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

Abstract

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. In order to integrate DNA in a sensor one needs to develop specific immobilization protocols to tether DNA preferably covalently and in head-on configuration onto a transducer surface. The platform of the developed DNA sensors is based on nano-crystalline diamond (NCD). In a first step, the diamond is hydrogenated. After hydrogenation, fatty acid is attached to surface covalently. Once the fatty acid is attached, it is functionalized with a probe DNA sequence by means of an 1–ethyl–3-[3-dimethylaminopropyl]-carbodiimide (EDC) reaction and in a last step this probe DNA sequence is hybridized with either a full matching target sequence or a sequence containing a mutation at one or several base pairs. ...