Label-free detection of tuberculosis DNA with capacitive field-effect biosensors

The detection of pathogens from sample material from infected patients is the basis on which a considerable medical diagnosis can be made. Pathogens can be clearly identified based on their genomic material (DNA). A large number of different DNA detection methods with individual advantages and disadvantages have been established. If such methods should be used for certain applications, e.g. for point-of-care measurements, there are a number of requirements which should be considered: A measurement must be performed very fast, inexpensive, simple and reliable. It has been shown that label-free detection principles in particular the field-effect based detection-methods, meet the given requirements. In this thesis, the development of a new measuring method for the detection of DNA (with sequences from Mycobacterium tuberculosis) using a field-effect sensor, is described. The electrolyte-insulator-semiconductor (EIS) structure was selected as the basis for the sensor chip because it has the simplest structure of all field-effect sensors and is inexpensive to manufacture. EIS sensors are capacitive structures that can be read out using an impedance analyzer. The measured value is directly related to the surface potential of the sensor. If the DNA, which is negatively charged in solution, is brought close to the sensor surface, this causes a change in the surface potential via a change in the charge situation on the chip surface. This change in potential can be read out with the help of the EIS sensors: The detection method is based on the detection of a hybridization event on the sensor surface. The surface is modified with a probe single-strand DNA (ssDNA) which has a known sequence that is complementary to the ssDNA that is intended to be detected. As soon as the target ssDNA reaches the surface, a hybridization can occur, whereby a signal shift can be measured. The signal shift is caused by the additional negative charge of the hybridized target DNA molecules. In the case of non-complementary target DNA (ncDNA), there is no hybridization and the signal remains constant. The immobilization of the catcher ssDNA was carried out with by a surface modification using positively-charged polyelectrolyte (poly (allylamine hydrochloride), PAH). Compared to other immobilization strategies that are described in the literature, the capture ssDNA binds adsorptively to the sensor surface, which simplifies the preparation and can be carried out quickly and cheaply. The main topics of this thesis cover the selection of the sensor layout (EIS sensor with SiO2 as surface oxide), the selection and optimization of the surface modification (using PAH), the verification of the forming of double-stranded DNA and the evaluation of the measurement data acquisition by means of capacitive measurements. Due to the adsorptive binding, the DNA strands likely lie flat on the sensor surface. This means that the negative charge of the DNA is located closely to the surface, which means that a high measurement signal can be recorded. The developed protocol was also used with light-addressable potentiometric sensors (LAPS). LAPS are structurally very similar to EIS sensors and have the advantage that they can detect changes in the surface potential in a spatially resolved manner. This makes it possible, for example, to arrange an array so that several DNA experiments can be carried out simultaneously on one chip. However, the measurement setup is more complex because of the necessity of a light source. The measurement of the DNA hybridization on the sensor surface was realized by using the developed method: PAH/ssDNA-modified EIS chips were brought into contact with cDNA solutions. Measurable surface potential changes could show that the hybridization was successful. In direct comparison with experiments where ncDNA was applied to the modified sensor, signal differences of about 11 times higher were measured for cDNA than for ncDNA. The developed method also allows a very simple reuse of the chip by just a repeating of the modification steps on an already used chip. This reusability of the sensors was investigated by performing up to five repetitive surface modification and DNA attachment experiments sequentially with just one chip. A steady decrease in the sensor signal could be observed after each additional layer (PAH or DNA); however, this observation is related to the Debye screening effect. Finally, the developed biosensor was used to detect PCR-amplified cDNA. A detection of the target cDNA was successful and significant, although the additional (interfering) substances in the solution, that were necessary for the PCR process (enzymes, etc.), disturbed the measurement signal. Measurements in which a concentration series of cDNA were used to determine the lower detection limit (0.3 nM) and the sensitivity (7.2 mV / decade). Extracted and amplified target DNA from Mycobacterium tuberculosis-spiked human saliva-samples was also examined using the method. A clear differentiation between positive and negative material could be recognized with the help of the PAH / ssDNA-modified EIS sensor chips. All developed process steps were validated using fluorescence measurements as a reference method. With the PAH-modified capacitive field-effect biosensor, that was developed in this thesis, a quick, simple and inexpensive measurement platform for the DNA hybridization reaction is given. The detection of amplified genomic DNA from real Mycobacterium tuberculosis-spiked saliva samples underlines the potential of this procedure as a sensor approach for pathogen detection for medical applications

Fate and Transport of Vertebrate DNA in Surface Water Environments: Developing a Basis for Quantification through Environmental DNA Monitoring

Many environmental engineering applications require sampling of DNA to be effective. Organisms shed DNA into the environment, and that environmental DNA (eDNA) can be collected, extracted, amplified, and quantified to provide information about the shedding organisms. Regardless of the target organism, shed eDNA is subject to environmental degradation and partitioning into various compartments. Fate and transport of eDNA in aquatic systems was examined using two vertebrate organisms (invasive bigheaded carps, Hypophthalmichthys spp., and endangered Topeka shiners, Notropis topeka). Target eDNA concentrations were higher in sediment than the overlying water column and were correlated with biomass density of the target fish. Target eDNA in sediments was detectable and quantifiable at least 132 days after removal of the fish, suggesting eDNA persistence in aquatic systems may be significantly longer than previously reported. Degradation rates were highly correlated with initial eDNA concentrations in ponds with bioturbation and reduced macrophytes, suggesting degradation may be dominated by enzymatic hydrolysis in those systems. N. topeka DNA from museum specimens was extracted and sequenced for cytochrome oxidase 1 (COI), cytochrome oxidase b (CytB), NAD dehydrogenase 2 (ND2), and the control region (D-loop). Such data are rare, and these tissue extractions and sequences represent important contributions to preservation of an endangered species. Both detection probability and concentration of N. topeka eDNA in water samples increased at biomass densities two to three times larger than naturally occurring schools (80 versus 20 to 30 fish). After an initial spike, fish eDNA in water dropped below detection limits within 7 days, regardless of stocking density. However, at 14 days detection in high density tanks increased to initial spike levels and remained so at 26 days after stocking. Fish eDNA was detected in water 27 days after fish removal, regardless of density, though at lower detection probability than during fish presence. Despite consistent detection, water column concentrations of fish eDNA did not exceed 20% of the initial spike over the course of 335 days. eDNA monitoring must account for partitioning and differential fate in the environment. Therefore, future studies should focus on models incorporating resuspension, ecological condition, and the mechanisms of degradation