formation-biomembrane-microarrays-with-squeegee-based-assembly

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

University of Minnesota

Inexpensive, reproducible, and high-throughput fabrication of nanometric apertures in metallic films can benefit many applications in plasmonics, sensing, spectroscopy, lithography, and imaging. Here we use template-stripping to pattern periodic nanohole arrays in optically thick, smooth Ag films with a silicon template made via nanoimprint lithography. Ag is a low-cost material with good optical properties, but it suffers from poor chemical stability and biocompatibility. However, a thin silica shell encapsulating our template-stripped Ag nanoholes facilitates biosensing applications by protecting the Ag from oxidation as well as providing a robust surface that can be readily modified with a variety of biomolecules using well-established silane chemistry. The thickness of the conformal silica shell can be precisely tuned by atomic layer deposition, and a 15 nm thick silica shell can effectively prevent fluorophore quenching. The Ag nanohole arrays with silica shells can also be bonded to polydimethylsiloxane (PDMS) microfluidic channels for fluorescence imaging, formation of supported lipid bilayers, and real-time, label-free SPR sensing. Additionally, the smooth surfaces of the template-stripped Ag films enhance refractive index sensitivity compared with as-deposited, rough Ag films. Because nearly centimeter-sized nanohole arrays can be produced inexpensively without using any additional lithography, etching, or lift-off, this method can facilitate widespread applications of metallic nanohole arrays for plasmonics and biosensing.

Template-stripped smooth Ag nanohole arrays with silica shells for surface plasmon resonance biosensing.

We demonstrate precise three-dimensional integration of smooth bumps, grooves, and apertures in optically thick metal films using template stripping. Patterned silicon wafers are used as high-quality, reusable templates. The heights or depths of the metallic features are controlled to within 2 nm, giving continuously tunable optical properties with sharp and intense plasmonic resonances. Furthermore, we demonstrate a pick-and-place template stripping method in situ, enabling versatile three-dimensional micromanipulation, imaging, and characterization of nanoscale devices.

Monolithic integration of continuously tunable plasmonic nanostructures.

Microarray technology has facilitated many powerful high-throughput studies in the fields of genetics and proteomics, among others. However, preparation of microarrays composed of cell-derived membranes with embedded receptors has proven difficult. Here we describe a new method for forming microarrays composed of synthetic lipid vesicles and natural cell membranes. The method is based upon assembly of vesicles and natural membranes into recessed micro- and nanowells and using a polydimethylsiloxane (PDMS) block as a "squeegee." This method is used to assemble phospholipid vesicles into arrays with micrometer and nanoscale dimensions. Native myelin and neuronal lipid raft arrays are also formed in 30 min or less. We show the natural membrane arrays can be used for sensing lipid-protein interactions by detecting cholera toxin binding to ganglioside GM1 in neuronal lipid rafts. In multicomponent arrays myelin can be distinguished from neuronal rafts by antibody binding to cell-specific surface antigens. Finally, myelin arrays formed in gold nanowells are used for surface plasmon resonance sensing. This assembly approach is simple, broadly applicable, and opens up new avenues of research not easily accomplished with standard microarray technology.

Facile assembly of micro- and nanoarrays for sensing with natural cell membranes.

With a template-stripping fabrication technique, we demonstrate the mass fabrication of high-quality, uniform, ultrasharp (10 nm) metallic probes suitable for single-molecule fluorescence imaging, tip-enhanced Raman spectroscopy (TERS), and other near-field imaging techniques. We achieve reproducible single-molecule imaging with sub-20-nm spatial resolution and an enhancement in the detected fluorescence signal of up to 200. Similar results are obtained for TERS imaging of carbon nanotubes. We show that the large apex angle (70.5°) of our pyramidal tip is well suited to scatter the near-field optical signal into the far-field, leading to larger emission enhancement and hence to a larger quantum yield. Each gold or silver pyramidal probe is used on-demand, one at a time, and the unused tips can be stored for extended times without degradation or contamination. The high yield (>95%), reproducibility, durability, and massively parallel fabrication (1.5 million identical probes over a wafer) of the probes hold promise for reliable optical sensing and detection and for cementing near-field optical imaging and spectroscopy as a routine characterization technique.

Highly reproducible near-field optical imaging with sub-20-nm resolution based on template-stripped gold pyramids.

Lipid bilayer membranes found in nature are heterogeneous mixtures of lipids and proteins. Model systems, such as supported lipid bilayers (SLBs), are often employed to simplify experimental systems while mimicking the properties of natural lipid bilayers. Here, we demonstrate a new method to form SLB arrays by first forming spherical supported lipid bilayers (SSLBs) on submicrometer-diameter SiO(2) beads. The SSLBs are then arrayed into microwells using a simple physical assembly method that requires no chemical modification of the substrate nor modification of the lipid membrane with recognition moieties. The resulting arrays have submicrometer SSLBs with 3 μm periodicity where >75% of the microwells are occupied by an individual SSLB. Because the arrays have high density, fluorescence from >1000 discrete SSLBs can be acquired with a single image capture. We show that 2-component random arrays can be formed, and we also use the arrays to determine the equilibrium dissociation constant for cholera toxin binding to ganglioside GM1. SSLB arrays are robust and are stable for at least one week in buffer.

High-density arrays of submicron spherical supported lipid bilayers.

Self-assembled plasmonic nanoring cavity arrays are formed alongside the curvature of highly packed metallic nanosphere gratings. The sub-10-nm gap size is precisely tuned via atomic layer deposition and highly ordered arrays are produced over a cm-sized area. The resulting hybrid nanostructure boosts coupling efficiency of light into plasmons, and shows an improved SERS detection limit. These substrates are used for SERS detection of the biological analyte, adenine, followed by concurrent localized surface plasmon resonance sensing.

Self-assembled plasmonic nanoring cavity arrays for SERS and LSPR biosensing.

We demonstrate the design, fabrication and characterization of a near-field plasmonic nanofocusing probe with a hybrid tip-plus-aperture design. By combining template stripping with focused ion beam lithography, a variety of aperture-based near-field probes can be fabricated with high optical performance. In particular, the combination of large transmission through a C-shaped aperture aligned to the sharp apex (<10 nm radius) of a template-stripped metallic pyramid allows the efficient delivery of light--via the C-shaped aperture--while providing a nanometric hotspot determined by the sharpness of the tip itself.

Plasmonic nanofocusing with a metallic pyramid and an integrated C-shaped aperture.

Solid-state quantum emitters, such as artificially engineered quantum dots or naturally occurring defects in solids, are being investigated for applications ranging from quantum information science and optoelectronics to biomedical imaging. Recently, these same systems have also been studied from the perspective of nanoscale metrology. In this letter, we study the near-field optical properties of a diamond nanocrystal hosting a single nitrogen vacancy center. We find that the nitrogen vacancy center is a sensitive probe of the surrounding electromagnetic mode structure. We exploit this sensitivity to demonstrate nanoscale fluorescence lifetime imaging microscopy (FLIM) with a single nitrogen vacancy center by imaging the local density of states of an optical antenna.

Nanoscale fluorescence lifetime imaging of an optical antenna with a single diamond NV center.

We demonstrate a novel scheme for plasmonic nanofocusing with internally illuminated asymmetric metallic pyramidal tips using linearly polarized light. A wafer-scale array of sharp metallic pyramids is fabricated via template stripping with films of different thicknesses on opposing pyramid facets. This structural asymmetry is achieved through a one-step angled metal deposition that does not require any additional lithography processing and when internally illuminated enables the generation of plasmons using a Kretschmann-like coupling method on only one side of the pyramids. Plasmons traveling toward the tip on one side will converge at the apex, forming a nanoscale "hotspot." The asymmetry is necessary for these focusing effects since symmetric pyramids display destructive plasmon interference at the tip. Computer simulations confirm that internal illumination with linearly polarized light at normal incidence on these asymmetric pyramids will focus optical energy into nanoscale volumes. Far-field optical experiments demonstrate large field enhancements as well as angle-dependent spectral tuning of the reradiated light. Because of the low background light levels, wafer-scale fabrication, and a straightforward excitation scheme, these asymmetric pyramidal tips will find applications in near-field optical microscopy and array-based optical trapping.

Template-stripped asymmetric metallic pyramids for tunable plasmonic nanofocusing.

We present a new plasmonic device architecture based on ultrasmooth metallic surfaces with buried plasmonic nanostructures. Using template-stripping techniques, ultrathin gold films with less than 5 Å surface roughness are optically coupled to an arbitrary arrangement of buried metallic gratings, rings, and nanodots. As a prototypical example, we present linear plasmonic gratings buried under an ultrasmooth 20 nm thick gold surface for biosensing. The optical illumination and collection are completely decoupled from the microfluidic delivery of liquid samples due to the backside, reflection-mode geometry. This allows for sensing with opaque or highly scattering liquids. With the buried nanostructure design, we maintain high sensitivity and decoupled backside (reflective) optical access as with traditional prism-based surface plasmon resonance (SPR) sensors. In addition, we also gain the benefits offered by nanoplasmonic sensors such as spectral tunability and high-resolution, wide-field SPR imaging with normal-incidence epi-illumination that is simple to construct and align. Beyond sensing, our buried plasmonic nanostructures with ultrasmooth metallic surfaces can benefit nanophotonic waveguides, surface-enhanced spectroscopy, nanolithography, and optical trapping.

Ultrasmooth metallic films with buried nanostructures for backside reflection-mode plasmonic biosensing.

In this paper we demonstrate that rigorous high-order perturbation of surfaces (HOPS) methods coupled with analytic continuation mechanisms are particularly well-suited for the assessment and design of nanoscale devices (e.g., biosensors) that operate based on surface plasmon resonances generated through the interaction of light with a periodic (metallic) grating. In this connection we explain that the characteristics of the latter are perfectly aligned with the optimal domain of applicability of HOPS schemes, as these procedures can be shown to be the methods of choice for low to moderate wavelengths of radiation and grating roughness that is representable by a few (e.g., tens of) Fourier coefficients. We argue that, in this context, the method can, for instance, produce full and precise reflectivity maps in computational times that are orders of magnitude faster than those of alternative numerical schemes (e.g., the popular "C-method," finite differences, integral equations or finite elements). In this initial study we concentrate on the description of the basic principles that underlie the solution scheme, including those that relate to analytic continuation procedures. Within this framework, we explain how, in spite of conventional wisdom to the contrary, the resulting perturbative techniques can provide a most valuable tool for practical investigations in plasmonics. We demonstrate this with some examples that have been previously discussed in the literature (including treatments of the reflectivity and band gap structure of some simple geometries) and extend this to demonstrate the wider applicability of the proposed approach.

A fast and high-order accurate surface perturbation method for nanoplasmonic simulations: basic concepts, analytic continuation and applications.

We experimentally demonstrate dielectrophoretic concentration of biological analytes on the surface of a gold nanohole array, which concurrently acts as a nanoplasmonic sensor and gradient force generator. The combination of nanohole-enhanced dielectrophoresis, electroosmosis, and extraordinary optical transmission through the periodic gold nanohole array enables real-time label-free detection of analyte molecules in a 5 μL droplet using concentrations as low as 1 pM within a few minutes, which is more than 1000 times faster than purely diffusion-based binding. The nanohole-based optofluidic platform demonstrated here is straightforward to construct, applicable to both charged and neutral molecules, and performs a novel function that cannot be accomplished using conventional surface plasmon resonance sensors.

Article	Year
Template-stripped smooth Ag nanohole arrays with silica shells for surface plasmon resonance biosensing. ACS nano\| PubMed ID: 21770414	2011
Monolithic integration of continuously tunable plasmonic nanostructures. Nano letters\| PubMed ID: 21834564	2011
Facile assembly of micro- and nanoarrays for sensing with natural cell membranes. ACS nano\| PubMed ID: 21842844	2011
Highly reproducible near-field optical imaging with sub-20-nm resolution based on template-stripped gold pyramids. ACS nano\| PubMed ID: 22938087	2012
High-density arrays of submicron spherical supported lipid bilayers. Analytical chemistry\| PubMed ID: 22967217	2012
Self-assembled plasmonic nanoring cavity arrays for SERS and LSPR biosensing. Advanced materials (Deerfield Beach, Fla.)\| PubMed ID: 23436239	2013
Plasmonic nanofocusing with a metallic pyramid and an integrated C-shaped aperture. Scientific reports\| PubMed ID: 23676841	2013
Nanoscale fluorescence lifetime imaging of an optical antenna with a single diamond NV center. Nano letters\| PubMed ID: 23815462	2013
Template-stripped asymmetric metallic pyramids for tunable plasmonic nanofocusing. Nano letters\| PubMed ID: 24144067	2013
Ultrasmooth metallic films with buried nanostructures for backside reflection-mode plasmonic biosensing. Annalen der Physik\| PubMed ID: 24159227	2012
A fast and high-order accurate surface perturbation method for nanoplasmonic simulations: basic concepts, analytic continuation and applications. Journal of the Optical Society of America. A, Optics, image science, and vision\| PubMed ID: 24322914	2013
Dielectrophoresis-enhanced plasmonic sensing with gold nanohole arrays. Nano letters\| PubMed ID: 24646075	2014

Timothy W. Johnson

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