PCR-Slide: a modular and cascadable platform for DNA sample processing with integrated nanoliter dosage
Subject of this thesis was the conception, development and validation of a novel and modular platform for performance of DNA-related multistep reactions based on ther-mal cycling. Compared to the state of the art, the expenditure of laboratory equipment for DNA-based assays was reduced significantly. The platform that was developed within the scope of this thesis consists of three components: 1. a microfluidic chip (PCR-Slide) with the outer dimensions of a microscopic slide with up to 24 parallel reaction cavities 2. a dispensing station for the simultaneous and parallel extraction of nanoliter quan-tities from the reaction cavities 3. a thermal cycler for parallel thermal sample processing The PCR-Slide is a micro-structured chip made of polycarbonate, consisting merely of simple microfludic structures that can be produced cost-effectively. This means that the PCR-Slide can be realized as a low-cost disposable. Despite the simplicity of the structures, they evolve a high degree of functionality, enabling the parallel extrac-tion of reagents in nanoliter quantities with a high accuracy. For example, it could be shown that by simple pneumatic actuation aliquots of 60 nl could be extracted from the cavities with a standard deviation of 4 %. The structures of the PCR-Slide are arranged in a pitch corresponding to the pitch of the 384 well microtiter plates (4.5 mm). This allows the transfer of sample liquid into the wells of such a microtiter plate. As the reaction cavities of the PCR-Slide are also arranged in the same way, it is possible to dispense from one PCR-Slide into another one, allowing for the performance of several assay steps of a multistep assay on the PCR-Slide platform. For the actuation of the functional structures being integrated in the PCR-Slide two devices are needed, a thermal cycler and a dispensing station. The thermal cycler has two individually controllable plates, which can reach temperatures ranging from 4 °C to 120 °C. With the dispensing station the dispensing event is initiated by means of a pressure pulse. The applied dispensing principle is based on the Dispensing Well Plate technique, which allows to dispense liquid in the range of several nanoliters in a free jet and with high accuracy. Within the scope of this thesis it turned out that the dispensed volume of a Dispensing Well Plate dispenser is only independent from the actuation parame-ters for certain geometries of the dispenser's structure. By means of a developed mathematical model design rules for the layout of a dispensing geometry could be formulated. The functionality of the PCR-Slide was proofed by performing two different assays on the chip, a hybridization assay and a genotyping assay. The hybridization assay consisted of four different steps: 1. PCR 2. sample post-processing based on exonuclease digestion 3. sample liquid transfer by means of the inbuilt nanoliter dosage (transfer of 5 times 100 nl = 500 nl of the post-processed sample into 1.5 µl hybridization buffer in a microarray) 4. hybridization and detection The genotyping assay included several reaction steps, of which two were performed subsequently and successfully on the PCR-Slide. The genotype specific product was detected using capillary electrophoresis.
Nanoliterdosierung, Polymerasekettenreaktion (PCR), polymerase chain reaction, microfluidic, nanoliter dosage, process platform
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