Wolfgang Knoll

Biotechnology Applications of Tethered Lipid Bilayer Membranes

The importance of cell membranes in biological systems has prompted the development of model membrane platforms that recapitulate fundamental aspects of membrane biology, especially the lipid bilayer environment. Tethered lipid bilayers represent one of the most promising classes of model membranes and are based on the immobilization of a planar lipid bilayer on a solid support that enables characterization by a wide range of surface-sensitive analytical techniques. Moreover, as the result of molecular engineering inspired by biology, tethered bilayers are increasingly able to mimic fundamental properties of natural cell membranes, including fluidity, electrical sealing and hosting transmembrane proteins. At the same time, new methods have been employed to improve the durability of tethered bilayers, with shelf-lives now reaching the order of weeks and months. Taken together, the capabilities of tethered lipid bilayers have opened the door to biotechnology applications in healthcare, environmental monitoring and energy storage. In this review, several examples of such applications are presented. Beyond the particulars of each example, the focus of this review is on the emerging design and characterization strategies that made these applications possible. By drawing connections between these strategies and promising research results, future opportunities for tethered lipid bilayers within the biotechnology field are discussed.

Bloch surface wave-enhanced fluorescence biosensor

A new approach to signal amplification in fluorescence-based assays for sensitive detection of molecular analytes is reported. It relies on a sensor chip carrying a one-dimensional photonic crystal (1DPC) composed of two piled up segments which are designed to increase simultaneously the excitation rate and the collection efficiency of fluorescence light. The top segment supports Bloch surface waves (BSWs) at the excitation wavelength and the bottom segment serves as a Bragg mirror for the emission wavelength of used fluorophore labels. The enhancement of the excitation rate on the sensor surface is achieved through the resonant coupling to BSWs that is associated with strong increase of the field intensity. The increasing of collection efficiency of fluorescence light emitted from the sensor surface is pursued by using the Bragg mirror that minimizes its leakage into a substrate and provides its beaming toward a detector. In order to exploit the whole evanescent field of BSW, extended three-dimensional hydrogel-based binding matrix that is functionalized with catcher molecules is attached to 1DPC for capturing of target analyte from a sample. Simulations supported by experiments are presented to illustrate the design and determined the performance characteristics of BSW-enhanced fluorescence spectroscopy. A model immunoassay experiment demonstrates that the reported approach enables increasing signal to noise ratio, resulting in about one order of magnitude improved limit of detection (LOD) with respect to regular total internal reflection fluorescence (TIRF) configuration.

A facile route for the preparation of azide-terminated polymers. "Clicking" polyelectrolyte brushes on planar surfaces and nanochannels

In this work we describe the facile preparation of azide-terminated polymers by conventional radical polymerization (cRP) using azo initiators bearing azide groups. We show that cRP provides a convenient avenue for the preparation of azide end-functional polymers in a one-step process. The versatility of this chemical methodology was demonstrated by the synthesis of unprecedented azide end group-functionalized sodium polystyrene sulfonate (PSSNa) and poly(2-methacryloyloxyethyl-trimethylammonium chloride) (PMETAC) which were then “clicked” onto alkyne-terminated silicon surfaces and polyethylene terephthalate nanochannels to form polyelectrolyte brush layers. The facile synthesis of the end-functionalized macromolecular building blocks will enable the creation of a wide variety of “clickable” architectures using very simple synthetic tools. We are confident that these results will constitute a key element in the “click” chemistry toolbox and, as such, will have strong implications for the molecular design of interfaces using macromolecular architectures.

Long range surface plasmon and hydrogel optical waveguide field-enhanced fluorescence biosensor with 3D hydrogel binding matrix: On the role of diffusion mass transfer

An implementation of evanescent wave affinity biosensor with a large-capacity three-dimensional binding matrix for ultra-sensitive detection of molecular analytes is investigated. In the experimental part of the work, highly swollen carboxylated poly(N-isopropylacryamide) (NIPAAm) hydrogel with up to micrometer thickness was grafted to a sensor surface, functionalized with antibody recognition elements and employed for immunoassay-based detection of target molecules contained in a liquid sample. Molecular binding events were detected by long range surface plasmon (LRSP) and hydrogel optical waveguide (HOW) field-enhanced fluorescence spectroscopy. These novel methods allowed probing an extended three-dimensional biointerface with an evanescent field reaching up to several micrometers from the sensor surface. The resonant excitation of LRSP and HOW modes provided strong enhancement of intensity of electromagnetic field that is directly translated into an increased fluorescence signal associated with the binding of fluorophore-labeled molecules. Experimental observations were supported by numerical simulations of mass transfer and affinity binding of target molecules in the hydrogel. Through the optimization of the hydrogel thickness and profile of the probing evanescent wave, low femtomolar limit of detection was achieved.

Compact surface plasmon-enhanced fluorescence biochip

A new concept of compact biochip for surface plasmon-enhanced fluorescence assays is reported. It takes advantage of the amplification of fluorescence signal through the coupling of fluorophore labels with confined and strongly enhanced field intensity of surface plasmons. In order to efficiently excite and collect the emitted fluorescence light via surface plasmons on a metallic sensor surface, (reverse) Kretschmann configuration is combined with diffractive optical elements embedded on the chip surface. These include a concentric relief grating for the imaging of highly directional surface plasmon-coupled emission to a detector. Additional linear grating is used for the generating of surface plasmons at the excitation wavelength on the sensor surface in order to increase the fluorescence excitation rate. The reported approach offers the increased intensity of fluorescence signal, reduced background, and compatibility with nanoimprint lithography for cost-effective preparation of sensor chip. The presented approach was implemented for biosensing in a model immunoassay experiment in which the limit of detection of 11 pM was achieved.

Surface plasmon-coupled emission on plasmonic Bragg gratings

Surface plasmon-coupled emission (SPCE) from emitters in a close proximity to a plasmonic Bragg grating is investigated. In this study, the directional fluorescence emission mediated by Bragg-scattered surface plasmons and surface plasmons diffraction cross-coupled through a thin metallic film is observed by using the reverse Kretschmann configuration. We show that controlling of dispersion relation of these surface plasmon modes by tuning the refractive index at upper and lower interfaces of a dense sub-wavelength metallic grating enables selective reducing or increasing the intensity of the light emitted to certain directions. These observations may provide important leads for design of advanced plasmonic structures in applications areas of plasmon-enhanced fluorescence spectroscopy and nanoscale optical sources.

Electronic Olfactory Sensor Based on A. mellifera Odorant‐Binding Protein 14 on a Reduced Graphene Oxide Field‐Effect Transistor

Abstract An olfactory biosensor based on a reduced graphene oxide (rGO) field‐effect transistor (FET), functionalized by the odorant‐binding proteinu200514 (OBP14) from the honey bee (Apis mellifera) has been designed for the inu2005situ and real‐time monitoring of a broad spectrum of odorants in aqueous solutions known to be attractants for bees. The electrical measurements of the binding of all tested odorants are shown to follow the Langmuir model for ligand–receptor interactions. The results demonstrate that OBP14 is able to bind odorants even after immobilization on rGO and can discriminate between ligands binding within a range of dissociation constants from K d=4u2005μm to K d=3.3u2005mm. The strongest ligands, such as homovanillic acid, eugenol, and methyl vanillate all contain a hydroxy group which is apparently important for the strong interaction with the protein.

Probing mobility and structural inhomogeneities in grafted hydrogel films by fluorescence correlation spectroscopy

We employed fluorescence correlation spectroscopy to investigate the effect of crosslinking density, annealing in the dry state, temperature, and solvent quality on the one-dimensional swelling, permeability, and mobility of tracer molecules in thermoresponsive hydrogel films. These consist of a carboxylated poly(N-isopropylacrylamide) derivative (PNIPAAm) covalently anchored to glass substrates. Upon increasing the temperature beyond the collapse transition at about 32 °C, the gels shrunk from the swollen to a collapsed state. A molecular dye (Alexa 647) and green fluorescent protein were chosen as tracers as they display only weak interaction with the carboxylated PNIPAAm. At large swelling ratios (low temperatures) the hydrogel layers are spatially homogeneous and both tracers show single Fickian diffusion. Diffusion coefficients scale with the PNIPAAm volume fraction. Upon temperature increase a qualitatively different behavior is observed already in the pretransition region (25–32 °C) concurrently with moderate swelling ratios (<4). This is manifested by an additional, faster Fickian diffusion and structural inhomogeneities, which are also found by optical waveguide mode spectroscopy. Above the collapse transition all diffusants are expelled from the hydrogels at a limiting swelling ratio ∼1.5. Subtle differences in the solvent quality influence the diffusion of tracers in the PNIPAAm hydrogel films. In the transition temperature range structural inhomogeneities at the nanoscale appeared.

Hydrogel-supported protein-tethered bilayer lipid membranes: a new approach toward polymer-supported lipid membranes

Polymer-supported bilayer lipid membranes offer great opportunities for the investigation of functional membrane proteins. Here we present a new approach in this direction by introducing a thin hydrogel layer as a soft ‘cushion’ on indium–tin oxide (ITO), providing a smooth, functional surface to form the protein-tethered BLM (ptBLM). ITO was used as a transparent electrode, enabling simultaneous implementation of electrochemical and optical waveguide techniques. The hydrogel poly(N-(2-hydroxyethyl)acrylamide-co-5-acrylamido-1-carboxypentyl-iminodiacetate-co-4-benzoylphenyl methacrylate) (P(HEAAm-co-NTAAAm-co-MABP)) was functionalized with the nickel chelating nitrilotriacetic acid (NTA) groups, to which cytochrome c oxidase (CcO) from Paracoccus denitrificans was bound in a well defined orientation via a his-tag attached to its subunit I. Given that the mesh size of P(HEAAm-co-NTAAAm-co-MABP) was smaller than the protein size, binding to the hydrogel occurred only on the top of the layer. The lipid bilayer was formed around the protein by in situdialysis. Electrochemical impedance spectroscopy showed good electrical sealing properties with a resistance of ∼1 MΩ cm2. Furthermore, surface plasmon resonance optical waveguide spectroscopy (SPR/OWS) indicated an increased anisotropy of the system after formation of the lipid bilayer. Cyclic voltammetry in the presence of reduced cytochrome c demonstrated that CcO was incorporated into the gel-supported ptBLM in a functionally active form.

Host–guest supramolecular chemistry in solid-state nanopores: potassium-driven modulation of ionic transport in nanofluidic diodes

We describe the use of asymmetric nanopores decorated with crown ethers for constructing robust signal-responsive chemical devices. The modification of single conical nanopores with 18-crown-6 units led to a nanodevice whose electronic readout, derived from the transmembrane ion current, can be finely tuned over a wide range of K(+) concentrations. The electrostatic characteristics of the nanopore environment arising from host-guest ion-recognition processes taking place on the pore walls are responsible for tuning the transmembrane ionic transport and the rectification properties of the pore. This work illustrates the potential and versatility of host-guest chemistry, in combination with nanofluidic elements, as a key enabler to achieve addressable chemical nanodevices mimicking the ion transport properties and gating functions of specific biological channels.