Impedimetric sensor for label-free multiplex detection of ß-lactam antibiotic resistance genes employing Rolling Circle Amplification

Abstract

The aim of this work was the development and testing of an impedimetric based biosensor for the detection of antibiotic resistance genes using Rolling Circle Amplification (RCA) as the method of choice for isothermal amplification. Before an own sensor ("Biosensor_RCA_bla_AIT") was developed, a biosensor ("Biosensor_Virus") provided by Infineon, Villach, was tested. This kind of system with interdigitated electrodes (IDEs) is normally used for another biological application, but initially it should be verified if this kind of sensor would also work in the case of the impedimetric detection of isothermal amplified deoxyribonucleic acid (DNA). Because of the fast growing resistance to antibiotics by the extensive and very often unnecessary use in non-human fields, for instance in agriculture and the veterinary sector a rapid detection method is essential. The -golden- standard techniques for the detection of pathogens such as ß-lactamases are based on cultivation methods in solid or liquid media. The advantages of disc diffusion tests or fully automated systems in clinical practice are that they are cheap and highly sensitive. But in some cases these methods are very time consuming: for instance the detection of mycobacterial growth, which can cause (among other diseases) tuberculosis, needs between four and eight weeks, which can be too long under some circumstances. Furthermore, potentially false-positive results are possible if the pathogens are exposed to wrong growing conditions. With this newly developed biosensor it is possible to detect differences between positive DNA amplification and negative control. By a positive amplification reaction the impedance is changed over time to higher values whereas at a negative control stays the same or nearly the same. These differences in impedance are more pronounced at higher frequencies. In this thesis it could be shown that the sensor is a potential candidate for the impedimetric detection of ß-lactam antibiotic resistance genes especially if this system is modified to smaller sample volumes so that the cost-benefit relationship becomes more attractive. This modification process is also important in order to get lower target-DNA concentrations. Now this system is able to detect ~1012 molecules per milliliter, which is a very high DNA concentration that can only be reached by a pre-amplification step or the use of synthetic target-DNA.