The aim of this project is to develop novel bioanalytical assays for the sensitivedetection of biomolecular interactions in a microarray format. Micro- and nanoobjects such as polymeric particles and phospholipid vesicles will be their main components. The particles can be used as carriers for the biorecognition reaction in solution, can contribute to increase the sensor loading capacity and if fluorescent, can act as labels for an increased signal intensity.
In a first assay type, fluorescent particles are used themselves as labels. In the Figure 1, fluorescent micro- or nanoparticles are functionalized with the capture element and are therefore used for detection instead of the free antibodies. Sedimented and specific bound particles are discriminated using fluidic forces.
Figure 1. Fluorescent particle assay. Micro- or nanoparticles are functionalized with the capture element (b) and are used instead of the free antibodies (a). Thus, when the assay is performed the protein array is incubated with a fluorescent particle solution. (c) Biotin-BSA spot after fluorescent beads (1 micrometer) functionalized with streptavidin after applying a laminar flow.
In another approach, nanoobjects such as particles or vesicles are sorted from solution onto a DNA microarray. Heterogeneous functional vesicle arrays were obtained with this approach (Figure 2). We are currently working on the immobilization of membrane proteins as well as on the functionalization of DNA-tagged vesicles with antibodies for biosensing applications.
Figure 2. (a) Schematic and (b) microarray reader image of the green and red labeled vesicles surface sorted via ssDNA hybridization. A Ta2O5 waveguiding chip is coated with PLL-g-PEG/PEGbiotin. In a second step, complexes of SA and biotinylated DNA are spotted onto the chip. Finally the surface is exposed to a solution containing two kinds of functionalized vesicles (LEFT ('E'): AVCy3 modified DOPC/DOPS vesicles; RIGHT ('T'): BSA loaded POPC vesicles) tagged with DNA via the maleimide - thiol linkage. In addition references are included: 'H' (left): SA532; 'H' (right): SA633 and the underline: unlabeled streptavidin. The DNA tagged vesicles are selectively decorating the corresponding spots on the surface. Scale bar: 100 micrometers.
Our research group has extensive experience in the use and application of optical waveguide techniques to sense in situ and quantitatively interfacial processes. The planar optical waveguide technology (PWG) combined with fluorescence detection has been developed into a commercial instrument and microarray products by Zeptosens, the ZeptoREADER (Figure 3). Current applications comprise DNA, RNA and protein bioaffinity assays on microarrays. The technology has been shown to provide the same or better results as competitive technologies but without amplification steps which might introduce a bias . Moreover, in comparison with alternative technologies that are in use today, it has proved to have the highest sensitivity for both DNA/RNA and protein assays, approaching zeptomole detection sensitivity in the presence of 80 % serum (corresponding to detection of 600 molecules). Customers have been able to identify marker molecules which were not detectable with other technologies.
Figure 3. The ZeptoREADER is based on a plana optical waveguide (PWG) technique. Light of a plane polarized laser is coupled into a waveguide with an optical grating. It then propagates via total internal reflection and produces an evanescent field which penetrates the vicinity of the waveguide up to 100 to 200 nm. Through a flow cell, the surface can be brought in contact with a liquid. The instrument is detecting fluorescence which is emitted when fluorescent dyes get excited by the evanescent field in the vicinity of the waveguide. It is therefore an in situ and highly surface sensitive technique.
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