{"id":{"repo_id":"freiburg-diss","oai_identifier":"oai:freidok.uni-freiburg.de:2316"},"canonical_url":"https://search.dev.ndltd.org/etd/freiburg-diss/oai:freidok.uni-freiburg.de:2316","repository":{"repo_id":"freiburg-diss","name":"University of Freiburg","base_url":"https://freidok.uni-freiburg.de/oai/oai2.php"},"display":{"title":"PCR-Slide: a modular and cascadable platform for DNA sample processing with integrated nanoliter dosage","abstract":"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.","abstract_html":"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. <br>The platform that was developed within the scope of this thesis consists of three components: <br>1. a microfluidic chip (PCR-Slide) with the outer dimensions of a microscopic slide with up to 24 parallel reaction cavities <br>2. a dispensing station for the simultaneous and parallel extraction of nanoliter quan-tities from the reaction cavities <br>3. a thermal cycler for parallel thermal sample processing <br>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 %. <br>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. <br>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. <br>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. <br>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: <br>1. PCR <br>2. sample post-processing based on exonuclease digestion <br>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) <br>4. hybridization and detection <br>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.","abstract_has_math":false,"creators":["Kaack, Rolf Markus"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Zengerle, Roland"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":null,"date_issued":"","date_published":null,"updated_at":"2026-07-24T02:22:40Z","subjects":["Nanoliterdosierung","Polymerasekettenreaktion (PCR)","polymerase chain reaction","microfluidic","nanoliter dosage","process platform"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://freidok.uni-freiburg.de/data/2316","outbound_label":"Repository record","outbound_source":"source_url"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Zengerle, Roland"]},{"key":"dc:creator","label":"Author","values":["Kaack, Rolf Markus"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:type","label":"Dc Type","values":["DoctoralThesis"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Nanoliterdosierung","Polymerasekettenreaktion (PCR)","polymerase chain reaction","microfluidic","nanoliter dosage","process platform"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.","Thema der vorliegenden Arbeit ist die Konzeption, Entwicklung und Validierung einer neuen, modularen Plattform, mit der mehrschrittige, thermisch basierte DNA-Testverfahren durchgeführt werden können. Gegenüber dem Stand der Technik wurde durch intelligente Miniaturisierung der übliche, gerätetechnische Aufwand für derartige Testverfahren deutlich reduziert.
Die im Rahmen der Arbeit entwickelte Plattform besteht aus drei Komponenten: 1. einem mikrofluidischen Chip (PCR-Slide) in der Größe eines Mikroskop-Objektträgers mit bis zu 24 parallelen Reaktionskavitäten
2. einer Dispensierstation zur simultanen und parallelen Extraktion von Nanolitermengen aus den Reaktionskavitäten
3. einem Thermocycler für die parallele thermische Probenprozessierung
Bei dem PCR-Slide handelt es sich um einen mikrostrukturierten Polycarbonat-Chip, der lediglich einfache, kostengünstig herstellbare, mikrofluidische Kanalstrukturen enthält und demzufolge als low-cost disposable ausgeführt werden kann. Nichtsdestotrotz entfalten diese einfachen Strukturen ein hohes Maß an Funktionalität. Sie ermöglichen die parallele Extraktion von Reagenzien in Nanolitermengen mit hoher Genauigkeit. Es konnte beispielsweise nachgewiesen werden, dass durch einfache pneumatische Anregung 60 nl mit einer Standardabweichung von 4 % extrahiert werden können.
Die auf dem PCR-Slide integrierten Strukturen wurden im Rastermaß der 384er Mikrotiterplatte (4,5 mm) angeordnet. Somit kann Probenflüssigkeit aus den Reaktionskavitäten des PCR-Slides in die Kavitäten von 384er Mikrotiterplatten mit der integrierten Nanoliter-Dosiereinheit transferiert werden. Da auch die Reaktionskavitäten des PCR-Slides in der gleichen Weise angeordnet sind, kann man auch von einem PCR-Slide in ein anderes dosieren und damit mehrere Schritte eines Assays auf der PCR-Slide-Plattform durchführen.
Zur Ansteuerung der im PCR-Slide integrierten funktionalen Strukturen bedarf es zweier Geräte, einem Thermocycler und einer Dispensierstation. Der Thermocycler hat zwei unabhängig voneinander temperierbare Platten, regelbar in einem Temperaturbereich von 4 °C bis 120 °C. Mit Hilfe der Dispensierstation wird der Dosiervorgang über einen pneumatischen Druckpuls ausgelöst.
Dem verwendeten Dosierprinzip liegt das Dispensing-Well-Plate-Verfahren zugrunde, mit dem Flüssigkeitsmengen im Nanoliterbereich mit hoher Präzision im Freistrahl abgegeben werden können. Im Rahmen dieser Arbeit hat sich gezeigt, dass das Dosiervolumen beim Dispensing-Well-Plate-Verfahren nur für bestimmte Geo-metrien der Dispenserstruktur unabhängig von der Ansteuerung ist. Mittels eines entwickelten mathematischen Modells des Dosiervorgangs konnten Designregeln für die Auslegung der Dispensergeometrie aufgestellt werden.
Die Funktionalität von PCR-Slide wurde bei der Ausführung zweier DNA-Testverfahren, einem Hybridisierungsassay und einem Genotypisierungsassay, erfolgreich gezeigt. Der Hybridisierungsassay bestand aus den folgenden vier Schritten:
1. PCR
2. Probenaufbereitung (Exonuclease-Verdau)
3. Probentransfer durch die integrierte Nanoliterdosierfunktionalität (es wurden 5 x 100 nl = 500 nL des aufbereiteten PCR-Produkts in 1.5 µl Hybridisierungspuffer auf einem Mikroarray transferiert)
4. Hybridisierung und Detektion des Produkts
Der Genotypisierungsassay beinhaltete mehrere Reaktionsschritte, von denen zwei hintereinander auf dem PCR-Slide erfolgreich durchgeführt wurden. Hierbei wurde das entstandene Produkt mittels Kapillarelektrophorese analysiert."]},{"key":"dc:format.medium","label":"Dc Format Medium","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["PCR-Slide: a modular and cascadable platform for DNA sample processing with integrated nanoliter dosage","PCR-Slide: eine modulare und kaskadierbare Plattform zur DNA-Probenprozessierung mit integrierter Nanoliterdosierung"]}]}],"canonical_facts":{"dc:contributor":["Zengerle, Roland"],"dc:creator":["Kaack, Rolf Markus"],"dc:description.abstract":["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.","Thema der vorliegenden Arbeit ist die Konzeption, Entwicklung und Validierung einer neuen, modularen Plattform, mit der mehrschrittige, thermisch basierte DNA-Testverfahren durchgeführt werden können. Gegenüber dem Stand der Technik wurde durch intelligente Miniaturisierung der übliche, gerätetechnische Aufwand für derartige Testverfahren deutlich reduziert.
Die im Rahmen der Arbeit entwickelte Plattform besteht aus drei Komponenten: 1. einem mikrofluidischen Chip (PCR-Slide) in der Größe eines Mikroskop-Objektträgers mit bis zu 24 parallelen Reaktionskavitäten
2. einer Dispensierstation zur simultanen und parallelen Extraktion von Nanolitermengen aus den Reaktionskavitäten
3. einem Thermocycler für die parallele thermische Probenprozessierung
Bei dem PCR-Slide handelt es sich um einen mikrostrukturierten Polycarbonat-Chip, der lediglich einfache, kostengünstig herstellbare, mikrofluidische Kanalstrukturen enthält und demzufolge als low-cost disposable ausgeführt werden kann. Nichtsdestotrotz entfalten diese einfachen Strukturen ein hohes Maß an Funktionalität. Sie ermöglichen die parallele Extraktion von Reagenzien in Nanolitermengen mit hoher Genauigkeit. Es konnte beispielsweise nachgewiesen werden, dass durch einfache pneumatische Anregung 60 nl mit einer Standardabweichung von 4 % extrahiert werden können.
Die auf dem PCR-Slide integrierten Strukturen wurden im Rastermaß der 384er Mikrotiterplatte (4,5 mm) angeordnet. Somit kann Probenflüssigkeit aus den Reaktionskavitäten des PCR-Slides in die Kavitäten von 384er Mikrotiterplatten mit der integrierten Nanoliter-Dosiereinheit transferiert werden. Da auch die Reaktionskavitäten des PCR-Slides in der gleichen Weise angeordnet sind, kann man auch von einem PCR-Slide in ein anderes dosieren und damit mehrere Schritte eines Assays auf der PCR-Slide-Plattform durchführen.
Zur Ansteuerung der im PCR-Slide integrierten funktionalen Strukturen bedarf es zweier Geräte, einem Thermocycler und einer Dispensierstation. Der Thermocycler hat zwei unabhängig voneinander temperierbare Platten, regelbar in einem Temperaturbereich von 4 °C bis 120 °C. Mit Hilfe der Dispensierstation wird der Dosiervorgang über einen pneumatischen Druckpuls ausgelöst.
Dem verwendeten Dosierprinzip liegt das Dispensing-Well-Plate-Verfahren zugrunde, mit dem Flüssigkeitsmengen im Nanoliterbereich mit hoher Präzision im Freistrahl abgegeben werden können. Im Rahmen dieser Arbeit hat sich gezeigt, dass das Dosiervolumen beim Dispensing-Well-Plate-Verfahren nur für bestimmte Geo-metrien der Dispenserstruktur unabhängig von der Ansteuerung ist. Mittels eines entwickelten mathematischen Modells des Dosiervorgangs konnten Designregeln für die Auslegung der Dispensergeometrie aufgestellt werden.
Die Funktionalität von PCR-Slide wurde bei der Ausführung zweier DNA-Testverfahren, einem Hybridisierungsassay und einem Genotypisierungsassay, erfolgreich gezeigt. Der Hybridisierungsassay bestand aus den folgenden vier Schritten:
1. PCR
2. Probenaufbereitung (Exonuclease-Verdau)
3. Probentransfer durch die integrierte Nanoliterdosierfunktionalität (es wurden 5 x 100 nl = 500 nL des aufbereiteten PCR-Produkts in 1.5 µl Hybridisierungspuffer auf einem Mikroarray transferiert)
4. Hybridisierung und Detektion des Produkts
Der Genotypisierungsassay beinhaltete mehrere Reaktionsschritte, von denen zwei hintereinander auf dem PCR-Slide erfolgreich durchgeführt wurden. Hierbei wurde das entstandene Produkt mittels Kapillarelektrophorese analysiert."],"dc:format.medium":["application/pdf"],"dc:subject":["Nanoliterdosierung","Polymerasekettenreaktion (PCR)","polymerase chain reaction","microfluidic","nanoliter dosage","process platform"],"dc:title":["PCR-Slide: a modular and cascadable platform for DNA sample processing with integrated nanoliter dosage","PCR-Slide: eine modulare und kaskadierbare Plattform zur DNA-Probenprozessierung mit integrierter Nanoliterdosierung"],"dc:type":["DoctoralThesis"]},"updated_at":"2026-07-24T02:22:40Z"}