Surface functionalization of amorphous hydrogenated silicon and polyimide evanescent waveguide based Mach-Zehnder interferometers to realize a biosensing platform

In dieser Diplomarbeit wurde die Funktionalisierung von auf amorphen hydrogenierten Silizium (a-Si:H) und Polyimid (PI) evanszenten Wellenleitern basierenden Mach-Zehnder Interferometern (MZI) realisiert und mittels Streptavidin-Biotin Model verifiziert. Weiters wurde eine biosensorische Plattform für DNA Experimente etabliert. Für die Realisierung dieses Projektes war vor allem die Etablierung von verschieden Oberflächencharakterisierungsmethoden entscheidend, mithilfe deren die Überwachung des Fortschritts der Funktionalisierung möglich wurde. Ebenso konnte sichergestellt werden, dass das empfindliche MZI Schichtsystem (a-Si:H-SU-8 und PI-Ormoclad) nicht angegriffen oder zerstört wurde. Die Anwendung der Photoelektronenspektroskopie (engl.: X-ray photoelectron spectrometer, XPS) ermöglichte die Charakterisierung der PI und a-Si:H Schichten. Nach dieser Charakterisierung wurden die Schichten mit verschieden chemischen Gruppen (Carboxyl-, Amino- und Sulfhydryl Gruppen) versehen. Das Protokoll für die Funktionalisierung mit Sulfhydryl-Gruppen, gefolgt von einer Biotinylierung der PI und a-Si:H Substrate wurde schließlich gewählt, um das Protein Streptavidin zu immobilisieren. Dabei wurde das fluoreszenzmarkierte Chromeon 642-Streptavidin verwendet, das es erlaubt durch Fluoreszenzscans zusätzliche Informationen zur Oberflächenkonzentration zu erhalten. Dadurch war es möglich auf nicht geblockten biotinylierten PI Kontrollproben 144 +/- 20 fmol/mm2 und auf nicht geblockten biotinylierten a-Si:H Kontrollproben 27 +/- 2 fmol/mm2 Chromeon 642-Streptavidin nachzuweisen. Das blocken der biotinylierten Oberflächen mit Bovine Serum Albumin (BSA) verhinderte eine nicht spezifische Bindung und reduzierte die Konzentration von nun ausschließlich selektiv zu Biotin gebundenem Chromeon 642-Streptavidin auf 80 +/- 20 fmol/mm2 auf PI und 20 +/- 2 fmol/mm2 auf a-Si:H Kontrollproben. Analysen mithilfe von Rasterkraftmikroskopie (engl.: Atomic force microscopy, AFM) auf Kontrollproben zeigte, dass die dünnen PI und a-Si:H Schichten durch die einzelnen Funktionalisierungsschritte nicht angegriffen wurden und die mittlere Rauigkeit nicht über den Wert von Ra=0.60 stieg. Zusätzliche XPS Messungen der Proben erbrachten weitere Informationen bezüglich der Atomkomposition von oberflächennahen Regionen nach unterschiedlichen Funktionalisierungsschritten. Die Resultate der XPS-Messungen stützten die Resultate der Fluoreszenzscans und zeigten mögliche Reaktionswege auf. Nachdem erfolgreich ein Funktionalisierungsprotokoll auf Kontrollproben etabliert wurde, konnten oberflächensensitive MZI-Echtzeitmessungen durchgeführt werden. Diese Messungen wurden als Experimente so geplant, dass zusätzliche Informationen über die Sensorstabilität und Reproduzierbarkeit, sowie die Sensitivität und die Selektivität der chemischen Bindung erhalten wurden. Dabei konnte gezeigt werden das BSA blocken alle nicht spezifischen Interaktionen mit den PI und a-Si:H Oberflächen verhindert und das Chromeon 642-Streptavidin selektiv angebunden werden kann. Allerdings wird dadurch auch das Sensorsignal bei PI-MZI Sensoren um 40% und bei a-Si:H MZI Sensoren um 26% verringert. Diese Resultate entsprechen den Ergebnissen aus den Fluoreszenzmessungen an geblockten und nicht geblockten biotinylierten Kontrollproben, bei denen die Änderung der Oberflächenkonzentration des Chromeon 642-Streptavidins 44% bei PI und 26% bei a-Si:H Substraten betrug. Die kleinste gemessene Konzentration von Chromeon 642-Streptavidin mit den MZI Sensoren war 1,6 nM (0,1 µg/ml), was eine Phasenverschiebung von 0,1pi auf PI und 0,5pi auf a-Si:H-MZI Sensoren erzeugte. Der linearen Bereich liegt bei a-Si:H-MZI Sensoren zwischen 1,6 und 416 nM mit einer chemischen Sensitivität von 0,0026pi/nM und bei PI-MZI Sensoren zwischen 16 und 166 nM mit einer chemischen Sensitivität von 0,0008pi/nM. Dieses Verhalten zeigt, dass für die Messung von Chromeon 642-Streptavidin a-Si:H-MZI Sensoren 3.6 mal sensitiver sind als PI-MZI Sensoren. Neben diesen Messungen wurde auch oberflächensensitive DNA Experimente durchgeführt. Dazu wurden die biotinylierten Sensoren mit einem nicht markierten Streptavidin beschichtet. Die ersten DNA Bindungsexperimente wurden als Referenzmessungen deklariert und zeigten dass der an die Streptavidinoberfläche bindet. Eine nicht spezifische Bindung des komplementären und nicht komplementären DNA Einzelstrangs konnte ausgeschlossen werden, da während den Messungen keinerlei signifikante Signaländerung auftrat. Dies zeigt, dass der Biotin getagte DNA Einzelstrang spezifisch an die Streptavidinoberfläche bindet. Die Hybridisierungsversuche zeigten, dass der komplementäre DNA Einzelstrang an den oberflächengebunden Biotin getagten DNA Einzelstrang bindet. Die resultierende Phasenverschiebung während der Hybridisierung war 2,3 +/- 0,2pi bei a-Si:H-MZI Sensoren und 0.26 +/- 0.02pi für PI-MZI Sensoren. Zusätzlich wurde während eines vergleichbaren Versuches mit nicht komplementären DNA keine Phasenverschiebung und daraus folgend keine Bindung gemessen. Das zeigte wiederum die Selektivität der Bindung des komplementären DNA Einzelstrangs an den oberflächengebundenen DNA Einzelstrang. Diese Resultate ermöglichen nun verschiedene DNA Hybridisierungsexperimente auf a-Si:H-MZI Sensoren. Die Phasenverschiebung auf PI-MZI Sensoren ist für weiterführende DNA Experimente zu gering und muss über andere Strategien erhöht werden. Dies kann beispielsweise über eine Erhöhung der Oberflächenkonzentration an gebundener Einzelstrang DNA mit einer anderen Funktionalisierungsstrategie oder durch Verwendung von DNA Oligonukleotiden mit einer höheren Anzahl an Basen (z.B. 70 statt 25) erreicht werden. Zusammenfassend kann gesagt werden, dass diese Diplomarbeit zwar nur eine limitierte Zahl an möglichen Oberflächenmodifikationen wieder gibt, im Gegenzug jedoch eine große Zahl an weiteren die Möglichkeiten der Oberflächenchemie eröffnet, die zur Entwicklung einer Vielzahl von anwendungsorientierten Biosensoren führen kann.In this diploma thesis, the surface functionalization of amorphous hydrogenated silicon (a-Si:H) and polyimide (PI) evanescent waveguide based Mach-Zehnder interferometers (MZIs) was realized and verified with the model of streptavidin-biotin binding. In addition, a biosensing platform for DNA experiments was established. Important for the successful realization of this project was the implementation of several surface characterization methods, such as the X-ray photoelectron spectroscopy (XPS), the surface fluorescence scans and atomic force microscopy (AFM). These methods enable the analysis of the progress of the functionalization and the monitoring of vital layer characteristics such as layer thickness or surface roughness. The XPS allowed to characterize the PI and a-Si:H substrates with respect to their atom composition. Surface modifications with different chemical groups (i.e. carboxyl-, amine- and sulfhydryl-groups) could be realized. The protocol of the sulfhydryl functionalization followed by the biotinylation was then chosen for the streptavidin immobilization. The use of Chromeon 642-streptavidin allowed for fluorescence scans in order to measure the surface concentration. It was found that on unblocked biotinylated PI control samples 144 ± 20 fmol/mm2 Chromeon 642-streptavidin and on unblocked biotinylated a-Si:H control samples 27 ± 2 fmol/mm2 Chromeon 642-streptavidin can bind to the surface. Blocking with bovine serum albumin (BSA) hinders the binding of the Chromeon 642-streptavidin to unspecific binding sites. The specific binding of streptavidin on the biotinylated BSA blocked PI and a-Si:H surfaces amounts to approximately 80 ± 20 fmol/mm2 and 20 ± 2 fmol/mm2 respectively. Atomic force microscopy (AFM) measurements indicated that the thin PI and a-Si:H layers are not damaged in course of the surface reactions and that the surface roughness stays below a mean roughness of Ra =0.60 nm. XPS measurements provided further information, such as the atom composition at the layer surfaces before and after several functionalization steps. These analysis support the results from previous fluorescence scans and indicate possible reaction paths. The progress of the surface modification was analyzed on control samples, and after their successful functionalization, surface sensing real-time measurements on the MZI sensors were performed. These measurements were planned as experiments that provide additional information about the sensor stability, reproducibility and sensitivity of sensors as well as the selectivity of the chemical binding. These measurements showed that the characteristics of a-Si:H- and the PI-MZI sensors were not influenced by chemical treatments during the functionalization and it could be proven that the BSA blocking completely suppresses unspecific interactions of Chromeon 642-streptavidin with the sensor surfaces. As a consequence of the BSA blocking a reduction of the phase shifts by about 40% for PI-MZI sensors and by about 26% for a-Si:H-MZI sensors was found. These results were supported by fluorescence measurements on blocked and unblocked samples, where the bound Chromeon 642-streptavidin concentration was reduced by about 44% on PI and 26% on a-Si:H surfaces. The lowest measured Chromeon 642-streptavidin concentration on the PI- and a-Si:H-MZI sensors was 1.6 nM (0.1 µg/ml), which caused a phase shift of 0.1π and 0.5π on PI and a-Si:H-MZI sensors. The linear range for a-Si:H-MZIs is between 1.6 and 416 nM with a chemical sensitivity (SC) of 0.026 pi/nM and for PI-MZI sensors between 16 and 166 nM with a chemical sensitivity 0.008 pi/nM. Therefore, a-Si:H-MZI sensors have a 3.6 higher SC than PI-MZI sensors for the measurement of Chromeon 642-streptavidin. Beside characterization of the MZI sensors by the Chromeon 642-streptavidin measurements, preliminary surface sensing DNA experiments on MZI sensor were performed. For this purpose unblocked biotinylated MZI sensors got functionalized with unlabeled streptavidin. The first DNA binding experiments, referred to as reference measurements, indicated that the biotin tagged single stranded DNA (ssDNA) bound to the streptavidin surface, because a phase shift corresponding to the binding process could be detected. On contrary, no unspecific interaction of the complementary and non complementary ssDNA strands to the streptavidin sensor surface could be found, which proves that the binding of the biotin tagged ssDNA was selective. The hybridization experiments showed that the complementary ssDNA strands bind to the surface bound biotin tagged ssDNA strand. The resulting phase shifts during hybridization were 2.3 +/- 0.2pi for a-Si:H-MZI sensors and 0.26 +/- 0.02pi for PI-MZI sensors respectively. In experiments with non complementary DNA no phase shift, and therefore, no unselective binding of non complementary DNA was detected, and that proves that the complementary DNA strands binds selective to the surface bound biotin tagged ssDNA strand. These results open up the way for different DNA hybridization experiments on a-Si:H-MZI sensors. For the PI-MZI sensors the remaining phase shifts are too low for advanced experiments and the sensors have to be improved, for example by the increase of the amount of surface bound single stranded DNA. Also the use of oligonucleotides with a higher number of base pairs (e.g. 70 instead of 25) may be possible in order to obtain larger phase shifts. For increasing the numbers of surface bound single stranded DNA, 3D-matrixes or dendrimers could be attached to the sensor surfaces. However, this diploma thesis gives a limited number of possible surface modification ways for the used sensor surfaces, but opens the door to an almost infinite number of surface chemistries, which can be used to create application-orientated biosensors

Seed-produced anti-globulin VHH-Fc antibodies retrieve globulin precursors in the insoluble fraction and modulate the Arabidopsis thaliana seed subcellular morphology

Key message Nanobody-heavy chain (VHH-Fc) antibody formats have the potential to immunomodulate even highly accumulating proteins and provide a valuable tool to experimentally modulate the subcellular distribution of seed storage proteins. Recombinant antibodies often obtain high accumulation levels in plants, and thus, besides being the actual end-product, antibodies targeting endogenous host proteins can be used to interfere with the localization and functioning of their corresponding antigens. Here, we compared the effect of a seed-expressed nanobody-heavy chain (VHH-Fc) antibody against the highly abundant Arabidopsis thaliana globulin seed storage protein cruciferin with that of a VHH-Fc antibody without endogenous target. Both antibodies reached high accumulation levels of around 10% of total soluble protein, but strikingly, another significant part was present in the insoluble protein fraction and was recovered only after extraction under denaturing conditions. In seeds containing the anti-cruciferin antibodies but not the antibody without endogenous target, the amount of soluble, processed globulin subunits was severely reduced and a major part of the cruciferin molecules was found as precursor in the insoluble fraction. Moreover, in these seeds, aberrant vacuolar phenotypes were observed that were different from the effects caused by the depletion of globulins in knock-out seeds. Remarkably, the seeds with strongly reduced globulin amounts are fully viable and germinate with frequencies similar to wild type, illustrating how flexible seeds can retrieve amino acids from the stored proteins to start germination

Impact of c-MYC expression on proliferation, differentiation, and risk of neoplastic transformation of human mesenchymal stromal cells

Background: Mesenchymal stromal cells isolated from bone marrow (MSC) represent an attractive source of adult stem cells for regenerative medicine. However, thorough research is required into their clinical application safety issues concerning a risk of potential neoplastic degeneration in a process of MSC propagation in cell culture for therapeutic applications. Expansion protocols could preselect MSC with elevated levels of growth-promoting transcription factors with oncogenic potential, such as c-MYC. We addressed the question whether c-MYC expression affects the growth and differentiation potential of human MSC upon extensive passaging in cell culture and assessed a risk of tumorigenic transformation caused by MSC overexpressing c-MYC in vivo. Methods: MSC were subjected to retroviral transduction to induce expression of c-MYC, or GFP, as a control. Cells were expanded, and effects of c-MYC overexpression on osteogenesis, adipogenesis, and chondrogenesis were monitored. Ectopic bone formation properties were tested in SCID mice. A potential risk of tumorigenesis imposed by MSC with c-MYC overexpression was evaluated. Results: C-MYC levels accumulated during ex vivo passaging, and overexpression enabled the transformed MSC to significantly overgrow competing control cells in culture. C-MYC-MSC acquired enhanced biological functions of c-MYC: its increased DNA-binding activity, elevated expression of the c-MYC-binding partner MAX, and induction of antagonists P19ARF/P16INK4A. Overexpression of c-MYC stimulated MSC proliferation and reduced osteogenic, adipogenic, and chondrogenic differentiation. Surprisingly, c-MYC overexpression also caused an increased COL10A1/COL2A1 expression ratio upon chondrogenesis, suggesting a role in hypertrophic degeneration. However, the in vivo ectopic bone formation ability of c-MYC-transduced MSC remained comparable to control GFP-MSC. There was no indication of tumor growth in any tissue after transplantation of c-MYC-MSC in mice. Conclusions: C-MYC expression promoted high proliferation rates of MSC, attenuated but not abrogated their differentiation capacity, and did not immediately lead to tumor formation in the tested in vivo mouse model. However, upregulation of MYC antagonists P19ARF/P16INK4A promoting apoptosis and senescence, as well as an observed shift towards a hypertrophic collagen phenotype and cartilage degeneration, point to lack of safety for clinical application of MSC that were manipulated to overexpress c-MYC for their better expansion

Development and characterization of passivation methods for microneedle-based biosensors

Microneedles (MN) are short, sharp structures that have the ability to painlessly pierce the stratum corneum, the outermost layer of the skin, and interface with the dermal interstitial fluid that lies beneath. Because the interstitial fluid is rich in biomarkers, microneedle-based biosensors have the potential to be used in a wide range of diagnostic applications. To act as an electrochemical sensor, the tip or the body of the MN must be functionalized, while the substrate areas are generally passivated to block any unwanted background interference that may occur outside of the skin. This work presents four different passivation techniques, based on the application of SiO2, polymethyl methacrylate (PMMA), an adhesive film, and varnish to the substrate areas. Optical, SEM and electrochemical measurements were performed to quantitatively assess the performance of each film. The data shows that whilst manual application of varnish provided the highest level of electrical isolation, the spin-coating of a 5 μm thick layer of PMMA is likely to provide the best combination of performance and manufacturability. Clinical Relevance— Substrate passivation techniques will improve the performance of microneedle-based non-invasive continuous monitoring systems

In planta deglycosylation improves the SARS-CoV-2 neutralization activity of recombinant ACE2-Fc

SARS-CoV-2 infects human cells via binding of the viral spike glycoprotein to its main cellular receptor, angiotensin-converting enzyme 2 (ACE2). The spike protein-ACE2 receptor interaction is therefore a major target for the development of therapeutic or prophylactic drugs to combat coronavirus infections. Various engineered soluble ACE2 variants (decoys) have been designed and shown to exhibit virus neutralization capacity in cell-based assays and in vivo models. Human ACE2 is heavily glycosylated and some of its glycans impair binding to the SARS-CoV-2 spike protein. Therefore, glycan-engineered recombinant soluble ACE2 variants might display enhanced virus-neutralization potencies. Here, we transiently co-expressed the extracellular domain of ACE2 fused to human Fc (ACE2-Fc) with a bacterial endoglycosidase in Nicotiana benthamiana to produce ACE2-Fc decorated with N-glycans consisting of single GlcNAc residues. The endoglycosidase was targeted to the Golgi apparatus with the intention to avoid any interference of glycan removal with concomitant ACE2-Fc protein folding and quality control in the endoplasmic reticulum. The in vivo deglycosylated ACE2-Fc carrying single GlcNAc residues displayed increased affinity to the receptor-binding domain (RBD) of SARS-CoV-2 as well as improved virus neutralization activity and thus is a promising drug candidate to block coronavirus infection

Heritable Genomic Fragment Deletions and Small Indels in the Putative ENGase Gene Induced by CRISPR/Cas9 in Barley

Targeted genome editing with the CRISPR/Cas9 system has been used extensively for the selective mutation of plant genes. Here we used CRISPR/Cas9 to disrupt the putative barley (Hordeum vulgare cv. “Golden Promise”) endo-N-acetyl-β-D-glucosaminidase (ENGase) gene. Five single guide RNAs (sgRNAs) were designed for different target sites in the upstream part of the ENGase coding region. Targeted fragment deletions were induced by co-bombarding selected combinations of sgRNA with wild-type cas9 using separate plasmids, or by co-infection with separate Agrobacterium tumefaciens cultures. Genotype screening was carried out in the primary transformants (T0) and their T1 progeny to confirm the presence of site-specific small insertions and deletions (indels) and genomic fragment deletions between pairs of targets. Cas9-induced mutations were observed in 78% of the plants, a higher efficiency than previously reported in barley. Notably, there were differences in performance among the five sgRNAs. The induced indels and fragment deletions were transmitted to the T1 generation, and transgene free (sgRNA:cas9 negative) genome-edited homozygous ENGase knock outs were identified among the T1 progeny. We have therefore demonstrated that mutant barley lines with a disrupted endogenous ENGase and defined fragment deletions can be produced efficiently using the CRISPR/Cas9 system even when this requires co-transformation with multiple plasmids by bombardment or Agrobacterium-mediated transformation. We confirm the specificity and heritability of the mutations and the ability to efficiently generate homozygous mutant T1 plants

Electromagnetic Characterization and Simulation of a Carbonate Buffer System on a Microwave Biosensor

In order to develop a fast, sensitive and easy-to-produce biosensor, a high-quality microwave split-ring resonator is going to be developed. In the final sensing device, a blood sample will be placed as a droplet on the sensitive area of the sensor. In case of specific target biomolecules binding a shift in resonance frequency will be induced due to the effective permittivity change. This shift in resonance frequency depends on the concentration of biomolecules and is therefore quantitative. The aim of this work is to find a position for the bio-functionalization that providesa measurable frequency shift when the analyte is added. Different areas are tested experimentally and via simulations. Two buffer solutions are used which have to be characterized in terms of its electromagnetic properties in advance. This preliminary study should pave the way for the measurements in real human samples such as serum or plasma

Design of a Photonic Crystal Defect Waveguide Biosensor Operating in Aqueous Solutions at 1.34 µm

A two-dimensional photonic crystal defect waveguide sensor based on CMOS-compatible silicon-on-insulator technology was designed for operation in aqueous solutions at a wavelength of 1.34 µm, by the use of the 3D Plane Wave Expansion and the Finite Difference Time Domain simulation method. An operation under water in this wavelength regime allows for a significantly smaller propagation loss in contrast to the state-of-the-art operation wavelength of photonic crystals at 1.55 µm. The sensor working principle is label-free and based on evanescent wave sensing exploiting the local refractive index change induced by the specific binding of target molecules to a capture molecules immobilized on the surface of the phontonic crystal structure. We experimentally proved the theoretical predications of our simulations and demonstrated the sensing functionality of the photonic crystal defect waveguide using the biotin-straptavidin binding system

Electrochemical Diffusion Study in Poly(Ethylene Glycol) Dimethacrylate-Based Hydrogels

Hydrogels are of great importance for functionalizing sensors and microfluidics, and poly(ethylene glycol) dimethacrylate (PEG-DMA) is often used as a viscosifier for printable hydrogel precursor inks. In this study, 1–10 kDa PEG-DMA based hydrogels were characterized by gravimetric and electrochemical methods to investigate the diffusivity of small molecules and proteins. Swelling ratios (SRs) of 14.43–9.24, as well as mesh sizes ξ of 3.58–6.91 nm were calculated, and it was found that the SR correlates with the molar concentration of PEG-DMA in the ink (MCI) (SR = 0.1127 × MCI + 8.3256, R2 = 0.9692) and ξ correlates with the molecular weight (Mw) (ξ = 0.3382 × Mw + 3.638, R2 = 0.9451). To investigate the sensing properties, methylene blue (MB) and MB-conjugated proteins were measured on electrochemical sensors with and without hydrogel coating. It was found that on sensors with 10 kDa PEG-DMA hydrogel modification, the DPV peak currents were reduced to 92 % for MB, 73 % for MB-BSA, and 23 % for MB-IgG. To investigate the diffusion properties of MB(-conjugates) in hydrogels with 1–10 kDa PEG-DMA, diffusivity was calculated from the current equation. It was found that diffusivity increases with increasing ξ. Finally, the release of MB-BSA was detected after drying the MB-BSA-containing hydrogel, which is a promising result for the development of hydrogel-based reagent reservoirs for biosensing. This work received funding from the Austrian Research Promotion Agency (FFG) under the HydroChip2 (grant no. 883914) and the Predict project (grant no. 870027) as well as from the European Union’s Horizon 2020 research and innovation program under Grant Agreement No. 761000 (GREENSENSE)</p