6 research outputs found
Functional 3D Nanostructures : Bio Applications for STED Lithography and Multiphoton Polymerization
This thesis is dedicated to method development of super-resolution far-field lithography for 3D polymer structuring. Multiphoton polymerization (MPP) and stimulated emission depletion (STED) lithography were used to create structures with the highest resolution achievable, structures with additional chemical functionality and to develop a new platform for protein assays. MPP enables the fabrication of polymeric structures in complex 3D geometries, with a wide range of chemical and physical functionality. However, the resolution of MPP is limited by diffraction. STED controls the spatial distribution of excited photoinitiators, thereby confining the polymerization volume. Hence, combining MPP with STED improves the resolution beyond the diffraction limit. Record resolutions of 120 nm in the lateral direction and 275 nm in the axial direction were achieved by STED lithography. Single features with 55 nm width were demonstrated. MPP enables fabrication of composite structures with different chemical functionality. To combine the functional versatility of MPP with the superior resolution of STED, new functional photoresists were employed. Acrylate monomers with additional carboxyl- or thiol groups were used to write reactive nano-structures with 60 nm feature size. Two- and three-dimensional composite structures were written and orthogonally functionalized with different fluorophores. An axial resolution of 550 nm between differently functionalized layers was achieved. The carboxyl-photoresist enables fabrication of structures with improved protein adhesiveness. A 3D composite platform was fabricated to immobilize functional capture proteins onto binding sites, which were elevated from the substrate surface. Molecular recognition of fluorescently labeled proteins was shown on those binding sites. The first 3D protein immunoassay for confocal readout was demonstrated, with signal to noise ratios exeeding 10.Die vorliegende Arbeit ist der Methodenentwicklung fĂŒr die höchstauflösende Fernfeld-Lithographie gewidmet. Mittels Multiphotonen Polymerisierung (MPP) und Stimulated Emission Depletion (STED) Lithographie wurden Polymerstrukturen in der höchstmöglichen Auflösung erzeugt. ZusĂ€tzlich wurden Strukturen mit chemischer FunktionalitĂ€t hergestellt, die in weiterer Folge kovalent modifiziert wurden oder auch als Plattform fĂŒr Protein-Assays genutzt wurden. Mittels MPP können komplexe 3D Polymerstrukturen mit vielseitiger FunktionalitĂ€t erzeugt werden. MPP ist jedoch beugungsbegrenzt. STED kontrolliert die rĂ€umliche Verteilung angeregter Photoinitiatoren und schrĂ€nkt dadurch das Polymerisationsvolumen ein. Eine Kombination von MPP mit STED ermöglicht weit unter dem Beugungslimit zu strukturieren. Auflösungen von 120 nm in der lateralen Richtung bzw. 275 nm in der axialen Richtung konnten gezeigt werden. Um die funktionelle Vielseitigkeit von MPP mit der ĂŒberlegenen Auflösung der STED Lithographie zu verbinden, wurden neue Photolacke eingesetzt. Acryl-Monomere mit zusĂ€tzlichen Carboxyl- oder Thiol-Gruppen wurden zur Erzeugung von reaktiven Nanostrukturen mit 60 nm StrukturgröĂe verwendet. 2D- und 3D Verbundstrukturen wurden erzeugt und mittels orthogonaler Chemie mit verschiedenen Fluorophoren funktionalisiert. Die axiale Auflösung zwischen verschieden funktionellen Schichten konnte auf 550 nm verbessert werden. Der Carboxyl-Photolack ermöglicht die Herstellung von Strukturen mit verbesserter Proteinanhaftung. Eine 3D Verbundstruktur wurde verwendet, um FĂ€nger-Proteine an definierten Bindungsstellen anzuhaften. Diese Bindungsstellen befinden sich ĂŒber der SubstratoberflĂ€che. Auf diesen Bindungsstellen wurde molekulare Erkennung von fluoreszent markierten Proteinen gezeigt. Mittels konfokaler Mikroskopie wurde somit der erste 3D Protein Assay gezeigt, mit einem Signal-Rausch-VerhĂ€ltnis von ĂŒber 10.submitted by Richard WollhofenUniversitĂ€t Linz, Dissertation, 2017OeBB(VLID)224624
Nano-Anchors with Single Protein Capacity Produced with STED Lithography
Acrylate
nanoanchors of subdiffraction-limited diameter are written
with optical stimulated emission depletion (STED) lithography. After
incubation, 98% of all nanoanchors are loaded quickly with fluorescently
labeled antibodies. Controlling the size of the nanoanchors allows
for limiting the number of the antibodies. Direct stochastic optical
reconstruction microscopy (dSTORM) imaging, statistical distribution
of fluorescence, quantitative fluorescence readout, and single molecule
blinking consistently prove that 80% of the nanoanchors with a 65
nm diameter are carrying only one antibody each, which are functional
as confirmed with live erythrocytes
Nano-Anchors with Single Protein Capacity Produced with STED Lithography
Acrylate
nanoanchors of subdiffraction-limited diameter are written
with optical stimulated emission depletion (STED) lithography. After
incubation, 98% of all nanoanchors are loaded quickly with fluorescently
labeled antibodies. Controlling the size of the nanoanchors allows
for limiting the number of the antibodies. Direct stochastic optical
reconstruction microscopy (dSTORM) imaging, statistical distribution
of fluorescence, quantitative fluorescence readout, and single molecule
blinking consistently prove that 80% of the nanoanchors with a 65
nm diameter are carrying only one antibody each, which are functional
as confirmed with live erythrocytes
Multiphoton-Polymerized 3D Protein Assay
Multiphoton
polymerization (MPP) enables 3D fabrication of micro- and nanoscale
devices with complex geometries. Using MPP, we create a 3D platform
for protein assays. Elevating the protein-binding sites above the
substrate surface allows an optically sectioned readout, minimizing
the inevitable background signal from nonspecific protein adsorption
at the substrate surface. Two fluorescence-linked immunosorbent assays
are demonstrated, the first one relying on streptavidinâbiotin
recognition and the second one on antibody recognition of apolipoprotein
A1, a major constituent of high-density lipoprotein particles. Signal-to-noise
ratios exceeding 1000 were achieved. The platform has high potential
for 3D multiplexed recognition assays with an increased binding surface
for on-chip flow cells
Stimulated Emission Depletion Lithography with Mercapto-Functional Polymers
Surface
reactive nanostructures were fabricated using stimulated
emission depletion (STED) lithography. The functionalization of the
nanostructures was realized by copolymerization of a bifunctional
metal oxo cluster in the presence of a triacrylate monomer. Ligands
of the cluster surface cross-link to the monomer during the lithographic
process, whereas unreacted mercapto functionalized ligands are transferred
to the polymer and remain reactive after polymer formation of the
surface of the nanostructure. The depletion efficiency in dependence
of the cluster loading was investigated and full depletion of the
STED effect was observed with a cluster loading exceeding 4 wt %.
A feature size by λ/11 was achieved by using a donut-shaped
depletion beam. The reactivity of the mercapto groups on the surface
of the nanostructure was tested by incubation with mercapto-reactive
fluorophores