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...

Watch this Scientific Journal Video about Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes at JoVE.com

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.

Research

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Bioengineering

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