We would also like to thank Xiaoyu Miao and Ben Wilson for developing the most of the methods described within. This work was funded by National Science Foundation (DBI 0454324) and the National Institute of Health (R21 EB005183) and by PHS NRSA T32 GM07270 from NIGMS to ECK.

1. Random Au Nanoparticle Array Fabrication 8,10,12,14
<ol>
	<li>The Au nanoparticle array is formed by first creating a template that is made of a dense layer of randomly adsorbed latex spheres with mean diameter of 454 nm. This is achieved by first evaporating gold on a glass coverslip to a thickness of 20 nm using chromium as the adhesion layer.</li>
	<li>The polystyrene sphere monolayer is then self-assembled by exposing the gold-coated substrate to a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), latex sphere suspension and de-ionized water.</li>
	<li>The adsorption process is allowed to last for about one hour and the non-absorbed spheres are washed away with a copious amount of water.</li>
	<li>The formed monolayer is allowed to air dry.</li>
	<li>Finally, another 20 nm of gold is evaporated on the latex sphere monolayer to form the random gold nanoparticle array.</li>
	<li>If an SEM is available, the AuNP array can be seen under the SEM to look like Figure 1 and a diagram of the process is shown in figure 8.</li>
</ol>
2. Biological Sample Preparation9,11
<ol>
	<li>Sample preparation for optically trapping mouse cell nuclei is now shown.</li>
	<li>3T3 Mouse cell nuclei tagged with Acridine Orange dye were obtained from the Tewari group at the Fred Hutchinson Cancer Research Center.</li>
	<li>10% Bovine Serum Albumin (BSA) in phosphate buffered saline (PBS) is added to the mouse cell nuclei in a concentration of approximately 1 : 10 (BSA : Mouse Cell Nuclei). The BSA helps to prevent the nuclei from sticking to the substrate.</li>
	<li>Mix the solution using sonication.</li>
	<li>5 uL of our solution is deposited onto the aluminum grating array coverslip. It is better to perform this step with the aluminum grating on the microscope stage so that you do not have to transport the sample after the solution is deposited.</li>
	<li>Two stacks of two 1"by 1" coverslips are used to support a fifth coverslip through which the sample is viewed.</li>
	<li>Position the sample under the microscope for viewing.</li>
</ol>
3. Method for Trapping
<ol>
	<li>The optical tweezers are constructed by sending a 35 mW helium neon laser through a Zeiss Axio Imager.D1M equipped with a GFP 17 filter set which is modified to allow for 633 nm laser radiation to reach the sample.</li>
	<li>A Zeiss LD EC Epiplan - NEOFLUAR 50x objective is used to image the cell nuclei which are approximately 5 micron in diameter.</li>
	<li>After the sample is placed under the objective, focus the microscope on the gold nanoparticle array or diffraction grating.</li>
	<li>Translate microscope vertically until focus is achieved on the nuclei which you desire to trap.</li>
	<li>Position laser trap spot over particle and the particle should then maintain its position in the laser spot even when the stage is translated.</li>
</ol>
4. Representative Results:
The random gold nanoparticle array procedures should deposit a monolayer of AuNP's that can be viewed under an SEM to look like Figure 1. The trapping force created by these plasmonic tweezers can be 10-20 times the force generated by standard optical tweezers. The minimum intensities required by the plasmonic tweezers to achieve particle confinement are shown for particles of various sizes in Figure 4.9,10 The diffraction grating achieved alignment and trapping with 20 times higher trapping efficiency than the gold nanodots and could achieve trapping with as little as 17 uW/um2 (Figure 7).11
<img alt="Figure 1" src="/files/ftp_upload/3390/3390fig1.jpg" /> 
Figure 110 (a) SEM micrograph of the self-assembled gold nanoparticles. The diameter of individual gold nanoparticles is about 450 nm. (b) NSOM image of the plasmonic substrate where the nanoparticle distribution is sparse, showing the near-field radiation. The wavelength of the excitation laser is 633 nm. (c) High magnification view of the area marked with the red square in (b). (d) Scattering efficiency spectrum of the plasmonic substrate, showing the peak at 624 nm. (e) Absorption efficiency spectrum of the plasmonic substrate, showing the peak at 668 nm.
<img alt="Figure 2" src="/files/ftp_upload/3390/3390fig2.jpg" /> 
Figure 213 (a) Au nanospheres randomly distributed on a 2D domain 1 x 1 &mu;m2. Each blue dot represent the center of the nanosphere (a = 60 nm). Scattering field distributions on observation planes which are parallel to the random nanosphere array are shown in (b) - (e). The nanosphere array is uniformly illuminated by a plane wave at the wavelength of 540 nm. The refractive index of the surrounding medium is 1.33. The polarization direction of the plane wave points along the X-axis (horizontal in (a)). The magnitude of the incident electric field is assumed to be 1 in the calculation. The separation between the observation plane and the nanosphere array is defined as h. b) h = a. c) h = 2a. d) h = &lambda;. e) h = 2&lambda;.
<img alt="Figure 3" src="/files/ftp_upload/3390/3390fig3.jpg" /> 
Figure 39 Schematic of customized fluorescence microscope configuration including a bypassed excitation filter and a replaced dichroic beam-splitter. This is the configuration used for simultaneous trapping and fluorescence imaging.
<img alt="Figure 4" src="/files/ftp_upload/3390/3390fig4.jpg" /> 
Figure 410 The minimum laser intensity to maintain the trap as a function of flow rate of surrounding fluid utilizing plasmonic trapping. All the optical intensities are measured at the sample plane under the microscope objective. (a) - (f) show the measurement results for single polystyrene beads with diameter 7.3, 6.3 (non-spherical), 5.0, 3.9, 2.5 and 1.1 &mu;m, respectively. The insets show the corresponding microscopic images of particles. The scale bars in all images represent 5 &mu;m in length.
<img alt="Figure 5" src="/files/ftp_upload/3390/3390fig5.jpg" /> 
Figure 510 The slope of the fitted line through origin in Fig. 4 versus particle size for plasmonic trapping. The error bars show the standard deviations of the linear fits. The slope of the fitted line (ratio between the optical intensity threshold and flow rate) in Fig. 4 has an approximately linear relationship with particle size as shown in this figure, indicating the advantage of plasmonic trapping especially for smaller particles.
<img alt="Figure 6" src="/files/ftp_upload/3390/3390fig6.jpg" /> 
Figure 611 (a) Schematic drawing of the enhanced optical trapping utilizing 1-D periodic nanostructures. The incident beam is diffracted by the periodic nanostructure at far field. (b) The intensity distribution of light with two orthogonal polarizations nanostructure at far field. (b) The intensity distribution of light with two orthogonal polarizations at the surface of an aluminum grating with a 417 nm period obtained using FDTD simulations. The distribution is normalized to the intensity on a flat aluminum surface. (c) and (d) Trapping potential for particles directly above the grating surface versus location of the particle for (c) a 350 nm polystyrene bead and (d) a 1 &mu;m polystyrene bead. The white circles illustrate the sizes of the particles. Insets show the trapping potential above a flat aluminum surface for the same particle size as comparisons. The values are normalized for each particle size. For all FDTD simulation figures the field of view is 10 x 8 &mu;m2.
<img alt="Figure 7" src="/files/ftp_upload/3390/3390fig7.jpg" /> 
Figure 711 (a) Trap efficiency and minimum trapping intensity measured for polystyrene beads of various sizes with beam polarization perpendicular to grating lines. Inset shows trap asymmetry in trapping efficiency for translating a 3.87 um polystyrene bead perpendicular and parallel to the rules of the grating. The solid line (large asymmetry) is obtained with incident light polarized perpendicular to the grating, and the dash line (small asymmetry) is obtained with incident light polarized parallel to the grating. The unit is in (pN[mW/&mu;m2]-1). (b)-(d) Trapping demonstration of a fluorescent 590 nm polystyrene bead. The red circle indicates the position of the laser spot as the laser light was too dim to be seen. At first the particle is trapped within the spot at higher power, as the power is lowered the Brownian motion of the particle overcomes the trapping force, allowing the particle to escape. (e)-(g) Trapping demonstration of a fluorescent ovarian cancer cell nucleus. The minimum intensity required to initiate trapping was 16 &mu;W/&mu;m2 obtained using a 20x objective lens.
<img alt="Figure 8" src="/files/ftp_upload/3390/3390fig8.jpg" /> 
Figure 814 Fabrication procedure of the cap-shaped gold nanoparticles: a) Evaporation of the Cr and Au thin layer on the glass coverslip. b) Exposure to the polystyrene sphere suspension and adsorption of spheres for 1 hour. c) Removal of non-adsorbed polystyrene spheres and drying of the surface. d) Evaporation of another layer of Au on top of the template spheres. e) Schematic of the cap-shaped Au nanoparticle array, where Au only covers the top side of the template spheres.

<ol>
	<li>Jones, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electromechanics of Particles. , (1995).</li><li>Ashkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping+and+manipulation+of+neutral+particles+using+lasers.">Optical trapping and manipulation of neutral particles using lasers.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 4853-4853 (1997).</li><li>Neuman, K. C., Chadd, E. H., Liou, G. F., Bergman, K., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+photodamage+to+Escherichia+coli+in+optical+traps.">Characterization of photodamage to Escherichia coli in optical traps.</a> Biophys. J. 77, 2856-2856 (1999).</li><li>Chiou, P. C., Ohta, A. T., Wu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massively+parallel+manipulation+of+single+cells+and+microparticles+using+optical+images.">Massively parallel manipulation of single cells and microparticles using optical images.</a> Nature. 436, 370-370 (2005).</li><li>Hsu, H. Y., Ohta, A. T., Chiou, P. Y., Jamshidi, A., Nealea, S. L., Wua, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototransistor-based+optoelectronic+tweezers+for+dynamic+cell+manipulation+in+cell+culture+media.">Phototransistor-based optoelectronic tweezers for dynamic cell manipulation in cell culture media.</a> Lab Chip. 10, 165-172 (2010).</li><li>Righini, M., Ghenuche, P. S., Cherukulappurath, V., Myroshnychenko, F. J., Garcia de Abajo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quidant+Nano-optical+Trapping+of+Rayleigh+Particles+Escherichia+coli+Bacteria+with+Resonant+Optical+Antennas.">Quidant Nano-optical Trapping of Rayleigh Particles Escherichia coli Bacteria with Resonant Optical Antennas.</a> Nano Letters. 9, 3387-3391 (2009).</li><li>Righini, M., Zelenina, A. S., Girard, C., Quidant, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+and+Selective+Trapping+in+a+Patterned+Plasmonic+Landscape.">Parallel and Selective Trapping in a Patterned Plasmonic Landscape.</a> Nature Physics. 3, 477-480 (2007).</li><li>Miao, X., Lin, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+dielectrophoresis+force+and+torque+induced+by+localized+surface+plasmon+resonance+of+a+cap-shaped+Au+nanoparticle+array.">Large dielectrophoresis force and torque induced by localized surface plasmon resonance of a cap-shaped Au nanoparticle array.</a> Opt. Lett. 32, 295-297 (2007).</li><li>Wilson, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulation of Nanoparticles and Biological Samples through Enhanced Optical Forces [dissertation]. , (2009).</li><li>Miao, X. Y., Wilson, B. K., Pun, S. H., Lin, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+manipulation+of+micron/submicron+sized+particles+and+biomolecules+through+plasmonics.">Optical manipulation of micron/submicron sized particles and biomolecules through plasmonics.</a> Optics Exp. 16, 13517-13525 (2008).</li><li>Wilson, B. K., Mentele, T., Bachar, S., Knouf, E., Bendoraite, A., Tewari, M., Pun, S. H., Lin, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructure-enhanced+laser+tweezers+for+efficient+trapping+and+alignment+of+particles.">Nanostructure-enhanced laser tweezers for efficient trapping and alignment of particles.</a> Optics. Exp. 18, 16005-16013 (2010).</li><li>Miao, X., Wilson, B. K., Cao, G., Pun, S. H., Lin, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+and+Rotation+of+Nanowires+Assisted+by+Surface+Plasmons.">Trapping and Rotation of Nanowires Assisted by Surface Plasmons.</a> IEEE Journal of Selected Topics in Quantum Electronics. 15, 1515-1520 (2009).</li><li>Miao, X. Y., Lin, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+and+manipulation+of+biological+particles+through+a+plasmonic+platform.">Trapping and manipulation of biological particles through a plasmonic platform.</a> IEEE Journal of Selected Topics in Quantum Electronics. 13, 1655-1662 (2007).</li><li>Miao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plasmonics for Micro/Nano Manipulation and Optofluidics [dissertation]. , (2008).</li></ol>

No conflicts of interest declared.

The significance of these methods of trapping is that they decrease the optical intensity necessary for sustained trapping from somewhere on the order of 103 &mu;W/&mu;m2 to somewhere on the order of 10 &mu;W/&mu;m2.10,11 The limitations on these techniques are that the gold nanoparticle array experiences heating issues that must be overcome. To overcome this issue, a 2D photonic crystal structure that is composed of a dielectric material can be used. Such a structure could the...

<table border="1">
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalog Number</td>
<td>Comment</td>
</tr>
<tr>
<td>Axio Imager Microscope</td>
<td>D1M</td>
<td>Zeiss</td>
<td>D1M</td>
<td>Zeiss Axio Imager.D1M</td>
</tr>
<tr>
<td>Microscope Objective</td>
<td>50x/0.55</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>LD EC Epiplan - NEOFLUAR 50x/0.55 HD DIC</td>
</tr>
<tr>
<td>Zeiss Microscope Camera</td>
<td>AxioCam MRc</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Helium Neon Laser</td>
<td>35 mW</td>
<td>Research Electro-Optics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Variable Attenuator</td>
<td>Continuously Variable ND</td>
<td>ThorLabs</td>
<td>NDC-100C-4M</td>
<td>For adjusting microscope intensity</td>
</tr>
<tr>
<td>Zeiss Filter Set</td>
<td>Filter Set #17</td>
<td>Zeiss</td>
<td>488017-9901-000</td>
<td>Filter Set #17</td>
</tr>
<tr>
<td>Microscope Slides</td>
<td>0.5 mm thickness</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>3T3 mouse cell nuclei</td>
<td>&nbsp;</td>
<td colspan="2">Fred Hutchinson Cancer Research Center</td>
<td>Store as cold as possible</td>
</tr>
<tr>
<td>Acridine Orange dye</td>
<td>&nbsp;</td>
<td colspan="2">Fred Hutchinson Cancer Research Center</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Bovine Serum Albumin</td>
<td>1 to 10 ration in PBS</td>
<td colspan="2">Fred Hutchinson Cancer Research Center</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>454 nm polystyrene latex spheres</td>
<td>&nbsp;</td>
<td> Polysciences, Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>carbodiimide hydrochloride (EDC)</td>
<td>1-ethyl-3-(3-dimethylaminopropyl)</td>
<td>G-Biosciences</td>
<td>BC25-1</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>gold (for deposition)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Reflective ruled diffraction grating</td>
<td>&nbsp;</td>
<td>Edmund Optics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Phosphate Buffered Saline (PBS)</td>
<td>Dulbecco&#x2019;s Phosphate-Buffered Saline (D-PBS) (1X)</td>
<td>Invitrogen</td>
<td>14190-144</td>
<td>&nbsp;</td>
</tr>
</table>

utilization of plasmonic and photonic crystal nanostructures for enhanced micro- and nanoparticle manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Watch this Scientific Journal Video about Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation at JoVE.com

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

utilization-plasmonic-photonic-crystal-nanostructures-for-enhanced

Research

JoVE Journal

Bioengineering

12.2K Views.  University of Washington. Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

Video: Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs &#8212; polysiloxane-based and epoxy-based &#8212; are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 &#176;C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...