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

Summary

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.

Abstract

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.

Introduction

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

Protocol

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

1. Preparation of 100 mM NaCl solution
   1. Dissolve 584 mg of NaCl in 80 mL of deionized water. Add deionized water to bring total volume to 100 mL.

Results

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 (

Discussion

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 .......

Materials

List of materials used in this article

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