Name: engineering molecular recognition with bio-mimetic polymers on single walled carbon nanotubes
Uploaded: 2017-01-10T14:00:00
Description: Watch this Scientific Journal Video about Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes at JoVE.com

This work was supported by Burroughs Wellcome Fund Career Award at the Scientific Interface (CASI), a Simons Foundation grant, and a Brain and Behavior Research foundation young investigator grant.

Caution: Please consult all relevant material safety data sheets (SDS) before use. Nanomaterials may have additional hazards compared to their bulk material counterpart. Use all appropriate safety practices including engineering controls (fume hood, noise enclosure) and personal protective equipment (safety glasses, goggles, lab coat, full length pants, closed-toe shoes).
1. Preparation of Buffer, Surfactant, and Polymer Solutions
<ol>
	<li>Preparation of 100 mM NaCl solution</strong...

<ol>
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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individually+Suspended+Single-Walled+Carbon+Nanotubes+in+Various+Surfactants.">Individually Suspended Single-Walled Carbon Nanotubes in Various Surfactants.</a> Nano Letters. 3 (10), 1379-1382 (2003).</li><li>Tu, X., Zheng, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA-based+approach+to+the+carbon+nanotube+sorting+problem.">A DNA-based approach to the carbon nanotube sorting problem.</a> Nano Research. 1 (3), 185-194 (2008).</li><li>Mangalum, A., Rahman, M., Norton, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-Specific+Immobilization+of+Single-Walled+Carbon+Nanotubes+onto+Single+and+One-Dimensional+DNA+Origami.">Site-Specific Immobilization of Single-Walled Carbon Nanotubes onto Single and One-Dimensional DNA Origami.</a> J Am Chem Soc. 135 (7), 2451-2454 (2013).</li><li>Monopoli, M. P., &#197;berg, C., Salvati, A., Dawson, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomolecular+coronas+provide+the+biological+identity+of+nanosized+materials.">Biomolecular coronas provide the biological identity of nanosized materials.</a> Nature Nano. 7 (12), 779-786 (2012).</li><li>Jeng, E. S., Moll, A. E., Roy, A. C., Gastala, J. B., Strano, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+DNA+Hybridization+Using+the+Near-Infrared+Band-Gap+Fluorescence+of+Single-Walled+Carbon+Nanotubes.">Detection of DNA Hybridization Using the Near-Infrared Band-Gap Fluorescence of Single-Walled Carbon Nanotubes.</a> Nano Letters. 6 (3), 371-375 (2006).</li><li>Yang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+Nanotube-Quenched+Fluorescent+Oligonucleotides:+Probes+that+Fluoresce+upon+Hybridization.">Carbon Nanotube-Quenched Fluorescent Oligonucleotides: Probes that Fluoresce upon Hybridization.</a> J Am Chem Soc. 130 (26), 8351-8358 (2008).</li><li>Yum, K., Ahn, J., McNicholas, T., Barone, P., Mu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boronic+acid+library+for+selective,+reversible+near-infrared+fluorescence+quenching+of+surfactant+suspended+single-walled+carbon+nanotubes+in+response+to+glucose.">Boronic acid library for selective, reversible near-infrared fluorescence quenching of surfactant suspended single-walled carbon nanotubes in response to glucose.</a> Acs Nano. 6 (1), 819-830 (2011).</li><li>Kim, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Luciferase/Single-Walled+Carbon+Nanotube+Conjugate+for+Near-Infrared+Fluorescent+Detection+of+Cellular+ATP.">A Luciferase/Single-Walled Carbon Nanotube Conjugate for Near-Infrared Fluorescent Detection of Cellular ATP.</a> Ange Chem Int Ed. 49 (8), 1456-1459 (2010).</li><li>Jin, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+single-molecule+H2O2+signalling+from+epidermal+growth+factor+receptor+using+fluorescent+single-walled+carbon+nanotubes.">Detection of single-molecule H2O2 signalling from epidermal growth factor receptor using fluorescent single-walled carbon nanotubes.</a> Nat Nano. 5 (4), 302-309 (2010).</li><li>Kim, J. 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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugated+Polymer-Assisted+Dispersion+of+Single-Wall+Carbon+Nanotubes:+The+Power+of+Polymer+Wrapping.">Conjugated Polymer-Assisted Dispersion of Single-Wall Carbon Nanotubes: The Power of Polymer Wrapping.</a> Acc Chem Res. 47 (8), 2446-2456 (2014).</li><li>Zou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispersion+of+Pristine+Carbon+Nanotubes+Using+Conjugated+Block+Copolymers.">Dispersion of Pristine Carbon Nanotubes Using Conjugated Block Copolymers.</a> Adv Mat. 20 (11), 2055-2060 (2008).</li><li>Zheng, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-Based+Carbon+Nanotube+Sorting+by+Sequence-Dependent+DNA+Assembly.">Structure-Based Carbon Nanotube Sorting by Sequence-Dependent DNA Assembly.</a> Science. 302 (5650), (2003).</li><li>Strano, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+Nature+of+the+DNA-Assisted+Separation+of+Single-Walled+Carbon+Nanotubes+Using+Fluorescence+and+Raman+Spectroscopy.">Understanding the Nature of the DNA-Assisted Separation of Single-Walled Carbon Nanotubes Using Fluorescence and Raman Spectroscopy.</a> Nano Lett. 4 (4), 543-550 (2004).</li><li>Bisker, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-targeted+corona+phase+molecular+recognition.">Protein-targeted corona phase molecular recognition.</a> Nat Comm. 7, 10241 (2016).</li><li>Nelson, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Immobilized+Protein+A+Binding+to+Immunoglobulin+G+on+Nanosensor+Array+Surfaces.">Mechanism of Immobilized Protein A Binding to Immunoglobulin G on Nanosensor Array Surfaces.</a> Anal Chem. 87 (16), 8186-8193 (2015).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+recognition+using+corona+phase+complexes+made+of+synthetic+polymers+adsorbed+on+carbon+nanotubes.">Molecular recognition using corona phase complexes made of synthetic polymers adsorbed on carbon nanotubes.</a> Nat nanotechnol. 8 (12), 959-968 (2013).</li><li>Kruss, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotransmitter+detection+using+corona+phase+molecular+recognition+on+fluorescent+single-walled+carbon+nanotube+sensors.">Neurotransmitter detection using corona phase molecular recognition on fluorescent single-walled carbon nanotube sensors.</a> J Am Chem Soc. 136 (2), 713-724 (2014).</li><li>Salem, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chirality+dependent+corona+phase+molecular+recognition+of+DNA-wrapped+carbon+nanotubes.">Chirality dependent corona phase molecular recognition of DNA-wrapped carbon nanotubes.</a> Carbon. 97, 147-153 (2016).</li><li>Giraldo, J. 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SWNTs are readily suspended in aqueous solution via direct sonication with SDS or ssDNA, as indicated by an increase in optical density provided by the colloidal dispersion of the resulting SWNT-polymer hybrid. SDS and ssDNA disperses and solubilizes bundles of SWNTs by adsorbing onto the SWNT surface through hydrophobic or pi-pi interactions. Additionally, other polymers, such as genomic DNA, amphiphilic polymers, conjugated polymers and lipids, can be adsorbed onto the surface of SWNTs by dialysis of samples suspended ...

Single-walled carbon nanotubes (SWNTs) are atomically thin layers of carbon atoms rolled into long, thin cylinders that exhibit unique electronic and optical properties.1 Such properties include a band-gap producing near infrared (nIR) fluorescence emission via exciton recombination that is highly sensitive to its local environment. The nIR emission of SWNTs falls within the near infrared window in which the penetration depth of light is maximal for biological tissue.2,3 Additionally, SWNTs exhibit several unique features atypical in contrast to organic fluorophores: SWNT exhibit a large Stokes shift, do not photobleach, and do not blink.4 Recently, exploiting these characteristics has led to the development of an assortment of novel molecular sensors with applications to biology.5,6 Unmodified, however, SWNTs are insoluble in water, and obtaining suspensions of individual SWNTs can be a challenge.7,8 Bundling and aggregation of SWNTs in solution can obfuscate their band-gap fluorescence,2 rendering them unsuitable for sensing applications.
Dispersing individual carbon nanotubes in aqueous solution requires modifying their surface to prevent hydrophobicity-driven aggregation.9 While covalent modification can render SWNTs water-soluble,10 as well as impart specific binding chemistry, defect sites in the SWNT lattice reduce or abate their fluorescence emission. Instead, SWNT functionalization can be accomplished by using surfactants, lipids, polymers and DNA9,11-13 that adsorb to the nanotube surface through hydrophobic and pi-pi stacking interactions. The resulting chemical environment surrounding surface-functionalized SWNTs is referred to as its corona phase. Perturbations to the corona phase can have a large impact on excitons traveling on the nanotube surface, causing modulations to SWNT fluorescence emission. It is this sensitive relationship between the corona phase and SWNT fluorescence that can be exploited to develop new molecular sensors by incorporating specific binding modalities onto the large surface area of SWNT. Perturbations to the SWNT corona phase upon binding analyte can lead to changes in the local dielectric environment, charge transfer, or introduce lattice defects, all of which can modulate the fluorescence emission of the SWNTs to serve as a signal transduction mechanism.14 This approach is used in the development of novel fluorescent sensors for the detection of many different classes of molecules including DNA,15,16 glucose17 and small molecules such as ATP,18 reactive oxygen species19 and nitric oxide.20,21 However, these approaches are limited in that they rely on the existence of a known binding modality for the target analyte.
Recently, a more generic approach to designing fluorescent sensors was developed using SWNTs non-covalently functionalized with amphiphilic heteropolymers, phospholipids, and polynucleic acids. These molecules adsorb to carbon nanotube surfaces to produce highly stable suspensions of individual SWNTs22-25 with unique corona phases that can specifically bind proteins26,27 or small molecules including the neurotransmitter dopamine.28-30 Engineering the corona phase to disperse SWNTs and specifically bind target analytes is referred to as corona phase molecular recognition (CoPhMoRe).28 The small size, low toxicity, high stability and unbleaching nIR fluorescence of CoPhMoRe SWNT sensors make them excellent candidates for in vivo sensing for extended time-resolved measurements.6 Recent work has shown their applications in plant tissues for optical detection of reactive nitrogen and oxygen species.31 A particularly exciting application for CoPhMoRe SWNT sensors is the potential for label free detection of neurotransmitters such as dopamine in vivo, where other techniques, such as electrochemical sensing or immunohistochemistry, suffer from a lack of spatial resolution, temporal resolution, and specificity.
Designing and discovering CoPhMoRe SWNT sensors has so far been restrained by the size and chemical diversity of the dispersant library, limiting the likelihood of finding a sensor for a particular target. To date, researchers have only scratched the surface of available conjugated, co-block, biological and biomimetic polymers that could serve as functionally active dispersants for SWNT sensors. Here, we present different methods for both dispersing SWNTs and characterizing their fluorescence for high throughput screening and for single SWNT sensor analysis. Specifically, we outline the procedure for coating SWNTs with polynucleic acid oligomers using direct sonication as well as how to functionalize SWNT with amphiphilic polymers through surfactant exchange by dialysis. We use (GT)15-DNA and polyethylene glycol functionalized with rhodamine isothiocyanate (RITC-PEG-RITC) as examples. We demonstrate the use of (GT)15-DNA SWNTs as a CoPhMoRe sensor for the detection of dopamine. Lastly, we outline procedures for performing single molecule sensor measurements, which can be used for characterization or single molecule sensing.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-1</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium dodecyl sulfate</td>
			<td>Sigma Aldrich</td>
			<td>L6026</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium cholate hydrate</td>
			<td>Sigma Aldrich</td>
			<td>C6445</td>
			<td></td>
		</tr>
		<tr>
			<td>tris base (Trizma base)</td>
			<td>Sigma Aldrich</td>
			<td>93362</td>
			<td></td>
		</tr>
		<tr>
			<td>hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Amine-PEG-amine,NH2-PEG-NH2</td>
			<td>Nanocs Inc</td>
			<td>PG2-AM-5k</td>
			<td></td>
		</tr>
		<tr>
			<td>rhodamine B isothiocyanate</td>
			<td>Sigma Aldrich</td>
			<td>283924</td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescein isothiocyanate</td>
			<td>Sigma Aldrich</td>
			<td>F7250</td>
			<td></td>
		</tr>
		<tr>
			<td>dichloromethane</td>
			<td>Sigma Aldrich</td>
			<td>676853</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethylformamide</td>
			<td>Sigma Aldrich</td>
			<td>D4551</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-diisopropylethylamine&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D125806</td>
			<td></td>
		</tr>
		<tr>
			<td>diethyl ether</td>
			<td>Sigma Aldrich</td>
			<td>673811</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine hydrochloride&#160;</td>
			<td>Sigma Aldrich</td>
			<td>C4706&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>5&#8217;-thiol-modified DNA&#160;</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>methoxypolyethylene glycol maleimide</td>
			<td>Sigma Aldrich</td>
			<td>63187</td>
			<td></td>
		</tr>
		<tr>
			<td>100k Da spin filters</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HiPCO Super purified single walled carbon nanotubes</td>
			<td>Integris</td>
			<td>HiPco SuperPurified</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>P5493</td>
			<td></td>
		</tr>
		<tr>
			<td>anti static gun</td>
			<td>Milty</td>
			<td>Milty Zerostat 3</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Eppendorf</td>
			<td>5415 D</td>
			<td></td>
		</tr>
		<tr>
			<td>ultra sonicator</td>
			<td>Cole Parmer</td>
			<td>CV18</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis cassettes</td>
			<td>Thermo scientific</td>
			<td>Slide-A-Lyzer G2 87722</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA-biotin</td>
			<td>Thermo scientific</td>
			<td>29130</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutravidin protein</td>
			<td>Thermo scientific</td>
			<td>31000</td>
			<td></td>
		</tr>
		<tr>
			<td>(3-Aminopropyl)triethoxysilane (APTES)</td>
			<td>Sigma Aldrich</td>
			<td>440140</td>
			<td></td>
		</tr>
		<tr>
			<td>inverted microscope</td>
			<td>Zeiss</td>
			<td>Axio Observer.Z1</td>
			<td></td>
		</tr>
		<tr>
			<td>kinematic mirrors</td>
			<td>ThorLabs</td>
			<td>KM200-E03</td>
			<td></td>
		</tr>
		<tr>
			<td>periscope</td>
			<td>ThorLabs</td>
			<td>RS99</td>
			<td></td>
		</tr>
		<tr>
			<td>immersion oil</td>
			<td>Zeiss</td>
			<td>Immersol 518f</td>
			<td></td>
		</tr>
		<tr>
			<td>100X objective</td>
			<td>Zeiss</td>
			<td>Plan-apochromat 100X oil, 1.4NA, PH3, 420791-9911-000</td>
			<td></td>
		</tr>
		<tr>
			<td>20X objective</td>
			<td>Zeiss</td>
			<td>N-Achroplan 0.45 NA, 420953-9901-000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>cover glass</td>
			<td>Healthrow Scientific</td>
			<td>HS159879H</td>
			<td></td>
		</tr>
		<tr>
			<td>dopamine hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>H8502&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>infrared 2d array camera</td>
			<td>Princeton Instruments</td>
			<td>NIRvana</td>
			<td></td>
		</tr>
		<tr>
			<td>infrared 1d sensor array</td>
			<td>Princeton Instruments</td>
			<td>PyLoN IR</td>
			<td></td>
		</tr>
		<tr>
			<td>nIR spectrograph</td>
			<td>Princeton Instruments</td>
			<td>SCT-320</td>
			<td></td>
		</tr>
		<tr>
			<td>planoconvex lens</td>
			<td>ThorLabs</td>
			<td>LA1384</td>
			<td></td>
		</tr>
		<tr>
			<td>wellplates (glass bottom)</td>
			<td>Corning</td>
			<td>4580</td>
			<td></td>
		</tr>
	</tbody>
</table>

engineering molecular recognition with bio-mimetic polymers on single walled carbon nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

SWNTs were suspended in aqueous solution using both surfactants and amphiphilic polymers by direct sonication and by dialysis exchange. Figure 1 shows SWNTs, grown using the iron carbonyl catalyzed method (HiPCO), suspended using SC, RITC-PEF20-RITC, and (GT)15-DNA. The optical density of a SWNTs with SDS (or polymer) increases dramatically after sonication and decreases upon removal of aggregates and contaminants through purification by centrifugation (<strong...

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Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

The overall goal of this procedure is to prepare near-infrared fluorescent single walled carbon nanotubes for molecular sensing that have been functionalized using biomimetic polymers. These nanosensors can enable time resolved imaging in living tissue. For example, they can be used to detect dopamine in the brain.The main advantage of these sensors is their inherent near infrared fluorescence, which enables imaging deeper into tissue. Synthetic sensors enable the detection of molecules without natural molecular recognition elements making this generic method of sensors development especially useful for small molecule detection. To prepare a nucleic acid suspension of SWNTs, first dissolve nucleic acids in 0.1 molar sodium chloride to a concentration of 100 milligrams per milliliter.In a fume hood, remove static electricity from the disposable spatula, microcentrifuge tubes, and the SWMT stock, using an antistatic gun. Next, add 20 microliters of the nucleic acid solution to 980 microliters of 0.1 molar sodium chloride. Then add 1 milligram of washed SWNTs to the resulting nucleic acid solution before sonicating the mixture.Positioning the probe tip during sonication is critical. If foaming occurs, reposition the probe tip, and try lowering the amplitude. Alternatively, you can try pulses of sonication or bath sonication if the suspension remains poor.Using an ultrasonicator with a 3 millimeter diameter tip, sonicate the solution for 10 minutes at 40%amplitude in an ice bath. Centrifuge the DNA SWNT solution twice for 90 minutes at 16, 100 xg. Collect and keep the supernatant, being careful not to disturb the pellet containing carbon nanotube bundles and aggregates.Discard the pellet in accordance with institutional hazardous waste procedures appropriate for nanomaterials. Measure the solution absorbance at 632 nanometers using a UV-vis spectrophotometer to determine the approximate concentration of suspended SWNTs, in accordance to Lambert-Beer's law, and the appropriate dilution factor. For amphiphilic polymer suspensions, first remove the static electricity from the disposable spatula, microcentrifuge tubes, and SWNT stock.In a fume hood, add five milligrams of SWNTs to five milliliters of 2%sodium cholate solution. Using an ultrasonicator with a six millimeter diameter tip, sonicate the solution for one hour at 40%amplitude in an ice bath. Centrifuge the sample at 150, 000 xg for four hours using an ultracentrifuge, before carefully collecting the supernatant.Next, dissolve 1%weight of amphiphilic polymer in the scSWNT solution. Then, dialyze the polymer scSWNT solution using a 3.5 kilodalton dialysis membrane against one liter of deionized water or buffer for five days. Change out the water or buffer at hour two, and at hour four.Measure the solution absorbance at 632 nanometers using a UV-Vis spectrophotometer to determine the approximate concentration of suspended SWNTs. To prepare the BSA-biotin coated slides, clean a microscope slide and 0.17 millimeter cover glass with deionized water, followed by methanol, acetone, and a final rinse of deionized water. Create channels by placing several pieces of double sided tape approximately five millimeters apart on the clean microscope slide.Seal the channels by taking a long glass cover slip and pressing it onto the top of the double sided tape. Be sure to center it on the microscope slide so that the edges of the cover slip and slide are not flush. Next, add 100 microliters of BSA-biotin stock solution to 900 microliters of 1x Tris buffer to a final concentration of one milligram per milliliter.Flow 50 microliters of the BSA-biotin solution into the channel by pipetting the solution into one end and wicking away the solution at the other end using a tissue. After incubating the solution for five minutes, perform three to five flushes with 50 microliters of 0.1 molar sodium chloride. Next, dilute 40 microliters of five milligrams per milliliters NAV stock solution into 960 microliters of 1x Tris buffer, to a final concentration of 0.2 milligrams per milliliter.Flow 50 microliters of the NAV solution into the channel by pipetting the solution into one end and wicking away the solution at the other end using a tissue. After incubating the slide for two to five minutes, perform three to five flushes with 50 microliters of 0.1 molar sodium chloride. Then, dilute the stock solutions of suspended SWNTs and imaging buffer to a concentration of 1 to 10 milligrams per liter.Flow 50 microliters of this solution into the channel and incubate for five minutes. Gently rinse away excess SWNTs using 50 microliters of imaging buffer. To measure the reversible response of GT15 DNA-SWNTs to dopamine, first mount the sensor coated channels onto the fluorescence microscope stage.Bring the channels into focus using a 100X Oil Objective and indium gallium arsenide camera. Then, add 50 microliters of 100 micromolar dopamine solution in phosphate buffered saline to the flow channel, and record the change in fluorescence intensity. Wash out the dopamine solution using phosphate buffered saline, and observe the change in fluorescence of the individual SWNT sensors.To perform the well plate screening for analyte response of biomimetic polymer SWNTs, pipette equal volumes of the suspended SWNT sensor into each well. Use enough volume to cover the bottom of the well uniformly. Then, pipette analytes from the screening library into the well plates.Prepare each analyte in triplicate to account for potential well to well variation and fluctuations of excitation intensity, or temperature. Raise the objective while monitoring the emissions spectra using the spectrometer and indium gallium arsenide array. Optimal position of the objective will put the focal plane approximately in the middle of the sample volume in the well, which should correspond to a maximum in measured intensity.For each sample well, record a one to 10 second exposure to collect a spectrum. Compare the emissions spectra for each well to a control containing just SWNTs without additional analyte, to quantify the fluorescence response. Absorbance spectrum of GT15 DNA nanotube sensors shows multiple peaks corresponding to well dispersed carbon nanotubes of different chirality.The near infrared fluorescence of GT15 DNA nanotube sensors is shown. Interestingly, fluorescence intensity approximately doubles in response to dopamine. In this figure, near infrared fluorescence microscopy reveals single GT15 DNA nanotube sensors immobilized on the surface of a glass cover slip.Moving forward it is important to test the sensor for in vivo use, for example expanding the analyte library to include molecular competitors, testing for linearity of response in the biologically relevant concentration regime, and also testing for reversibility. Once mastered, this approach can be used to make a library of different synthetic sensors from molecules without known molecular recognition elements.

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Research

JoVE Journal

Bioengineering

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very promising application for molecular sensors, field effect transistor and super thin and flexible electronic devices1-4. Anodic arc discharge supported by the erosion of the anode material is one of the most practical and efficient methods, which can provide specific non-equilibrium processes and a high influx of carbon material to the developing structures at relatively higher temperature, and consequently the as-synthesized products have few structural defects and better crystallinity.
 To further improve the controllability and flexibility of the synthesis of carbon nanostructures in arc discharge, magnetic fields can be applied during the synthesis process according to the strong magnetic responses of arc plasmas. It was demonstrated that the magnetically-enhanced arc discharge can increase the average length of SWCNT 5, narrow the diameter distribution of metallic catalyst particles and carbon nanotubes 6, and change the ratio of metallic and semiconducting carbon nanotubes 7, as well as lead to graphene synthesis 8. 
 Furthermore, it is worthwhile to remark that when we introduce a non-uniform magnetic field with the component normal to the current in arc, the Lorentz force along the J&times;B direction can generate the plasmas jet and make effective delivery of carbon ion particles and heat flux to samples. As a result, large-scale graphene flakes and high-purity single-walled carbon nanotubes were simultaneously generated by such new magnetically-enhanced anodic arc method. Arc imaging, scanning electron microscope (SEM), transmission electron microscope (TEM) and Raman spectroscopy were employed to analyze the characterization of carbon nanostructures. These findings indicate a wide spectrum of opportunities to manipulate with the properties of nanostructures produced in plasmas by means of controlling the arc conditions.

Anodic arc discharge is one of the most practical and efficient methods to synthesize various carbon nanostructures. To increase the arc controllability and flexibility, a non-uniform magnetic field was introduced to process the one-step synthesis of large-scale graphene flakes and high-purity single-walled carbon nanotubes.

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment2.
Coagulation Factor VIII (FVIII)3 is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A&#160;-&#160;a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting4. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete5. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa6,7.
In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.&#160;The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.

We present a combination of Cryo-electron microscopy, lipid nanotechnology, and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory&#160;to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...