ultra-sensitve and high-resolution microarray scanner

The goal of this project is the development of a compact fluorescence based microarray scanning device with single molecule sensitivity. The system is based on a technology that for the first time enables high-throughput scanning of large areas with single molecule sensitivity and diffraction limited resolution. With single molecule sensitivity and a total dynamic range of 8 orders of magnitude, the device will be superior to currently available microarray scanners and thus allow revealing information currently not accessible in microarray-analysis. Microarrays permit the characterization of gene expression, DNA sequence variation, protein levels, tissues, cells and other biological and chemical molecules in a massively parallel format. The recent availability of advanced commercial RNA expression platforms and the milestone of over 5,000 published microarray papers demonstrate the rapid inroads made by this technology into basic and applied research. As a consequence of this enormous scientific potential the global market for microarrays is poised to grow rapidly (DNA-microarrays: from $596 million in 2003 to $937 million in 2010, protein microarrays: from $122 million in 2002 to more than $500 million in 2008). Without question, microarray technology offers an enormous potential. However, there are still limitations. In the case of DNA microarrays, error prone and time consuming amplification steps hinder obtaining fast and reliable results, in particular when only minute amounts of sample are available. For protein microarrays, the lack of an adequate amplification procedure to polymerase chain reaction even prohibits to obtain any results in case of small sample amounts. With the development of an ultra-sensitive compact scanner in this project, we provide the possibility to obtain reliable results from microarrays without the necessity for any amplification step. The system will allow detecting single hybridized biomolecules within a microarray spot and at the same time enabling precise quantification of up to 108 molecules bound. By increasing the dynamic range by 5 orders of magnitude compared to currently available microarray scanners, the device will open up new perspectives in biomedical research and diagnostics.

Within the project we have developed an analysis platform that allows analyzing the function of individual living cells and can be used to determine the composition of the pre-characterized cells in a correlated manner. In particular interesting is this kind of analysis approach for the investigation of heterogeneous cell populations. Heterogeneities are for example known from tumour tissue that can be treated by chemotherapy. However, often a small number of cancer cells -the cancer stem cells- survive the treatment and can give rise to tumour relapse. A single cell analysis approach can help characterizing such small cell populations thereby enabling the development of therapies that specifically address the small subpopulations. The analysis platform is based on a so called Lab-on-a-chip (LOC) system which is used as a miniaturized wet lab and allows to process samples volumes in the nanolitre range. The system allows to capture from single up to 100 cells and to observe their function by means of optical microscopy. After the functional characterization, cells are lysed and the target molecules are specifically stained using a fluorescent marker. For quantification, a parallel analysis of multiple target molecules is implemented on the basis of microarray technologies. While usage of the LOC prevents sample dilution and thereby increases the sensitivity of the following analysis of the cellular composition, ultra-sensitive fluorescence readout ensures the reliable detection of all molecules that are bound to the microarray surface. Due to the digital nature of single molecule detection, the signals can be quantified utilizing counting approaches thus preventing signal distortions due to variations in the labelling of target molecules. Additionally, spectroscopic parameters of the detected signals can be determined and are used to exclude non- specific signals from the data analysis. The performance of the developed platform has been determined on a model system of synthetic DNA. Unlabeled target molecules were specifically detected down to 100 femtomolar; at this concentration only approximately 2100 target molecules are present in the volume of 35 nanolitre of the LOC system. With this sensitivity the platform is well suited for analyzing small samples of 10 cells, in special cases even single cells.