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THESIS DR. BART VAN GRINSVEN - SYNOPSIS

"Fast genetic assays based on label-free electronic and thermal read-out strategies."

One of the central challenges in genomics is the detection and identification of single nucleotide polymorphisms (SNPs). This importance stems from several reasons: first, SNPs are involved in hundreds of genetic disorders such as Alzheimer, mucoviscidosis, phenylketonuria, and several types of breast- and colon cancer. Second, SNPs in the so-called ADME (absorption, distribution, metabolism, excretion) genes significantly influence the effectiveness of treatment and this is a major topic in the field of theranostics. Established technologies exist to identify these SNPs, but several disadvantages need consideration. Nearly all technologies need a lab environment, lack in speed (reaction times at the scale of at least 16 hours), are unable to provide dynamic information on the DNA binding kinetics (end point measurement) and need fluorescent labelling of the target DNA which induces the necessity of sophisticated optical readout techniques. Due to these reasons a general interest in fast, label-free, low-cost and user-friendly DNA sensors has emerged. This thesis reports on the development of DNA sensors that respond to these specifications.


Two read-out technologies were developed to distinguish between these full matching and mutated sequences. The first technology is based on impedance spectroscopy. But before starting genomic experiments we have first build an impedance spectroscopy unit, fully customized to be an essential part of this label-free, diamond based DNA sensor. We investigated if we could distinguish between different buffer solutions at different molarities and temperatures, commonly used in genomic research. After it was clear that we could regulate and understand these environmental variables we introduced the setup to genomic research and used the findings as preconditions for the experiments.


At this point we started the first experiments where we monitored the chemical denaturation of DNA (by inducing 0.1 M NaOH) electronically in combination with fluorescence spectroscopy. We found that the impedimetrical results could be separated into a time constant for NaOH exposure and a time constant for denaturation. This denaturation time can be used as a measure for the stability of the DNA duplex. When inducing a single mutation into a 29 base-pair duplex, the denaturation time is almost halved. It is even possible to give an indication of the position of the mutation as we can distinguish between denaturation times of identical mutations, located elsewhere in the sequence. This electronic method requires minimal instrumentation, is label-free and fast (within a time scale of minutes). These elements suggest that the monitoring of chemically-induced denaturation is an interesting method to measure DNA duplex stability and might be used as a tool in SNP analysis.


The mentioned method does produce results within the wanted SNP regime, but the raw data still needs mathematical processing. For automation purposes this is a drawback. Therefore we started thermal denaturation experiments with an electronic read–out. We considered it a possibility to make use of thresholds when denaturing thermally. This process would eliminate the need for a medium exchange. Impedance would be measured as a function of temperature instead of function of time and medium. However, when conducting first experiments a new finding surfaced. This new finding was in line, but not related to the impedimetrical data. A temperature anomaly occurred at the solid to liquid interface upon denaturation. We found that DNA in its double stranded form has a lower thermal resistance than in its single stranded form. This transfer from low to high thermal resistance could be linked to the denaturation process by confocal fluorescence spectroscopy. The transition occurred at a lower temperature when denaturing a mutated sequence when compared to a full matching sequence, meaning that this method allows identifying melting temperatures by using an adjustable heat source in combination with two thermocouples.