Label-free detection of tuberculosis DNA with capacitive field-effect biosensors Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Pharmazie der Philipps-Universität Marburg vorgelegt von Thomas Stefan Bronder aus Mönchengladbach Marburg/Lahn 2020 Gutachter: Prof. Dr. Michael Josef Schöning Gutachter: Prof. Dr. Michael Keusgen Eingereicht am: 20.10.2020 Tag der mündlichen Prüfung am: 08.12.2020 Hochschulkennziffer: 1180 I E R K L Ä R U N G Ich versichere, dass ich meine Dissertation Label-free detection of tuberculosis DNA with capacitive field-effect biosensors selbständig ohne unerlaubte Hilfe angefertigt und mich dabei keiner anderen als der von mir ausdrücklich bezeichneten Quellen bedient habe. Alle vollständig oder sinngemäß übernommenen Zitate sind als solche gekennzeichnet. Die Dissertation wurde in der jetzigen oder einer ähnlichen Form noch bei keiner anderen Hochschule eingereicht und hat noch keinen sonstigen Prüfungszwecken gedient. Marburg, den........................... ....................................................... (Unterschrift mit Vor- und Zuname) II Everything is possible with the right DNA. III Abstract A novel label-free DNA-detection method based on polyelectrolyte-modified electrolyte-insulator-semiconductor (EIS) sensor chips is developed in this thesis. This approach is motivated by the increasing demand on simple, easy to operate, cheap and reliable sensor platforms for the point-of-care detection of DNA from pathogens such as mycobacteria. Field-effect EIS sensors are chosen because of their ability to detect surface-potential changes with high sensitivity; with EIS sensors, the binding of charged molecules such as single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) can be monitored without a complex setup. The SiO2 surface-modification process of the EIS chips is carried out via adsorption of positively charged poly(allylamine hydrochloride) (PAH) on which the negatively charged DNA can bind by electrostatic attraction between the positive PAH charge and the negative DNA backbone resulting in a PAH/DNA bilayer. Compared to other binding methods, the adsorptive binding leads to a flat orientation of the DNA molecules, thus, the detectable intrinsic negative charge of the DNA is located closer to the sensor surface resulting in a higher contribution of signal generation. Results from electrochemical measurements of capacitance-voltage and constant-capacitance characteristics have been used as indicators for the respective surface-modification steps. A modification protocol is first established for the binding of positively charged PAH as well as the subsequent binding of dsDNA molecules. Both binding events of the charged molecules lead to a surface-potential change, which could be successfully monitored by electrochemical measurements. The developed protocol is also used to detect dsDNA molecules with light-addressable potentiometric sensors (LAPS), which belong to the group of EIS sensors. The LAPS technology allows to measure surface-potential changes at defined locations on the oxide layer, but requires a light source to focus to these respective regions. The dsDNA adsorption could also be monitored with LAPS, here a lower detection limit of 0.1 nM was determined. In order to monitor the hybridization reaction, a probe ssDNA is first immobilized onto the PAH-modified EIS-sensor surface. Then, the chip is exposed to solutions with target single-stranded complementary DNA (cDNA) and non-complementary DNA (ncDNA). In the case of cDNA, a hybridization reaction leads to a further change of the surface potential, which could be monitored by the EIS-sensor setup. Comparisons between incubation in solutions containing cDNA and ncDNA shows signal differences with a factor of 11. It was also investigated to reuse the sensor surface by simple repeating of the surface-modification steps without any kind of removing of the previous layers. It is possible to detect signal changes up to five PAH/DNA layers. The signal differences decrease by increasing the number of layers. This effect can be explained by the Debye charge-screening effect. To prove the assumption of the charge screening, additional experiments have been performed, in which the dependence of the ionic strength of the measurement solution on the resulting measured sensor signal is investigated. In addition, experiments are carried out, in which solutions containing polymerase-chain-reaction (PCR)-amplified cDNA have been analyzed with the developed sensing method. These cDNA-containing PCR solutions have IV been used to mimic realistic point-of-care test conditions. A test series with different concentrations of the PCR samples was performed in order to determine the lower detection limit (0.3 nM) and the sensitivity (7.2 mV/decade). In final experiments, the electrochemical detection of extracted and amplified target DNA from tuberculosis-spiked (positive) and non-spiked (negative) human sputum samples has been carried out with the developed method. A clear difference between the signals of positive and negative samples proved the successful recognition and ability to distinguish both probes under realistic conditions. All results of the electrochemical investigations have been validated by fluorescence- microscopy measurements. Overall, the developed label-free method fulfills the requirements of a simple, easy to operate, cheap and reliable procedure for DNA sensing. The detection of amplified genomic DNA from real tuberculosis-spiked sputum samples underlines the potential for promising realizations of this technology as a basis for medical devices for identification of pathogens. Keywords: DNA sensing, label-free, field-effect biosensor, tuberculosis, hybridization detection V Content Abstract ............................................................................................................................. IV Abbreviations ..................................................................................................................... X 1 Introduction ................................................................................................................ 1 1.1 DNA as receptor molecule for (bio)sensing .................................................... 1 1.2 Chip-based DNA-detection techniques – short overview and state-of-the-art ................................................................................................ 2 1.2.1 Labeled DNA-detection methods .............................................................. 2 1.2.2 Label-free DNA-detection methods .......................................................... 4 1.2.3 Commercially available DNA-detection devices ...................................... 7 1.3 Motivation, aims and outline ........................................................................ 10 1.3.1 Motivation and aims of this thesis ........................................................... 10 1.3.2 Outline of this thesis ................................................................................ 11 References............................................................................................................... 14 2 Theory ........................................................................................................................ 19 2.1 Structure and properties of the DNA molecule ............................................. 19 2.2 The electrochemical double-layer at solid-liquid interfaces ......................... 21 2.3 Surface modification of a silicon dioxide-layer with polyelectrolytes............................................................................................. 22 2.4 Electrolyte-Insulator-Semiconductor (EIS) sensors and their ability to detect charged molecules without labeling ................................... 24 2.4.1 Fabrication of EIS-sensor chips and measurement setup for electrochemical detection ........................................................................ 25 2.4.2 Signal generation, capacitance/voltage- and constant- capacitance-operating modes of EIS devices .......................................... 26 2.4.3 Label-free detection of DNA using polyelectrolyte-modified EIS sensors .............................................................................................. 32 2.5 DNA detection with LAPS ........................................................................... 34 2.6 Fluorescence-based DNA detection as reference for electrochemical methods .............................................................................. 35 References............................................................................................................... 38 3 Label-free detection of double-stranded DNA molecules with polyelectrolyte-modified capacitive field-effect sensors (tm – Technisches Messen 84 (2017) 628–634) ................................................................. 43 Abstract ................................................................................................................... 44 Zusammenfassung .................................................................................................. 44 Keywords ................................................................................................................ 44 3.1 Introduction ................................................................................................... 45 VI 3.2 Chip fabrication and measurement setup .....................................................