Refine
Year of publication
- 2022 (43) (remove)
Document Type
- Doctoral Thesis (43) (remove)
Is part of the Bibliography
- yes (43)
Keywords
- Arabidopsis thaliana (3)
- Transkriptionsfaktoren (3)
- transcription factors (3)
- Bewegungsökologie (2)
- Epigenetik (2)
- Koexistenz (2)
- epigenetics (2)
- movement ecology (2)
- 7-Methylheptadecan (1)
- 7-methylheptadecane (1)
Institute
- Institut für Biochemie und Biologie (43) (remove)
Synthetische Transkriptionsfaktoren bestehen wie natürliche Transkriptionsfaktoren aus einer DNA-Bindedomäne, die sich spezifisch an die Bindestellensequenz vor dem Ziel-Gen anlagert, und einer Aktivierungsdomäne, die die Transkriptionsmaschinerie rekrutiert, sodass das Zielgen exprimiert wird. Der Unterschied zu den natürlichen Transkriptionsfaktoren ist, sowohl dass die DNA-Bindedomäne als auch die Aktivierungsdomäne wirtsfremd sein können und dadurch künstliche Stoffwechselwege im Wirt, größtenteils chemisch, induziert werden können. Optogenetische synthetische Transkriptionsfaktoren, die hier entwickelt wurden, gehen einen Schritt weiter. Dabei ist die DNA-Bindedomäne nicht mehr an die Aktivierungsdomäne, sondern mit dem Blaulicht-Photorezeptor CRY2 gekoppelt. Die Aktivierungsdomäne wurde mit dem Interaktionspartner CIB1 fusioniert. Unter Blaulichtbestrahlung dimerisieren CRY2 und CIB1 und damit einhergehend die beiden Domänen, sodass ein funktionsfähiger Transkriptionsfaktor entsteht. Dieses System wurde in die Saccharomyces cerevisiae genomisch integriert. Verifiziert wurde das konstruierte System mit Hilfe des Reporters yEGFP, welcher durchflusszytometrisch detektiert werden konnte. Es konnte gezeigt werden, dass die yEGFP Expression variabel gestaltet werden kann, indem unterschiedlich lange Blaulichtimpulse ausgesendet wurden, die DNA-Bindedomäne, die Aktivierungsdomäne oder die Anzahl der Bindestellen, an dem sich die DNA-Bindedomäne anlagert, verändert wurden. Um das System für industrielle Anwendungen attraktiv zu gestalten, wurde das System vom Deepwell-Maßstab auf Photobioreaktor-Maßstab hochskaliert. Außerdem erwies sich das Blaulichtsystem sowohl im Laborstamm YPH500 als auch im industriell oft verwendeten Hefestamm CEN.PK als funktional. Des Weiteren konnte ein industrierelevante Protein ebenso mit Hilfe des verifizierten Systems exprimiert werden. Schlussendlich konnte in dieser Arbeit das etablierte Blaulicht-System erfolgreich mit einem Rotlichtsystem kombiniert werden, was zuvor noch nicht beschrieben wurde.
Microplastics in the environments are estimated to increase in the near future due to increasing consumption of plastic product and also due to further fragmentation in small pieces. The fate and effects of MP once released into the freshwater environment are still scarcely studied, compared to the marine environment. In order to understand possible effect and interaction of MPs in freshwater environment, planktonic zooplankton organisms are very useful for their crucial trophic role. In particular freshwater rotifers are one of the most abundant organisms and they are the interface between primary producers and secondary consumers. The aim of my thesis was to investigate the ingestion and the effect of MPs in rotifers from a more natural scenario and to individuate processes such as the aggregation of MPs, the food dilution effect and the increasing concentrations of MPs that could influence the final outcome of MPs in the environment. In fact, in a near natural scenario MPs interaction with bacteria and algae, aggregations together with the size and concentration are considered drivers of ingestion and effect. The aggregation of MPs makes smaller MPs more available for rotifers and larger MPs less ingested. The negative effect caused by the ingestion of MPs was modulated by their size but also by the quantity and the quality of food that cause variable responses. In fact, rotifers in the environment are subjected to food limitation and the presence of MPs could exacerbate this condition and decrease the population and the reproduction input. Finally, in a scenario incorporating an entire zooplanktonic community, MPs were ingested by most individuals taking into account their feeding mode but also the concentration of MPs, which was found to be essential for the availability of MPs. This study highlights the importance to investigate MPs from a more environmental perspective, this in fact could provide an alternative and realistic view of effect of MPs in the ecosystem.
The increasing introduction of non-native plant species may pose a threat to local biodiversity. However, the basis of successful plant invasion is not conclusively understood, especially since these plant species can adapt to the new range within a short period of time despite impoverished genetic diversity of the starting populations. In this context, DNA methylation is considered promising to explain successful adaptation mechanisms in the new habitat. DNA methylation is a heritable variation in gene expression without changing the underlying genetic information. Thus, DNA methylation is considered a so-called epigenetic mechanism, but has been studied in mainly clonally reproducing plant species or genetic model plants. An understanding of this epigenetic mechanism in the context of non-native, predominantly sexually reproducing plant species might help to expand knowledge in biodiversity research on the interaction between plants and their habitats and, based on this, may enable more precise measures in conservation biology.
For my studies, I combined chemical DNA demethylation of field-collected seed material from predominantly sexually reproducing species and rearing offsping under common climatic conditions to examine DNA methylation in an ecological-evolutionary context. The contrast of chemically treated (demethylated) plants, whose variation in DNA methylation was artificially reduced, and untreated control plants of the same species allowed me to study the impact of this mechanism on adaptive trait differentiation and local adaptation. With this experimental background, I conducted three studies examining the effect of DNA methylation in non-native species along a climatic gradient and also between climatically divergent regions.
The first study focused on adaptive trait differentiation in two invasive perennial goldenrod species, Solidago canadensis sensu latu and S. gigantea AITON, along a climate gradient of more than 1000 km in length in Central Europe. I found population differences in flowering timing, plant height, and biomass in the temporally longer-established S. canadensis, but only in the number of regrowing shoots for S. gigantea. While S. canadensis did not show any population structure, I was able to identify three genetic groups along this climatic gradient in S. gigantea. Surprisingly, demethylated plants of both species showed no change in the majority of traits studied. In the subsequent second study, I focused on the longer-established goldenrod species S. canadensis and used molecular analyses to infer spatial epigenetic and genetic population differences in the same specimens from the previous study. I found weak genetic but no epigenetic spatial variation between populations. Additionally, I was able to identify one genetic marker and one epigenetic marker putatively susceptible to selection. However, the results of this study reconfirmed that the epigenetic mechanism of DNA methylation appears to be hardly involved in adaptive processes within the new range in S. canadensis.
Finally, I conducted a third study in which I reciprocally transplanted short-lived plant species between two climatically divergent regions in Germany to investigate local adaptation at the plant family level. For this purpose, I used four plant families (Amaranthaceae, Asteraceae, Plantaginaceae, Solanaceae) and here I additionally compared between non-native and native plant species. Seeds were transplanted to regions with a distance of more than 600 kilometers and had either a temperate-oceanic or a temperate-continental climate. In this study, some species were found to be maladapted to their own local conditions, both in non-native and native plant species alike. In demethylated individuals of the plant species studied, DNA methylation had inconsistent but species-specific effects on survival and biomass production. The results of this study highlight that DNA methylation did not make a substantial contribution to local adaptation in the non-native as well as native species studied.
In summary, my work showed that DNA methylation plays a negligible role in both adaptive trait variation along climatic gradients and local adaptation in non-native plant species that either exhibit a high degree of genetic variation or rely mainly on sexual reproduction with low clonal propagation. I was able to show that the adaptive success of these non-native plant species can hardly be explained by DNA methylation, but could be a possible consequence of multiple introductions, dispersal corridors and meta-population dynamics. Similarly, my results illustrate that the use of plant species that do not predominantly reproduce clonally and are not model plants is essential to characterize the effect size of epigenetic mechanisms in an ecological-evolutionary context.
Aldehyde oxidases (AOXs) (E.C. 1.2.3.1) are molybdoflavo-enzymes belonging to the xanthine oxidase (XO) family. AOXs in mammals contain one molybdenum cofactor (Moco), one flavin adenine dinucleotide (FAD) and two [2Fe-2S] clusters, the presence of which is essential for the activity of the enzyme. Human aldehyde oxidase (hAOX1) is a cytosolic enzyme mainly expressed in the liver. hAOX1is involved in the metabolism of xenobiotics. It oxidizes aldehydes to their corresponding carboxylic acids and hydroxylates N-heterocyclic compounds. Since these functional groups are widely present in therapeutics, understanding the behaviour of hAOX1 has important implications in medicine. During the catalytic cycle of hAOX1, the substrate is oxidized at Moco and electrons are internally transferred to FAD via the FeS clusters. An electron acceptor juxtaposed to the FAD receives the electrons and re-oxidizes the enzyme for the next catalytic cycle. Molecular oxygen is the endogenous electron acceptor of hAOX1 and in doing so it is reduced and produces reactive oxygen species (ROS) including hydrogen peroxide (H2O2) and superoxide (O2.-). The production of ROS has patho-physiological importance, as ROS can have a wide range of effects on cell components including the enzyme itself.
In this thesis, we have shown that hAOX1 loses its activity over multiple cycles of catalysis due to endogenous ROS production and have identified a cysteine rich motif that protects hAOX1 from the ROS damaging effects. We have also shown that a sulfido ligand, which is bound at Moco and is essential for the catalytic activity of the enzyme, is vulnerable during turnover. The ROS produced during the course of the reaction are also able to remove this sulfido ligand from Moco. ROS, in addition, oxidize particular cysteine residues. The combined effects of ROS on the sulfido ligand and on specific cysteine residues in the enzyme result in its inactivation. Furthermore, we report that small reducing agents containing reactive sulfhydryl groups, in a selective manner, inactivate some of the mammalian AOXs by modifying the sulfido ligand at Moco. The mechanism of ROS production by hAOX1 is another scope that has been investigated as part of the work in this thesis. We have shown that the ratio of type of ROS, i.e. hydrogen peroxide (H2O2) and superoxide (O2.-), produced by hAOX1 is determined by a particular position on a flexible loop that locates in close proximity of FAD. The size of the cavity at the ROS producing site, i.e. the N5 position of the FAD isoalloxazine ring, kinetically affects the amount of each type of ROS generated by hAOX1. Taken together, hAOX1 is an enzyme with emerging importance in pharmacological and medical studies, not only due to its involvement in drug metabolism, but also due to ROS production which has physiological and pathological implications.
More than a century ago the phenomenon of non-Mendelian inheritance (NMI), defined as any type of inheritance pattern in which traits do not segregate in accordance with Mendel’s laws, was first reported. In the plant kingdom three genomic compartments, the nucleus, chloroplast, and mitochondrion, can participate in such a phenomenon. High-throughput sequencing (HTS) proved to be a key technology to investigate NMI phenomena by assembling and/or resequencing entire genomes. However, generation, analysis and interpretation of such datasets remain challenging by the multi-layered biological complexity. To advance our knowledge in the field of NMI, I conducted three studies involving different HTS technologies and implemented two new algorithms to analyze them.
In the first study I implemented a novel post-assembly pipeline, called Semi-Automated Graph-Based Assembly Curator (SAGBAC), which visualizes non-graph-based assemblies as graphs, identifies recombinogenic repeat pairs (RRPs), and reconstructs plant mitochondrial genomes (PMG) in a semiautomated workflow. We applied this pipeline to assemblies of three Oenothera species resulting in a spatially folded and circularized model. This model was confirmed by PCR and Southern blot analyses and was used to predict a defined set of 70 PMG isoforms. With Illumina Mate Pair and PacBio RSII data, the stoichiometry of the RRPs was determined quantitatively differing up to three-fold.
In the second study I developed a post-multiple sequence alignment algorithm, called correlation mapping (CM), which correlates segment-wise numbers of nucleotide changes to a numeric ascertainable phenotype. We applied this algorithm to 14 wild type and 18 mutagenized plastome assemblies within the Oenothera genus and identified two genes, accD and ycf2 that may cause the competitive behavior of plastid genotypes as plastids can be biparental inherited in Oenothera. Moreover, lipid composition of the plastid envelope membrane is affected by polymorphisms within these two genes.
For the third study, I programmed a pipeline to investigate a NMI phenomenon, known as paramutation, in tomato by analyzing DNA and bisulfite sequencing data as well as microarray data. We identified the responsible gene (Solyc02g0005200) and were able to fully repress its caused phenotype by heterologous complementation with a paramutation insensitive transgene of the Arabidopsis thaliana orthologue. Additionally, a suppressor mutant shows a globally altered DNA methylation pattern and carries a large deletion leading to a gene fusion involving a histone deacetylase.
In conclusion, my developed and implemented algorithms and data analysis pipelines are suitable to investigate NMI and led to novel insights about such phenomena by reconstructing PMGs (SAGBAC) as a requirement to study mitochondria-associated phenotypes, by identifying genes (CM) causing interplastidial competition as well by applying a DNA/Bisulfite-seq analysis pipeline to shed light in a transgenerational epigenetic inheritance phenomenon.
Stimuli-promoted in situ formation of hydrogels with thiol/thioester containing peptide precursors
(2022)
Hydrogels are potential synthetic ECM-like substitutes since they provide functional and structural similarities compared to soft tissues. They can be prepared by crosslinking of macromolecules or by polymerizing suitable precursors. The crosslinks are not necessarily covalent bonds, but could also be formed by physical interactions such as π-π interactions, hydrophobic interactions, or H-bonding. On demand in situ forming hydrogels have garnered increased interest especially for biomedical applications over preformed gels due to the relative ease of in vivo delivery and filling of cavities. The thiol-Michael addition reaction provides a straightforward and robust strategy for in situ gel formation with its fast reaction kinetics and ability to proceed under physiological conditions. The incorporation of a trigger function into a crosslinking system becomes even more interesting since gelling can be controlled with stimulus of choice. The use of small molar mass crosslinker precursors with active groups orthogonal to thiol-Michael reaction type electrophile provides the opportunity to implement an on-demand in situ crosslinking without compromising the fast reaction kinetics.
It was postulated that short peptide sequences due to the broad range structural-function relations available with the different constituent amino acids, can be exploited for the realisation of stimuli-promoted in situ covalent crosslinking and gelation applications. The advantages of this system over conventional polymer-polymer hydrogel systems are the ability tune and predict material property at the molecular level.
The main aim of this work was to develop a simplified and biologically-friendly stimuli-promoted in situ crosslinking and hydrogelation system using peptide mimetics as latent crosslinkers. The approach aims at using a single thiodepsipeptide sequence to achieve separate pH- and enzyme-promoted gelation systems with little modification to the thiodepsipeptide sequence. The realization of this aim required the completion of three milestones.
In the first place, after deciding on the thiol-Michael reaction as an effective in situ crosslinking strategy, a thiodepsipeptide, Ac-Pro-Leu-Gly-SLeu-Leu-Gly-NEtSH (TDP) with expected propensity towards pH-dependent thiol-thioester exchange (TTE) activation, was proposed as a suitable crosslinker precursor for pH-promoted gelation system. Prior to the synthesis of the proposed peptide-mimetic, knowledge of the thiol-Michael reactivity of the would-be activated thiol moiety SH-Leu, which is internally embedded in the thiodepsipeptide was required. In line with pKa requirements for a successful TTE, the reactivity of a more acidic thiol, SH-Phe was also investigated to aid the selection of the best thiol to be incorporated in the thioester bearing peptide based crosslinker precursor. Using ‘pseudo’ 2D-NMR investigations, it was found that only reactions involving SH-Leu yielded the expected thiol-Michael product, an observation that was attributed to the steric hindrance of the bulkier nature of SH-Phe. The fast reaction rates and complete acrylate/maleimide conversion obtained with SH-Leu at pH 7.2 and higher aided the direct elimination of SH-Phe as a potential thiol for the synthesis of the peptide mimetic.
Based on the initial studies, for the pH-promoted gelation system, the proposed Ac-Pro-Leu-Gly-SLeu-Leu-Gly-NEtSH was kept unmodified. The subtle difference in pKa values between SH-Leu (thioester thiol) and the terminal cysteamine thiol from theoretical conditions should be enough to effect a ‘pseudo’ intramolecular TTE. In polar protic solvents and under basic aqueous conditions, TDP successfully undergoes a ‘pseudo’ intramolecular TTE reaction to yield an α,ω-dithiol tripeptide, HSLeu-Leu-Gly-NEtSH. The pH dependence of thiolate ion generation by the cysteamine thiol aided the incorporation of the needed stimulus (pH) for the overall success of TTE (activation step) – thiol-Michael addition (crosslinking) strategy.
Secondly, with potential biomedical applications in focus, the susceptibility of TDP, like other thioesters, to intermolecular TTE reaction was probed with a group of thiols of varying thiol pKa values, since biological milieu characteristically contain peptide/protein thiols. L-cysteine, which is a biologically relevant thiol, and a small molecular weight thiol, methylthioglycolate both with relatively similar thiol pKa, values, led to an increase concentration of the dithiol crosslinker when reacted with TDP. In the presence of acidic thiols (p-NTP and 4MBA), a decrease in the dithiol concentration was observed, an observation that can be attributed to the inability of the TTE tetrahedral intermediate to dissociate into exchange products and is in line with pKa requirements for successful TTE reaction. These results additionally makes TDP more attractive and the potentially the first crosslinker precursor for applications in biologically relevant media.
Finally, the ability of TDP to promote pH-sensitive in situ gel formation was probed with maleimide functionalized 4-arm polyethylene glycol polymers in tris-buffered media of varying pHs. When a 1:1 thiol: maleimide molar ratio was used, TDP-PEG4MAL hydrogels formed within 3, 12 and 24 hours at pH values of 8.5, 8.0 and 7.5 respectively. However, gelation times of 3, 5 and 30 mins were observed for the same pH trend when the thiol: maleimide molar was increased to 2:1.
A direct correlation of thiol content with G’ of the gels at each pH could also be drawn by comparing gels with thiol: maleimide ratios of 1:1 to those with 2:1 thiol: maleimide mole ratios. This is supported by the fact that the storage modulus (G') is linearly dependent on the crosslinking density of the polymer. The values of initial G′ for all gels ranged between (200 – 5000 Pa), which falls in the range of elasticities of certain tissue microenvironments for example brain tissue 200 – 1000 Pa and adipose tissue (2500 – 3500 Pa).
Knowledge so far gained from the study on the ability to design and tune the exchange reaction of thioester containing peptide mimetic will give those working in the field further insight into the development of new sequences tailored towards specific applications.
TTE substrate design using peptide mimetic as presented in this work has revealed interesting new insights considering the state-of-the-art. Using the results obtained as reference, the strategy provides a possibility to extend the concept to the controlled delivery of active molecules needed for other robust and high yielding crosslinking reactions for biomedical applications. Application for this sequentially coupled functional system could be seen e.g. in the treatment of inflamed tissues associated with urinary tract like bladder infections for which pH levels above 7 were reported. By the inclusion of cell adhesion peptide motifs, the hydrogel network formed at this pH could act as a new support layer for the healing of damage epithelium as shown in interfacial gel formation experiments using TDP and PEG4MAL droplets.
The versatility of the thiodepsipeptide sequence, Ac-Pro-Leu-Gly-SLeu-Leu-Gly-(TDPo) was extended for the design and synthesis of a MMP-sensitive 4-arm PEG-TDPo conjugate. The purported cleavage of TDPo at the Gly-SLeu bond yields active thiol units for subsequent reaction of orthogonal Michael acceptor moieties. One of the advantages of stimuli-promoted in situ crosslinking systems using short peptides should be the ease of design of required peptide molecules due to the predictability of peptide functions their sequence structure. Consequently the functionalisation of a 4-arm PEG core with the collagenase active TDPo sequence yielded an MMP-sensitive 4-arm thiodepsipeptide-PEG conjugate (PEG4TDPo) substrate.
Cleavage studies using thiol flourometric assay in the presence of MMPs -2 and -9 confirmed the susceptibility of PEG4TDPo towards these enzymes. The resulting time-dependent increase in fluorescence intensity in the presence of thiol assay signifies the successful cleavage of TDPo at the Gly-SLeu bond as expected. It was observed that the cleavage studies with thiol flourometric assay introduces a sigmoid non-Michaelis-Menten type kinetic profile, hence making it difficult to accurately determine the enzyme cycling parameters, kcat and KM .
Gelation studies with PEG4MAL at 10 % wt. concentrations revealed faster gelation with MMP-2 than MMP-9 with 28 and 40 min gelation times respectively. Possible contributions by hydrolytic cleavage of PEG4TDPo has resulted in the gelation of PEG4MAL blank samples but only after 60 minutes of reaction. From theoretical considerations, the simultaneous gelation reaction would be expected to more negatively impact the enzymatic than hydrolytic cleavage. The exact contributions from hydrolytic cleavage of PEG4TDPo would however require additional studies.
In summary this new and simplified in situ crosslinking system using peptide-based crosslinker precursors with tuneable properties exhibited in situ crosslinking gelation kinetics on similar levels with already active dithiols reported. The advantageous on-demand functionality associated with its pH-sensitivity and physiological compatibility makes it a strong candidate worth further research as biomedical applications in general and on-demand material synthesis is concerned.
Results from MMP-promoted gelation system unveils a simple but unexplored approach for in situ synthesis of covalently crosslinked soft materials, that could lead to the development of an alternative pathway in addressing cancer metastasis by making use of MMP overexpression as a trigger. This goal has so far not being reach with MMP inhibitors despite the extensive work this regard.
An important goal in biotechnology and (bio-) medical research is the isolation of single cells from a heterogeneous cell population. These specialised cells are of great interest for bioproduction, diagnostics, drug development, (cancer) therapy and research. To tackle emerging questions, an ever finer differentiation between target cells and non-target cells is required. This precise differentiation is a challenge for a growing number of available methods.
Since the physiological properties of the cells are closely linked to their morphology, it is beneficial to include their appearance in the sorting decision. For established methods, this represents a non addressable parameter, requiring new methods for the identification and isolation of target cells. Consequently, a variety of new flow-based methods have been developed and presented in recent years utilising 2D imaging data to identify target cells within a sample. As these methods aim for high throughput, the devices developed typically require highly complex fluid handling techniques, making them expensive while offering limited image quality.
In this work, a new continuous flow system for image-based cell sorting was developed that uses dielectrophoresis to precisely handle cells in a microchannel. Dielectrophoretic forces are exerted by inhomogeneous alternating electric fields on polarisable particles (here: cells). In the present system, the electric fields can be switched on and off precisely and quickly by a signal generator. In addition to the resulting simple and effective cell handling, the system is characterised by the outstanding quality of the image data generated and its compatibility with standard microscopes. These aspects result in low complexity, making it both affordable and user-friendly.
With the developed cell sorting system, cells could be sorted reliably and efficiently according to their cytosolic staining as well as morphological properties at different optical magnifications. The achieved purity of the target cell population was up to 95% and about 85% of the sorted cells could be recovered from the system. Good agreement was achieved between the results obtained and theoretical considerations. The achieved throughput of the system was up to 12,000 cells per hour. Cell viability studies indicated a high biocompatibility of the system.
The results presented demonstrate the potential of image-based cell sorting using dielectrophoresis. The outstanding image quality and highly precise yet gentle handling of the cells set the system apart from other technologies. This results in enormous potential for processing valuable and sensitive cell samples.
Fiber-based microfluidics has undergone many innovative developments in recent years, with exciting examples of portable, cost-effective and easy-to-use detection systems already being used in diagnostic and analytical applications. In water samples, Legionella are a serious risk as human pathogens. Infection occurs through inhalation of aerosols containing Legionella cells and can cause severe pneumonia and may even be fatal. In case of Legionella contamination of water-bearing systems or Legionella infection, it is essential to find the source of the contamination as quickly as possible to prevent further infections. In drinking, industrial and wastewater monitoring, the culture-based method is still the most commonly used technique to detect Legionella contamination. In order to improve the laboratory-dependent determination, the long analysis times of 10-14 days as well as the inaccuracy of the measured values in colony forming units (CFU), new innovative ideas are needed. In all areas of application, for example in public, commercial or private facilities, rapid and precise analysis is required, ideally on site.
In this PhD thesis, all necessary single steps for a rapid DNA-based detection of Legionella were developed and characterized on a fiber-based miniaturized platform. In the first step, a fast, simple and device-independent chemical lysis of the bacteria and extraction of genomic DNA was established. Subsequently, different materials were investigated with respect to their non-specific DNA retention. Glass fiber filters proved to be particularly suitable, as they allow recovery of the DNA sample from the fiber material in combination with dedicated buffers and exhibit low autofluorescence, which was important for fluorescence-based readout.
A fiber-based electrophoresis unit was developed to migrate different oligonucleotides within a fiber matrix by application of an electric field. A particular advantage over lateral flow assays is the targeted movement, even after the fiber is saturated with liquid. For this purpose, the entire process of fiber selection, fiber chip patterning, combination with printed electrodes, and testing of retention and migration of different DNA samples (single-stranded, double-stranded and genomic DNA) was performed. DNA could be pulled across the fiber chip in an electric field of 24 V/cm within 5 minutes, remained intact and could be used for subsequent detection assays e.g., polymerase chain reaction (PCR) or fluorescence in situ hybridization (FISH). Fiber electrophoresis could also be used to separate DNA from other components e.g., proteins or cell lysates or to pull DNA through multiple layers of the glass microfiber. In this way, different fragments experienced a moderate, size-dependent separation. Furthermore, this arrangement offers the possibility that different detection reactions could take place in different layers at a later time. Electric current and potential measurements were collected to investigate the local distribution of the sample during migration. While an increase in current signal at high concentrations indicated the presence of DNA samples, initial experiments with methylene blue stained DNA showed a temporal sequence of signals, indicating sample migration along the chip.
For the specific detection of a Legionella DNA, a FISH-based detection with a molecular beacon probe was tested on the glass microfiber. A specific region within the 16S rRNA gene of Legionella spp. served as a target. For this detection, suitable reaction conditions and a readout unit had to be set up first. Subsequently, the sensitivity of the probe was tested with the reverse complementary target sequence and the specificity with several DNA fragments that differed from the target sequence. Compared to other DNA sequences of similar length also found in Legionella pneumophila, only the target DNA was specifically detected on the glass microfiber. If a single base exchange is present or if two bases are changed, the probe can no longer distinguish between the DNA targets and non-targets. An analysis with this specificity can be achieved with other methods such as melting point determination, as was also briefly indicated here. The molecular beacon probe could be dried on the glass microfiber and stored at room temperature for more than three months, after which it was still capable of detecting the target sequence. Finally, the feasibility of fiber-based FISH detection for genomic Legionella DNA was tested. Without further processing, the probe was unable to detect its target sequence in the complex genomic DNA. However, after selecting and application of appropriate restriction enzymes, specific detection of Legionella DNA against other aquatic pathogens with similar fragment patterns as Acinetobacter haemolyticus was possible.