O. Kurtulus and D. Dimlioglu are supported by a University of California Laboratory Fees Research Program Award 12-LR-237197. P. Daggumati is supported by a University of California Davis Research Investments in the Sciences &amp; Engineering (RISE) Award. C.A. Chapman is supported by a Department of Education Graduate Assistance Areas of National Need Fellowship. This work was supported by UC Lab Fees Research Program, UC Davis RISE, and UC Davis College of Engineering start-up funds.

1. Nanoporous Gold Fabrication
<ol>
	<li>Clean substrates in Piranha solution
	<ol>
		<li>Add 25 ml hydrogen peroxide (30%) to 100 ml sulfuric acid (96%) in a crystallization dish and heat the mixture to 65 &deg;C on a hotplate. CAUTION: The liquids are extremely corrosive and must be handled with care. The spent solution should not be stored in a sealed container, as it may explode.</li>
		<li>Place 1-inch by 3-inch microscope slides into the mixture using acid-resistant forceps and clean them for 10 min. Use a porcelain immunostaining boat for batch-cleaning small coverslips. Treat small coverslips with air plasma at 10 W for 30 sec prior to immersion into the solution.</li>
		<li>Rinse the cleaned samples in crystallization dishes under running deionized (DI) water for 3 min. Blow-dry samples with a nitrogen gun over lint-free towels.</li>
	</ol>
	</li>
	<li>Prepare stencil mask (Method 1: Use this for creating millimeter-scale patterns)
	<ol>
		<li>Punch 250 &mu;m-thick silicone elastomer sheets with biopsy punches and/or incise out regions with a scalpel over a semi-hard plastic surface. Clean the processed elastomer sheets in 70% isopropanol and dry with nitrogen gun.</li>
		<li>Place the punched sheet on a lint-free cleanroom towel and align the piranha-cleaned coverslips over the stencil with the sample surface facing the stencil.</li>
	</ol>
	</li>
	<li>Pattern lift-off photoresist (Method 2: Use this for creating sub-millimeter-scale patterns)
	<ol>
		<li>Place a piranha-cleaned microscope slide on spinning chuck and blow off any particulates with nitrogen gun. Dispense 1.5 ml of adhesion promoter (hexamethyldisilazane) onto the glass slide using a plastic pipette. Spread the promoter by spinning the slide successively at 500 rpm for 5 sec and 1,500 rpm for 30 sec. Bake the slide on a hotplate at 115 &deg;C for 7 min and let it cool for 5 min.</li>
		<li>Dispense 4 ml of photoresist onto the glass slide (3-inch by 1-inch). Spread the photoresist by spinning the slide with the same protocol as for the adhesion promoter. Bake the photoresist on a hotplate at 115 &deg;C for 1.5 min and let it cool for 10 min.</li>
		<li>Expose the photoresist-coated slide with UV light (intensity: 22 mW/cm2) through a transparency mask for 15 sec. Bake the photoresist on a hotplate at 115 &deg;C for 1.5 min and wait for 45 min. Dissolve the exposed photoresist in the developer for at least 3.5 min. Rinse thoroughly with DI water. Inspect the developed patterns under an optical microscope.</li>
	</ol>
	</li>
	<li>Deposit precursor metals to produce np-Au thin films
	<ol>
		<li>Load the samples into a sputtering machine that can independently deposit gold, silver, and chrome. Sputter-clean the samples for 90 sec at 50 W under 25 mTorr argon processing atmosphere before starting metal deposition.</li>
		<li>Sputter chrome for 10 min at 300 W under 10 mTorr argon. Sputter gold for 90 sec at 400 W under 10 mTorr argon. Co-sputter gold and silver for 10 min with silver at 200 W and Au power at 100 W. Turn off gold sputter source approximately 10 sec before turning off the silver sputter source.</li>
	</ol>
	</li>
	<li>Obtain precursor metal micropatterns
	<ol>
		<li>Sonicate photoresist-coated samples in ~180 ml of photoresist stripper for 10 cycles of 20 sec sonication and 2 min pause between the cycles. Rinse the samples with DI water and dry with a nitrogen gun. Inspect the metal patterns under a microscope.</li>
		<li>Peel the elastomer stencil from non-photoresist coated samples using two tweezers to reveal the deposited metal.</li>
	</ol>
	</li>
	<li>Dealloy precursor metal and modify nanostructure via thermal treatment
	<ol>
		<li>Fill a 200 ml glass beaker with 170 ml of nitric acid (70%) and maintain the solution temperature at 55 &deg;C on a hotplate. CAUTION: The nitric acid is extremely corrosive and should be handled with appropriate protective equipment.</li>
		<li>Treat small coverslips with air plasma at 10 W for 30 sec prior to immersion into nitric acid. Place 1-inch by 3-inch microscope slides into the beaker using acid-resistant forceps and dealloy them for 15 min. Use a porcelain immunostaining boat for batch-dealloying small coverslips.</li>
		<li>Rinse the dealloyed samples by successively immersing them in two beakers filled with fresh DI water three times. Store samples in DI water and replace the water with fresh DI water every day for at least a week. Blow-dry samples with a nitrogen gun over lint-free towels before use.</li>
		<li>Load samples onto a clean silicon wafer in a rapid thermal processing equipment. Adjust the temperature to between 200 &deg;C and 450 &deg;C and the ramp rate to 10 &deg;C/sec. Expose the samples to the prescribed temperature for 10 min under nitrogen ambient. Let the chamber cool (&lt;100 &deg;C) and remove the samples. Alternatively, slowly place the samples on the hotplate for thermal treatment.</li>
	</ol>
	</li>
</ol>
2. Cell Culture
<ol>
	<li>Prepare np-Au samples for cell culture
	<ol>
		<li>Place np-Au samples in polystyrene dishes and treat with air plasma at 10 W for 30 sec and transfer the samples to 24-well tissue culture plates.</li>
		<li>Add 500 &mu;l complete culture media (Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and 1% penicillin/streptomycin) to each well. Store in a humidified incubator at 37 &deg;C and 5% CO2 until seeding the cells (&lt;1 hr).</li>
	</ol>
	</li>
	<li>Maintain, passage, and seed cells
	<ol>
		<li>Maintain cells of interest (in this case 3T3-NIH fibroblasts or murine astrocytes) in T75 flasks with culture media and passage when cells are 70% confluent.</li>
		<li>For passaging the cells, remove media from the flask, wash twice with 10 ml phosphate buffered saline (PBS), add 2 ml of 1x Trypsin/EDTA and incubate until the cells detach (~5 min). Add 3 ml fresh media and centrifuge at 1,200 rpm for 3 min. Aspirate supernatant and suspend the pellet in 2 ml media.</li>
		<li>Remove the spent media from the wells and seed the cells onto the glass coverslips at a density of 25,000 cells/cm2 with a final volume of 1 ml. Shake the culture plate forward-back and right-left to ensure a uniform coating of cells over the samples. Incubate the cells until analysis while inspecting daily.</li>
	</ol>
	</li>
</ol>
3. Cell and Material Analysis
<ol>
	<li>Stain cells to visualize cytoskeleton and nuclei
	<ol>
		<li>Remove spent media from wells and wash cells twice with PBS. Fix cells in 4% paraformaldehyde in PBS for 15 min.</li>
		<li>Prepare staining solution of 300 nM Alexa Fluor 488 phalloidin in PBS with 1% bovine serum albumin.</li>
		<li>Wash cells twice with PBS, and permeabilize them in 500 &mu;l 0.1% Triton X-100 in PBS for 5 min.</li>
		<li>Wash cells twice with PBS and transfer them to clean wells. Blot 50 - 200 &mu;l staining solution onto the samples and store in dark for 20 min.</li>
		<li>Wash cells with PBS and counter-stain with 3 nM of DAPI in PBS for 5 min.</li>
		<li>Wash cells with PBS, and dip in deionized water before mounting on glass cover slides with mounting media and seal with clear nail polish.</li>
	</ol>
	</li>
	<li>Acquire images of np-Au samples and cell cultures on np-Au surfaces
	<ol>
		<li>Image np-Au surfaces with scanning electron microscope (SEM) with 50,000X magnification at 10 kV electron energy using secondary electron detector.</li>
		<li>Capture composite cell images at different spots on the samples using an inverted fluorescence microscope at 10X magnification with the appropriate filter cubes.</li>
	</ol>
	</li>
	<li>Process images to determine pore and cell morphology
	<ol>
		<li>Open images in ImageJ and split into individual channels if applicable. Convert the images to 8-bit, subtract the background, and smooth them by median filtering. Adjust threshold either manually or by built-in thresholding algorithms to highlight the pores (voids) and cell bodies/nuclei.</li>
		<li>Use watershed command to separate merged pores or cells. Set particle analysis parameters and execute the command to extract number of particles, average area, and percent coverage by particles. Use DAPI-stained cell images for cell counting and phalloidin-stained cell images for quantifying percent cell coverage.</li>
		<li>Modify the included macro files to perform batch analysis of multiple images.</li>
	</ol>
	</li>
</ol>

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Authors have no conflicting financial interest.

We demonstrate two different techniques to micropattern np-Au films for expanding use of these films in microsystems and biological studies. Sputter-coating gold and silver is a versatile method to create np-Au patterns, as sputtering is compatible with conventional microfabrication processes and the alloy composition and thickness can be easily controlled by varying the individual sputtering gun powers (for gold and silver targets) and the deposition time respectively. Typical np-Au film thicknesses range from 200 nm to...

Materials with nano-scale features have shown promise in enhancing various applications, including fuel cells1, sensors2,3, and biomedical devices4,5. A relatively new material is nanoporous gold (np-Au), which is produced by a nano-scale self-assembly process. The precursor to np-Au is a gold alloy that most commonly consists of silver at 60% to 80% by atomic percentage. Briefly, the characteristic open-pore nanostructure is the result of rearrangement of gold atoms in clusters as silver is dissolved by a strong acid (e.g. nitric acid 70%) or under an electrochemical potential6-8. Np-Au benefits from several desirable attributes, including large effective surface area, high electrical conductivity, well-established surface functionalization techniques, and biocompatibility9. Even though there has been a rapid expansion of studies on np-Au, most of them focus on np-Au's mechanical properties10,11, catalytic activity12, and biomolecular sensing performance13-15. While the desirable attributes are highly useful for several biomedical tools16,17, the applications in this area have been limited. One possible reason for this is that most studies have predominantly used macro-scale specimens (e.g. sheets, foils, and ingots) and the techniques for incorporating np-Au in miniaturized systems have remained inadequate. In fact there are only a handful of examples of using conventional microfabrication techniques that employ np-Au films16-20. With the advent of miniaturization technology and the need for novel biomedical tools, it has become crucial to be able to integrate new materials into devices. This typically requires that the materials can be deposited and patterned with conventional microfabrication techniques. In addition, rapid quantification of cell-material interactions is commonly necessary to evaluate the biocompatibility of a new material. The goal of this paper is to demonstrate basic techniques to micropattern np-Au films and quantify both nanostructure and cell-material interactions via digital image processing.

<table><tbody><tr><td>Gold target</td><td>Lesker</td><td>EJTAUXX403A2</td><td>Precursor to alloy for producing np-Au</td></tr><tr><td>Chrome target</td><td>Lesker</td><td>EJTCRXX353A2</td><td>Adhesive layer</td></tr><tr><td>Silver target</td><td>Lesker</td><td>EJTAGXX403A2</td><td>Precursor to alloy for producing np-Au</td></tr><tr><td>Porcelain boat</td><td>Thomas Scientific</td><td>8542E40</td><td>Used for processing small samples</td></tr><tr><td>Nitric acid</td><td>Sigma-Aldrich</td><td>43873</td><td>Used at 70% for dealloying</td></tr><tr><td>Sulfuric acid</td><td>J.T Baker</td><td>7664-93-9</td><td>Used at 96% for piranha cleaning</td></tr><tr><td>Hydrogen peroxide</td><td>J.T Baker</td><td>7722-84-1</td><td>Used at 30% for piranha cleaning</td></tr><tr><td>Biopsy punches</td><td>Ted Pella</td><td>150xx</td><td>Available in several sizes</td></tr><tr><td>Silicone elastomer sheets</td><td>Rogers Corporation</td><td>HT 6240</td><td>Available in several thicknesses</td></tr><tr><td>Hexamethyldisilazane</td><td>Sigma-Aldrich</td><td>440191-100ML</td><td>Used as adhesion promoter for positive resist</td></tr><tr><td>Microposit MF CD26</td><td>Shipley</td><td>38490</td><td>Positive photoresist developer</td></tr><tr><td>PRS 3000</td><td>J.T Baker</td><td>JT6403-5</td><td>Positive photoresist stripper</td></tr><tr><td>Circular glass coverslips (12 mm)</td><td>Ted Pella</td><td>26023</td><td>Used as substrate for metal patterns and cell culture</td></tr><tr><td>Glass slides (1 x 3 inch)</td><td>Ted Pella</td><td>26007</td><td>Used as substrate for metal patterns</td></tr><tr><td>Kapton polyimide tape</td><td>VWR</td><td>82030-950</td><td>Used for securing elastomer</td></tr><tr><td>Transparency masks</td><td>Output City</td><td></td><td>Used in photolithography &lt;a href=&quot;http://www.outputcity.com/&quot; target=&quot;_blank&quot;&gt;http://www.outputcity.com/&lt;/a&gt;</td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-32G</td><td>Used for activating glass surfaces</td></tr><tr><td>Sputtering machine</td><td>Kurt J. Lesker</td><td>LAB18</td><td>Used for depositing metals</td></tr></tbody></table>

microfabrication of nanoporous gold patterns for cell-material interaction studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

Figure 1 outlines the major procedural steps, including creating the np-Au patterns, culturing cells, quantifying the nanostructure, and characterizing cell morphologies. The elastomer stencil shown in Figure 2a (top) is used for creating the np-Au patterns shown in the images underneath. Figure 2b is a photograph of the porcelain boat for batch processing specimens. Figure 2c displays the color change of the deposited metal patterns before and afte...

Watch this Scientific Journal Video about Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies at JoVE.com

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

microfabrication-nanoporous-gold-patterns-for-cell-material

Research

JoVE Journal

Bioengineering

11.3K Views.  University of California, Davis. We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

Video: Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Optical Trapping of Nanoparticles

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping and manipulating small biological particles. Ashkin and co-workers first demonstrated optical tweezers using a single focused beam1. The single beam trap can be described accurately using the perturbative gradient force formulation in the case of small Rayleigh regime particles1. In the perturbative regime, the optical power required for trapping a particle scales as the inverse fourth power of the particle size. High optical powers can damage dielectric particles and cause heating. For instance, trapped latex spheres of 109 nm in diameter were destroyed by a 15 mW beam in 25 sec1, which has serious implications for biological matter2,3.
A self-induced back-action (SIBA) optical trapping was proposed to trap 50 nm polystyrene spheres in the non-perturbative regime4. In a non-perturbative regime, even a small particle with little permittivity contrast to the background can influence significantly the ambient electromagnetic field and induce a large optical force. As a particle enters an illuminated aperture, light transmission increases dramatically because of dielectric loading. If the particle attempts to leave the aperture, decreased transmission causes a change in momentum outwards from the hole and, by Newton's Third Law, results in a force on the particle inwards into the hole, trapping the particle. The light transmission can be monitored; hence, the trap can become a sensor. The SIBA trapping technique can be further improved by using a double-nanohole structure.
The double-nanohole structure has been shown to give a strong local field enhancement5,6. Between the two sharp tips of the double-nanohole, a small particle can cause a large change in optical transmission, thereby inducing a large optical force. As a result, smaller nanoparticles can be trapped, such as 12 nm silicate spheres7 and 3.4 nm hydrodynamic radius bovine serum albumin proteins8. In this work, the experimental configuration used for nanoparticle trapping is outlined. First, we detail the assembly of the trapping setup which is based on a Thorlabs Optical Tweezer Kit. Next, we explain the nanofabrication procedure of the double-nanohole in a metal film, the fabrication of the microfluidic chamber and the sample preparation. Finally, we detail the data acquisition procedure and provide typical results for trapping 20 nm polystyrene nanospheres.

The following setup approach details low power optical trapping of dielectric nanoparticles using a double-nanohole in metal film.

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping ...

Engineering

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Plasmonic Trapping and Release of Nanoparticles in a Monitoring Environment

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few can observe immobilized particles. Moreover, a limited number of studies have experimentally measured the exertable forces on the particles. The designs can be classified as the protruding nanodisk type or the suppressed nanohole type. For the latter, microscopic observation is extremely challenging. In this paper, a new plasmonic tweezer system is introduced to monitor particles, both in directions parallel and orthogonal to the symmetric axis of a plasmonic nanohole structure. This feature enables us to observe the movement of each particle near the rim of the nanohole. Furthermore, we can quantitatively estimate the maximal trapping forces using a new fluidic channel.

A microchip fabrication process that incorporates plasmonic tweezers is presented here. The microchip enables the imaging of a trapped particle to measure maximal trapping forces.

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few ...

Fabrication of Periodic Gold Nanocup Arrays Using Colloidal Lithography

Within recent years, the field of plasmonics has exploded as researchers have demonstrated exciting applications related to chemical and optical sensing in combination with new nanofabrication techniques. A plasmon is a quantum of charge density oscillation that lends nanoscale metals such as gold and silver unique optical properties. In particular, gold and silver nanoparticles exhibit localized surface plasmon resonances-collective charge density oscillations on the surface of the nanoparticle-in the visible spectrum. Here, we focus on the fabrication of periodic arrays of anisotropic plasmonic nanostructures. These half-shell (or nanocup) structures can exhibit additional unique light-bending and polarization-dependent optical properties that simple isotropic nanostructures cannot. Researchers are interested in the fabrication of periodic arrays of nanocups for a wide variety of applications such as low-cost optical devices, surface-enhanced Raman scattering, and tamper indication. We present a scalable technique based on colloidal lithography in which it is possible to easily fabricate large periodic arrays of nanocups using spin-coating and self-assembled commercially available polymeric nanospheres. Electron microscopy and optical spectroscopy from the visible to near-infrared (near-IR) was performed to confirm successful nanocup fabrication. We conclude with a demonstration of the transfer of nanocups to a flexible, conformal adhesive film.

We demonstrate the fabrication of periodic gold nanocup arrays using colloidal lithographic techniques and discuss the importance of nanoplasmonic films.

Within recent years, the field of plasmonics has exploded as researchers have demonstrated exciting applications related to chemical and optical sensing ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Large Area Substrate-Based Nanofabrication of Controllable and Customizable Gold Nanoparticles Via Capped Dewetting

Recent scientific advances in the utilization of metallic nanoparticle for enhanced energy conversion efficiency, improved optical device performance, and high-density data storage have demonstrated the potential benefit of their use in industrial applications. These applications require precise control over nanoparticle size, spacing, and sometimes shape. These requirements have resulted in the use of time and cost intensive processing steps to produce nanoparticles, thus making the transition to industrial application unrealistic. This protocol will resolve this issue by providing a scalable and affordable method for the large-area production of nanoparticle films with improved nanoparticle control compared to the current techniques. In this article, the process will be demonstrated with gold, but other metals can also be used.

This protocol details a novel nano-manufacturing technique that can be used to make controllable and customizable nanoparticle films over large areas based on the self-assembly of dewetting of capped metal films.

Recent scientific advances in the utilization of metallic nanoparticle for enhanced energy conversion efficiency, improved optical device performance, and ...

Versatile Technique to Produce a Hierarchical Design in Nanoporous Gold

The potential to generate variable pore sizes, simplistic surface modification, and a breadth of commercial uses in the fields of biosensors, actuators, drug loading and release, and the development of catalysts have unquestionably accelerated the usage of nanoporous gold (NPG)-based nanomaterials in research and development. This article describes the process of the generation of hierarchical bimodal nanoporous gold (hb-NPG) by employing a step-wise procedure involving electrochemical alloying, chemical dealloying techniques, and annealing to create both macro- and mesopores. This is done to improve the utility of NPG by creating a bicontinuous solid/void morphology. The area available for surface modification is enhanced by smaller pores, while molecular transport benefits from the network of larger pores. The bimodal architecture, which is the result of a series of fabrication steps, is visualized using scanning electron microscopy (SEM) as a network of pores that are less than 100 nm in size and connected by ligaments to larger pores that are several hundred nanometers in size. The electrochemically active surface area of the hb-NPG is assessed using cyclic voltammetry (CV), with a focus on the critical roles that both dealloying and annealing play in creating the necessary structure. The adsorption of different proteins is measured by solution depletion technique, revealing the better performance of hb-NPG in terms of protein loading. By changing the surface area to volume ratio, the created hb-NPG electrode offers tremendous potential for biosensor development. The manuscript discusses a scalable method to create hb-NPG surface structures, as they offer a large surface area for the immobilization of small molecules and improved transport pathways for faster reactions.

Nanoporous gold with a hierarchical and bimodal pore size distribution can be produced by combining electrochemical and chemical dealloying. The composition of the alloy can be monitored via EDS-SEM examination as the dealloying process advances. The material's loading capacity can be determined by studying protein adsorption onto the material.

The potential to generate variable pore sizes, simplistic surface modification, and a breadth of commercial uses in the fields of biosensors, actuators, ...

Chemistry

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular reactions. However, it is difficult to determine the relation between the size of a nanostructure and the responses of cells owing to the lack of proper screening tools. Here, we show the development of reproducible and cost-effective gradient nanopattern plates for the manipulation of cellular responses. Using anodic aluminum oxide (AAO) as a master mold, gradient nanopattern plates with nanopillars of increasing diameter ranges [120-200&#8239;nm (GP 120/200), 200-280&#8239;nm (GP 200/280), and 280-360&#8239;nm (GP 280/360)] were fabricated by a thermal imprinting technique. These gradient nanopattern plates were designed to mimic the various sizes of nanotopography in the ECM and were used to screen the responses of human endothelial colony-forming cells (hECFCs). In this protocol, we describe the step-by-step procedure of fabricating gradient nanopattern plates for cell engineering, techniques of cultivating hECFCs from human peripheral blood, and culturing hECFCs on nanopattern plates.

Here, we present a protocol for the fabrication of gradient nanopattern plates via thermal nanoimprinting and the method of screening responses of human endothelial progenitor cells to the nanostructures. By using the described technology, it is possible to produce a scaffold that can manipulate cell behavior by physical stimuli.

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

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Rozdziały w tym wideo

Optical Trapping of Nanoparticles

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates

Plasmonic Trapping and Release of Nanoparticles in a Monitoring Environment

Fabrication of Periodic Gold Nanocup Arrays Using Colloidal Lithography

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Large Area Substrate-Based Nanofabrication of Controllable and Customizable Gold Nanoparticles Via Capped Dewetting

Versatile Technique to Produce a Hierarchical Design in Nanoporous Gold

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Optical Trapping of Nanoparticles

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Cell Patterning on Photolithographically Defined Parylene-C: SiO2 Substrates

Plasmonic Trapping and Release of Nanoparticles in a Monitoring Environment

Fabrication of Periodic Gold Nanocup Arrays Using Colloidal Lithography

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Large Area Substrate-Based Nanofabrication of Controllable and Customizable Gold Nanoparticles Via Capped Dewetting

Versatile Technique to Produce a Hierarchical Design in Nanoporous Gold

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates