Microfabricated systems applied for DNA amplification and for flow cytometry

Claus Riber Poulsen

    Research output: Book/ReportPh.D. thesisResearch

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    Abstract

    Since the early 1990’s, the ‘miniaturized total chemical analysis systems’ (μTAS) concept, also known as ‘Lab on Chip’ has evolved considerably. Sampling, detection, and read-out steps in a μTAS are ideally performed on a single microchip.

    The microchip format offers several benefits. These benefits can be utilized in biological applications of microfabricated analysis systems. In this thesis, applications for flow cytometry and polymerase chain reaction (PCR) are presented.

    Flow cytometry is used for the development of an acute cytotoxicity assay. A micromachined chip makes it possible to mix reagents and cells on-chip. Therefore, a cellular response can be observed from start to finish with fast acting reagents. The 25 cm detection channel makes on-chip dynamic assays possible. Results from the developed assay indicates that the Triton X-100 effect on cell membranes sets in between 0.2 and 25 seconds, and has not yet reached a steady state after 47 seconds.

    PCR is performed in a SU-8 based reaction chamber fabricated on a glass PCR chip. The chip has integrated thin film heaters and temperature sensor electrodes. In this design heating and cooling rates of 50°C/s and 30°C/s respectively have been obtained. It was required to passivate the SU-8 surface before PCR was possible to perform. However, after silanization an average PCR yield of 68% compared to standard PCR tubes was obtained.

    A practical use for the PCR chip was demonstrated by Campylobacter jejuni detection. The PCR chip proved successful in amplification of DNA from various sources. It was even possible to perform PCR directly on whole Campylobacter jejuni cells. The detection time has decreased significantly with the chip detection method, compared to conventional methods. The chip method can be performed in less than a day, where just under a week is needed for conventional detection methods.

    Finally, dielectrophoresis (DEP) is demonstrated as a sample pretreatment that can be integrated with PCR. In the presented example yeast cells were separated from the known PCR inhibitors haemoglobin or heparin. Subsequent PCR was only successfully if performed on cells treated in DEP chip. DEP microsystems is here demonstrated as an effective sample pretreatment for PCR.
    Original languageEnglish
    Place of PublicationKgs. Lyngby, Denmark
    PublisherMikroelektronik Centret, Technical University of Denmark
    Number of pages112
    ISBN (Print)87-89935-60-8
    Publication statusPublished - 2003

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