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
	<ol>
		<li>Dissolve 584 mg of NaCl in 80 mL of deionized water. Add deionized water to bring total volume to 100 mL.</li>
	</ol>
	</li>
	<li>Preparation of 3% sodium dodecyl sulfate (SDS) solution
	<ol>
		<li>Dissolve 3 g of SDS in 80 mL of deionized water. Add deionized water to bring total volume to 100 mL.</li>
	</ol>
	</li>
	<li>Preparation of 2% sodium cholate (SC) solution
	<ol>
		<li>Dissolve 2 g of sodium cholate hydrate in 80 mL of deionized water. Add deionized water to bring total volume to 100 mL.</li>
	</ol>
	</li>
	<li>Preparation of imaging buffer (1x Tris: 20 mM Tris, 100 mM NaCl)
	<ol>
		<li>Dissolve 22.23 g of Tris base and 58.44 g of NaCl in 500 mL of deionized water using a magnetic stir bar and plate.</li>
		<li>Carefully add concentrated HCl until a pH of 8.1 is reached.</li>
		<li>Add deionized water to reach a final volume of 1 L.</li>
	</ol>
	</li>
	<li>Synthesis of RITC-PEG-RITC polymer
	<ol>
		<li>Dissolve amine difunctionalized polyethylene glycol (PEG) (5 kDa or 20 kDa, 0.1 mol/L) and rhodamine isothiocyanate (RITC, 0.2 mol/L) in 1 mL of a 1:1 mixture of dichloromethane and dimethylformamide (DMF).</li>
		<li>Add 0.2 mol/L of N,N-diisopropylethylamine (DIEA).</li>
		<li>After 3 h, precipitate with 10x volume of diethyl ether followed by vacuum filtration.</li>
		<li>Redissolve in DMF and repeat ether precipitation followed by vacuum filtration. 
		NOTE: Other isothiocyanate modified molecules (e.g., fluorescein isothiocyanate, FITC) can be attached to PEG or other amine modified polymers using a similar method.</li>
	</ol>
	</li>
	<li>Preparation of pegylated-DNA (PEG-DNA)
	<ol>
		<li>Combine 100 &#181;L of Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution (0.5 M, pH 7.0) with 44.9 &#181;L of 5&#39;-thiol-modified DNA (1 mg/10 &#181;L in 0.1 M NaCl) and add to 4.855 mL of deionized water.</li>
		<li>Stir for 1 h.</li>
		<li>Dissolve 500 mg of methoxypolyethylene glycol maleimide in 5 mL of phosphate buffered saline.</li>
		<li>Combine DNA and PEG solutions (10 mL total) and stir for 24 h.</li>
	</ol>
	</li>
</ol>
2. Preparation of Single Walled Carbon Nanotube (SWNTs) Suspensions
<ol>
	<li>Wash of SWNTs to remove catalyst and impurities.
	<ol>
		<li>Add 200-300 mg of unwashed SWNTs into a plastic centrifuge tube containing 45 mL of deionized water.</li>
		<li>Vortex the solution for 2 min and sonicate using a bath sonicator for 5 min. Note that sonicator settings vary from instrument to instrument, so check that the optical density of the SWNT solution increases (i.e., turns black) to ensure that the SWNTs are being dispersed.</li>
		<li>Centrifuge the solution for 20 min at 16,100 x g and discard supernatant.</li>
		<li>Add approximately 45 mL of fresh deionized water.</li>
		<li>Repeat steps 2.1.2-2.1.4 up to 8 times.</li>
		<li>Remove as much water as possible being careful not to disturb pelleted SWNTs and allow SWNT pellet to air dry.</li>
	</ol>
	</li>
	<li>Nucleic acid suspensions of SWNTs
	<ol>
		<li>Dissolve nucleic acids (NA) in 0.1 M NaCl to a concentration of 100 mg/mL.</li>
		<li>Remove static electricity from disposable spatula, microcentrifuge tubes and SWNT stock using an anti-static gun. In a fume hood, add 20 &#181;L of the NA solution to 980 &#181;L of 0.1 M NaCl followed by the addition of 1 mg SWNTs.</li>
		<li>Using an ultrasonicator with 3 mm diameter tip, sonicate the solution for 10 min at 40% amplitude in an ice bath.</li>
		<li>Centrifuge the DNA-SWNT solution TWICE for 90 min at 16,100 x g. If using PEG-DNA, purify out excess and unreacted DNA and PEG using a 100 kDa spin-filter. Add enough PBS to fill the spin-filter and spin at 9,300 x g for 1.5 min, Repeat this wash step 3 times.</li>
		<li>Collect and keep the supernatant, being careful not to disturb the pellet containing CNT bundles and aggregates. Discard the pellet in accordance with institutional hazardous waste procedures appropriate for nanomaterials.</li>
		<li>Measure the solution absorbance at 632 nm using a UV/Vis spectrophotometer to determine approximate concentration of suspended SWNTs using &#949;=0.036 L/cm&#183;mg in accordance with Lambert-Beer&#39;s law and the appropriate dilution factor.</li>
	</ol>
	</li>
	<li>Amphiphilic polymer suspensions of SWNTs
	<ol>
		<li>Remove static electricity from disposable spatula, micro centrifuge tubes, and SWNT stock. In a fume hood, add 5 mg SWNTs to 5 mL of 2% SC solution (alternatively, SDS solution can be used).</li>
		<li>Using an ultrasonicator with 6 mm diameter tip, sonicate the solution for 1 h at 40% amplitude in an ice bath.</li>
		<li>Centrifuge sample at 150,000 x g for 4 h using an ultracentrifuge and carefully collect supernatant.</li>
		<li>Dissolve 1 wt % of amphiphilic polymer (e.g., RITC-PEG-RITC) in SC-SWNT solution.</li>
		<li>Dialyze the polymer-SC-SWNT solution using a 3.5 kDa dialysis membrane against 1 L of deionized water or buffer for 5 days. Change out the water or buffer after hour 2 and hour 4. A larger dialysis membrane can be used, so long as it allows removal of the surfactant of choice, but retention of both the SWNT and amphiphilic polymer.</li>
		<li>Measure the solution absorbance at 632 nm using a UV/Vis spectrophotometer to determine approximate concentration of suspended SWNTs using &#949;=0.036 L/cm*mg in accordance with Lambert-Beer&#39;s law and the appropriate dilution factor.</li>
	</ol>
	</li>
</ol>
3. Preparation of Surface Immobilized SWNT Sensors
<ol>
	<li>Preparation of BSA-biotin and NeutrAvidin stock solutions
	<ol>
		<li>Dissolve 10 mg lyophilized BSA-biotin in 1 mL deionized water to make a 10 mg/mL stock solution and store at 4 &#176;C.</li>
		<li>Dissolve 10 mg NeutrAvidin protein (NAV, deglycosylated avidin protein) in 2 mL of deionized water to make a 5 mg/mL stock solution. Store aliquots at -20 &#176;C. Thawed aliquots can be kept for several days at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare BSA-Biotin coated microscope slides
	<ol>
		<li>Clean a microscope slide and 0.17 mm cover glass (or as appropriate for the microscope objective) with deionized water, followed by methanol, acetone and a final rinse of deionized water.</li>
		<li>Create channels by placing several pieces of double-sided tape approximately 5 mm apart on the clean microscope slide. Seal the channels by taking a long glass coverslip and pressing it onto the top of the double sided tape. Be sure it is centered on the microscope slide so the edges of the cover slip and slide are not flush.</li>
		<li>Add 100 &#181;L of BSA-Biotin stock solution to 900 &#181;L of 1x Tris buffer to a final concentration of 1 mg/mL.</li>
		<li>Flow 50 &#181;L of the BSA-biotin solution into the channel by pipetting the solution into one end and wicking away solution at the other using a tissue. Incubate for 5 minutes followed by 3-5 flushes with 50 &#181;L of 0.1 M NaCl.</li>
		<li>Dilute 40 &#181;L of 5 mg/mL NAV stock in 960 &#181;L 1x Tris buffer to a final concentration of 0.2 mg/mL.</li>
		<li>Flow 50 &#181;L of the NAV solution into the channel by pipetting the solution into one end and wicking away solution at the other using a tissue. Incubate for 2-5 minutes followed by 3-5 flushes with 50 &#181;L of 0.1 M NaCl.</li>
		<li>Dilute stock solutions of suspended SWNTs in imaging buffer to a concentration of 1-10 mg/L and flow 50 &#181;L of the solution into the channel and incubate for 5 min.</li>
		<li>Gently rinse away excess SWNTs using 50 &#181;L of imaging buffer.</li>
	</ol>
	</li>
	<li>Preparation of APTES silanized microscope slides 
	NOTE: APTES silanized slides allow a way to immobilize negatively charged DNA-wrapped SWNTs to the surface of a glass substrate.
	<ol>
		<li>Prepare a 10% solution of (3-aminopropyl)triethoxysilane (APTES) in ethanol.</li>
		<li>Using channels made using the steps outlined in 3.2.2, flow in 100 &#181;L of 1x Tris buffer.</li>
		<li>Flush the channel with APTES solution and incubate for 5 min. Wash with 1x Tris buffer.</li>
		<li>Dilute stock solutions of suspended DNA-SWNTs in imaging buffer to a concentration of 1-10 mg/Land flow 50 &#181;L of the solution into the channel and incubate for 5 min.</li>
		<li>Gently rinse away excess SWNTs using 50 &#181;L of imaging buffer.</li>
	</ol>
	</li>
</ol>
4. Fluorescence Spectroscopy and Microscopy of SWNT Sensors
<ol>
	<li>nIR Fluorescence Microscopy
	<ol>
		<li>Perform imaging of surface immobilized SWNTs using laser excitation and an inverted microscope outfitted with an InGaAs sensor array for imaging.</li>
		<li>Direct the laser beam to enter the back illumination port of an inverted microscope using mirrors on adjustable kinematic mounts and a pair of post-mounted irises set to the port height. Ensure the beam is level and straight by confirming it passes through both irises when placed between subsequent mirrors and before it enters the illumination port. If necessary, adjust the beam height using a periscope assembly. Remove any short-pass heat filters on the illumination port that would attenuate the beam.</li>
		<li>Insert an appropriate filter cube into the microscope to reflect the excitation light into the objective and collect emission light. This typically consists of a dichroic long pass filter with a cutoff above the excitation wavelength, (e.g., 750 LP) and an emission long pass filter (e.g., 850 LP) to further minimize scattered excitation light from hitting the sensor. Additionally, the emission filter can be chosen to be selective for the emission of SWNTs of a particular chirality.</li>
		<li>Perform fine adjustments to the beam alignment by inserting an appropriate alignment cube replacing the objective with two offset, frosted discs containing 1 mm pinholes. Align the beam to the center of both the lower and upper alignment discs.</li>
		<li>Attach a 2D InGaAs sensor array to the side imaging port of the microscope using an appropriate adapter and a 0.5X lens if necessary to accommodate the sensor size.</li>
		<li>Using a 100X oil immersion objective (1.4 NA), apply fluorescence-free immersion oil and place the immobilized SWNT sample onto the microscope stage.</li>
		<li>Raise the objective until the oil contacts the bottom of the cover glass (#1.5, 170 &#181;m thickness). Make adjustments to the objective collar if necessary for imaging conditions, e.g., temperature, glass thickness, etc.</li>
		<li>Slowly raise the objective with the excitation source on and monitor the fluorescence signal of the InGaAs camera. Fluorescence intensity should gradually increase as the focal plane approaches the surface and the immobilized sensors come into focus.</li>
	</ol>
	</li>
	<li>nIR Fluorescence Spectroscopy 
	NOTE: Fluorescence spectroscopy can be performed using the same microscope setup but by directing the collected light out of the microscope body and into a spectrometer and InGaAs linear array detector.
	<ol>
		<li>Using kinematic mounts and mirrors rated for nIR, direct the light path towards the entrance slit of the spectrometer. Focus the light down to a point onto the entrance slit using a focusing lens (e.g., plano-convex, f = 150 mm). This alignment is accomplished by attenuating the laser power to &#60;1 mW and replacing the microscope objective with a 1&#34; mirror and using a 50/50 beam-splitting filter cube. The excitation light will then leave the microscope body through the exit port and can be used to adjust the mirrors and lens to focus the beam onto the entrance slit.</li>
		<li>After replacing the microscope objective and long pass filter, place a well plate onto the stage and record the spectra of an in-focus sample using the spectrometer and InGaAs array.</li>
	</ol>
	</li>
	<li>Measuring the reversible response of (GT)15 DNA-SWNTs to dopamine
	<ol>
		<li>Mount sensor coated channels onto the microscope stage and bring into focus using a 100X oil objective and InGaAs camera. Add 50 &#181;L of 100 &#181;M dopamine solution in phosphate buffered saline (PBS) to the flow channel and record the change in fluorescence intensity.</li>
		<li>Wash out the dopamine solution using phosphate buffered saline and observe the change in fluorescence of the individual SWNT sensors.</li>
	</ol>
	</li>
	<li>Well-plate screening for analyte response of SWNTs sensors 
	NOTE: Screening of different analytes can be performed using a transparent well plate controlled using a motorized stage. An ideal well plate is transparent to visible and IR and has black sidewalls to minimize crosstalk between the wells.
	<ol>
		<li>Pipette equal volumes of suspended SWNT sensor (e.g., 5 mg/L concentration) into each well, enough to cover the bottom of the well uniformly, typically &#62;100 &#181;L for a 96-well plate and &#62;30 &#181;L for a 384-well plate.</li>
		<li>Pipette analytes (e.g., 2 &#181;L, 100 &#181;M final volume) from screening library into the well plates. Prepare each analyte in triplicate to account for potential well-to-well variation, fluctuations of excitation intensity, or temperature.</li>
		<li>Raise the objective (e.g., 20X achroplan, 0.45 NA) while monitoring the emission spectra using the spectrometer and InGaAs 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.</li>
		<li>For each sample well, record a 1-10 s exposure to collect a spectrum.</li>
		<li>Compare the emission spectra for each well to a control containing just SWNTs without additional analyte to quantify the fluorescence response.</li>
	</ol>
	</li>
</ol>

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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. 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=A+Ratiometric+Sensor+Using+Single+Chirality+Near-Infrared+Fluorescent+Carbon+Nanotubes:+Application+to+In+Vivo+Monitoring.">A Ratiometric Sensor Using Single Chirality Near-Infrared Fluorescent Carbon Nanotubes: Application to In Vivo Monitoring.</a> Small. 11 (32), 3973-3984 (2015).</li><li>Karousis, N., Tagmatarchis, N., Tasis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Progress+on+the+Chemical+Modification+of+Carbon+Nanotubes.">Current Progress on the Chemical Modification of Carbon Nanotubes.</a> Chem Rev. 110 (9), 5366-5397 (2010).</li><li>Movia, D., Del Canto, E., Giordani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purified+and+Oxidized+Single-Walled+Carbon+Nanotubes+as+Robust+Near-IR+Fluorescent+Probes+for+Molecular+Imaging.">Purified and Oxidized Single-Walled Carbon Nanotubes as Robust Near-IR Fluorescent Probes for Molecular Imaging.</a> J Phys Chem C. 114 (43), 18407-18413 (2010).</li><li>Cognet, L., 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=Stepwise+quenching+of+exciton+fluorescence+in+carbon+nanotubes+by+single-molecule+reactions.">Stepwise quenching of exciton fluorescence in carbon nanotubes by single-molecule reactions.</a> Science. 316 (5830), 1465-1468 (2007).</li></ol>

The authors have nothing to disclose.

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 using SC or SDS. Hydrophilic polymers, such as PEG, can be end-modified with hydrophobic &#34;anchors&#34; such as RITC or FITC to enable surface adsorption of the block copolymer. For polymers that are susceptible to shearing or degradation upon exposure to the high powers of probe-tip sonication, or for polymers with low binding affinities to SWNT, dialysis is the best method to produce a stable SWNT-polymer suspension. Following polymer encapsulation, centrifugation removes large SWNT bundles, amorphous carbon, residual metal catalyst, and other insoluble contaminants to leave a uniformly dispersed sample. Typical final concentrations of dispersed SWNTs after centrifugation range between 10-100 mg/L.
Sonication power and duration can be adjusted and optimized for the particular choice of dispersant. This is a critical step in the procedure for coating SWNTs because too little or weak sonication can result in poor dispersal, while too much or too powerful sonication can lead to poor fluorescence. Typically, lower powers are necessary to minimize damage when using polymers susceptible to shearing such as DNA. Longer sonication durations or higher intensities can lead to reduction in the length of SWNTs, where SWNTs below the ~100 nm exciton recombination length become non-fluorescent. The size of the sonicator probe tip should also match the sample volume to avoid splashing and foaming for optimal results (typically provided by manufacturer). Avoid touching the probe tip to the sides of the container and place the solution on an ice block to minimize heating of the solution. Once dispersed, solutions of SWNTs are stable at room temperature indefinitely.
Absorbance and emission spectra of solutions of dispersed generated SWNTs contain multiple peaks, indicating the presence of a mixture of dispersed SWNTs of different chiralities. Alternative methods of SWNT generation or purification methods can change the chirality distribution, leading to different peak excitation and emission fluorescence spectra. Additionally, different synthesis methods can yield samples of SWNTs with different chirality population distributions. For example: CoMoCAT (cobalt-molybdenum catalyst) grown SWNTs are rich in (6,5) chirality, while HiPCO (high pressure with iron carbonyl catalyst) grown SWNTs are rich in (7,6) chirality, leading to differences in the absorption and photoluminescence spectra.
Certain adsorbed polymers enable the specific detection of analytes by modulating the fluorescence emission of the SWNT by changing the local environment at the tube surface. This approach offers the distinct advantage over covalent attachment of binding moieties by not permanently disrupting the SWNT lattice, which can reduce fluorescence emission intensity.6,32-34 Additionally, the CoPhMoRe approach has the potential for developing antibody-free sensors for targets where there may not already exist a known binding moiety.28 Specifically, (GT)15-DNA has been shown to selectively enhance the fluorescence emission of SWNTs in the presence of dopamine, enabling its use as a dopamine sensor. Bulk fluorescence measurements show an increase of as much as 80% in peak emission for certain SWNT chiralities. Immobilizing (GT)15-DNA SWNTs on a glass slide enables fluorescence response measurements of individual (GT)15-DNA functionalized SWNT, showing that fluorescence can increase by over 3-fold for single SWNT sensors in the presence of dopamine, without any appreciable photobleaching, under continuous laser illumination. The interaction between dopamine and the SWNT sensor is reversible, as evident by the recovery of the original fluorescent signal after washing dopamine out of the microfluidic chamber with buffer. Additionally, single-molecule measurements can be a powerful characterization technique for quantifying polymer adsorption (Figure 4) or the kinetics of binding events. Also, ratiometric sensing can be achieved by chemically isolating a singular SWNT chirality with a unique excitation-emission peak, functionalizing it with (GT)15-DNA, and isolating a second SWNT chirality to be insensitive to dopamine. Monitoring both SWNT chiralities provides a steady control fluorescence channel that can be compared to the modulating fluorescence of the (GT)15-DNA SWNTs. Combining different polymers (e.g., polyethylene glycol and (GT)15-DNA) can add additional functionality such as modifying diffusivity or cellular uptake characteristics, properties that are critical when performing in vivo experiments.
Currently, a limitation of the CoPhMoRe approach to sensor development include polymer library development. Because binding moieties are not known a priori, developing a sensor for a particular target can be time intensive and require a large number of chemically varied polymers for constructing the sensor library for screening. Additionally, stability and compatibility of sensors in in vivo environments can vary from sensor to sensor. However, once a candidate sensor has been identified, further modification strategies can be employed to optimize properties as necessary for in vivo applications.
Herein, we have demonstrated a methodology for dispersing SWNTs in aqueous solutions applicable to a wide variety of dispersing agents. This approach can be used to create libraries of dispersed SWNTs for the discovery of novel new nIR sensors for small molecules and biological markers. Of particular interest are sensors for the detection of neurotransmitters, which could enable real time, spatially precise detection of these molecules in complex biological environments.

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 (Figure 1). Measurements of absorbance at 632 nm quantified the concentration of suspended SWNT.28
The photophysical properties of the SWNT suspensions are characterized using absorbance and fluorescence spectroscopy. Figure 2 shows the absorbance and fluorescence emission spectra of SWNTs suspended using (GT)15-DNA and RITC-PEG20-RITC. The absorbance spectra are a superposition of the individual absorbance peaks for each distinct chirality of nanotube present in the sample. Similarly, each chirality exhibits its unique fluorescence emission peak. Differences in relative emission peak intensity are a result of differences in the population distribution of chiralities as well as differences in excitation efficiency using the 721 nm laser.
The fluorescence response of (GT)15DNA-SWNTs (where I0 is the initial SWNT fluorescence intensity and I is the SWNT intensity after dopamine addition) in the presence of different concentrations of dopamine is measured by monitoring the fluorescence of a sample using a spectrophotometer and InGaAs linear array (Figure 3). The total fluorescence of (GT)15-DNA-SWNTs increased with increasing dopamine concentration (Figure 3a). The fluorescence response is a function of the emission peak (Figure 3b), indicating that the response may be chirality specific. The fluorescence of the 1,044 nm and 1,078 nm peaks increase in intensity 2-fold as dopamine concentration approaches 2 &#181;M. Figure 3e shows the intensity of the entire emission spectra of PEG-(GT)15 DNA-SWNT increase in response to the addition of dopamine.
Individual SWNTs coated using an alternative DNA sequence, C26-DNA (prepared using the same methods at (GT)15), tethered to the surface of a microscope slide are measured under laser illumination using an InGaAs camera and 100X oil immersion objective (Figure 4). Monitoring single emitters tethered to a surface can be used to verify the reversibility of sensor response by washing target molecules away using buffer solution. Total internal reflection fluorescence (TIRF) microscopy can also be used to image dye-conjugated DNA adsorbed on the SWNTs to quantify the number of DNA molecules attached to each tube through photobleaching experiments. Figure 4 shows 3 distinct bleaching events of Cy3-labeled DNA inferred from the quantized steps of the fluorescence intensity trace of a single emitter. These results indicate that three DNA molecules are attached to the SWNT.
<img alt="Figure 1" src="/files/ftp_upload/55030/55030fig1.jpg" /> 
Figure 1: Polymer and Surfactant Suspended SWNTs. (a) Photograph of RITC-PEG20-RITC SWNTs suspended using 2% SC at various points of preparation. Left: SWNTs added directly to SC solution prior to sonication. Center: After 10 min of bath sonication followed by 10 min probe tip sonication at 90% amplitude followed by centrifugation. The optical density of the solution increases as SWNTs bundles are dispersed (~100 mg/L SWNT concentration). Right: After dialysis with RITC-PEG20-RITC polymer, the final concentration of RITC-PEG-RITC suspended SWNTs is ~20 mg/L. (b) SWNT suspension yield can vary depending on the polymer used to suspend the SWNT. The optical density of the solution provides a good estimate of solution-phase SWNT concentration. Pictured are different concentrations of (GT)15-DNA suspended tubes at different concentrations. From left to right: 100 mg/L, 10 mg/L, 1 mg/L, 0 mg/L. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55030/55030fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55030/55030fig2.jpg" /> 
Figure 2: Absorption and fluorescence emission spectra of surfactant and polymer suspended SWNTs. (a) Representative absorbance spectra of SWNTs suspended using (GT)15-DNA by direct sonication. The concentration of SWNT is 10 mg/L. Inset: The UV region of the absorbance spectra shows the characteristic DNA absorbance peak at 260 nm. (b) Absorbance spectra of RITC-PEG20-RITC SWNTs after exchange of SC by dialysis. Inset: Absorbance of a 10x diluted sample of RITC-PEG-RITC SWNTs shows the characteristic absorbance of rhodamine. (c) Representative nIR emission spectra of SWNTs suspended using (GT)15-DNA by direct sonication (785 nm excitation). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55030/55030fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55030/55030fig3.jpg" /> 
Figure 3: Fluorescence detection of dopamine using (GT)15-DNA wrapped SWNTs. (a) Fluorescence response of (GT)15-DNA wrapped SWNTs to the addition of dopamine. Samples of sensors at a concentration of 5 mg/L were excited using a 500 mW, 721 nm CW laser. The integrated fluorescence of sensor emission from 900-1,350 nm increases with added dopamine concentration 1 &#181;M to 250 &#181;M. (b) Fluorescence emission spectra of PEG-(GT)15-DNA wrapped SWNTs before and after addition of dopamine. The concentration of sensor is 10 mg/mL to which dopamine was added to a final concentration of 100 &#181;M. The samples were excited using a 500 mW, 721 nm CW laser. The two highest intensity peaks approximately double in intensity after addition of dopamine. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55030/55030fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55030/55030fig4.jpg" /> 
Figure 4: Fluorescence imaging of single surface-immobilized SWNTs. (a) Fluorescence emission of individual C26-DNA-SWNT (red arrows) immobilized onto a silica cover slip (#1.5) using an APTES silanization procedure and imaged using a 2D InGaAs sensor array, inverted microscope with a 100X oil immersion objective (plan apochromat, 1.4 NA), and a 500 mW, 721 nm CW laser. (b) Fluorescence bleaching experiment of C26-Cy3 DNA-SWNTs tethered to a surface using APTES. The DNA strands are 3&#39; terminally labeled with Cy3 prior to tube suspension. Tracking the incremental step-wise photobleaching (red fitted trace) of individual sensors is used to determine the number of DNA molecules adsorbed onto the surface of the SWNTs. Images were acquired using an inverted microscope in TIRF mode with a 100X oil immersion objective (plan apochromat, 1.4 NA), and 561 nm laser excitation. Scale bar: 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55030/55030fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

engineering-molecular-recognition-with-bio-mimetic-polymers-on-single

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.1K Views.  University of California Berkeley. 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.

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

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 ...

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

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 ...

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 ...