Fluorescence from Cy5-labeled cDNA that was hybridized to probe DNA on a microarray using the PLL-g-PEG coated chip surface; c) Stack plot of spots after hybridization shown in (b), demonstrating columnar spot profiles with high signal-to-noise ratios [3].

The nanometer-thin, soft polymer cushions presented here are selected examples that illustrate how engineered surfaces blaze the trail for genomics and proteomics applications.

Authors

Janos Vörös^a, Susan M. De Paul^a, Marcus Textor^a,
Andreas P. Abel^b, Michael Pawlak^b, Ekkehard Kauffmann^b, Roman Liedkte^b, Markus Ehrat^b

^aLaboratory for Surface Science and Technology, Department of Materials
^bZeptosens AG, Benkenstrasse 254, CH-4108 Witterswil, Switzerland

References

1. Hochempfindliche Detektionstechnologie zum Aufspüren “verborgener” Gene und Proteine, Bioworld 3 (2002) 46-47.
2. Poly(L-Lysine)-g-poly(ethylene glycol) Layers on Metal Oxide Surfaces: Surface Analytical Characterization and Resistance to Serum and Fibrinogen Adsorption, N.-P. Huang, R. Michel, J. Vörös, M. Textor, R. Hofer, A. Rossi, D.L. Elbert, J.A. Hubbell, N.D. Spencer, Langmuir 17 (2): 489–498 (2001)
3. Controlled Biosensor Surfaces for Optical DNA/RNA Sensors, SM De Paul et al., Proceedings of the Conference “Biosensors 2002”, Kyoto, Japan, May 2002; and S.M. De Paul, D. Falconnet, S. Pasche, M. Textor, A.P. Abel, E. Kauffmann, R. Liedkte, M. Ehrat (to be published).
4. Short version of an article to appear in Bioworld, 2003