Introduction

Biochemical approaches for coupling different molecules to the surface of single-walled carbon nanotubes (SWNTs) would expand the circle of researchers working on the life science applications of these nanostructures and would allow nanotechnology experimentation in biomedical research laboratories. Of particular importance is fluorescent labeling of SWNTs. Fluorescent SWNTs are essential for the development of nanophotonics, quantum computing, and probes for biomedical research. In spite of their nanometer-scale diameters, fluorescent SWNTs can be directly observed by optical microscopes, which will enable the construction of nanosize devices via optically controlled assembly. SWNTs can also make unique composite fluorophores because, due to their length, they permit the attachment of multiple fluorescent dyes to a single nanotube.

Two major obstacles need to be surmounted for successful enzyme-driven preparation of fluorescent nanotubes. First, enzymatic approaches to label SWNTs have to be developed. Secondly, fluorescence quenching of the nanotube-coupled fluorophores should be prevented, as it is known that the fluorophores directly attached to SWNTs are quenched via energy transfer (1,2).

Here we describe the first procedure for enzymatic fluorescent labeling of SWNTs. The procedure is based on peroxidase-driven biotinylation of SWNTs with subsequent reaction with streptavidin-conjugated fluorophores. The fluorescence quenching property of SWNTs is bypassed by using colloidal semiconductor nanocrystals, quantum dots (Q-dots), instead of conventional fluorophores.

Materials and Methods

Fluorescence Labeling of SWNTs

Biotinylation of SWNTs or direct ligation of fluorescent tyramides to the surface of nanotubes was performed using purified and filtered preparation of SWNTs stabilized in 1% sodium dodecyl sulfate (SDS) (HiPco tubes received from Dr. R.E. Smalley, Rice University, Houston, TX, USA) containing predominantly individual SWNTs and SWNT ropes (3). The reaction was initiated in 50 mM Tris-HCl, pH 8.0, containing 3 µg/mL SWNTs; 200 ng/µL horseradish peroxidase (HRP), 0.01% hydrogen peroxide, 0.2% SDS, and a 1:50 dilution of biotinyl tyramide reagent (from the TSA™ Biotin System; Perkin-Elmer Life Sciences, Boston, MA, USA) or a 1:50 dilution of Alexa-Fluor®-tagged tyramides (Molecular Probes, Eugene, OR, USA) Alexa Fluor-594 (red fluorescence), Alexa Fluor-488 (green fluorescence), and Alexa Fluor-350 (blue fluorescence) for 1 h at 37°C. Although SDS concentration in the labeling mixture fell below 1%, we did not see any precipitation of the functionalized nanotubes, even after 1-month storage at 4°C. After the reaction, the biotin-tagged SWNTs were combined with streptavidin-fluorescein isothiocyanate (FITC) or streptavidin-Q-dots by mixing at a 1:1 ratio (v/v).

Q-dots (Adirondack Green, COOH-terminated semiconductor nanocrystals) were purchased (Evident Technologies Troy, NY, USA) and then conjugated to streptavidin in our laboratory using the EviTags™ Protein Labeling Kit (Evident Technologies) exactly as recommended by the manufacturer.

Gel Electrophoresis of Labeled SWNTs

SWNTs labeled with fluorescent dyes were separated from unreacted conjugates using 0.3% agarose gel electrophoresis. Small aliquots of labeling mixture containing various preparations of fluorescently labeled SWNTs were loaded in 15% Ficoll® on a 0.3% agarose gel in 1× Tris-borate-EDTA (TBE), and run for 30 min to 1 h at 100 V. They were observed using an FBTIV-88 transilluminator (Fisher Scientific, Hampton, NH, USA). Above the 312 nm emission peak, transilluminator lamps provide sufficient spectral output for the visualization of fluorophores excited in green and red parts of spectrum (see (Figures 2) and (3)), which is routinely used for the evaluation of fluorescent molecular probes (probes.invitrogen. com/handbook). Gel images were captured on a PC 1015 Canon digital photo camera (Canon U.S.A., Lake Success, NY, USA) equipped with a 4-megapixel charge-coupled device (CCD) and processed in Adobe® Photoshop® 7.0.

Imaging of Labeled SWNTs

The fluorescence of labeled SWNTs deposited on a glass slide was assessed, and images were captured using an Olympus IX70 fluorescent microscope (Leeds Instruments, Irving, TX, USA) and a MicroMax digital video camera (Princeton Instruments, Trenton, NJ, USA). A bandpass filter set (Chroma Technology, Rockingham, VT, USA) was used (FITC and Alexa Fluor-488 excitation D490/40, emission 520/10; Alexa Fluor-594 excitation D560/40, emission 620/30; Alexa Fluor-350 excitation D360/40, emission 460/20). Composite images were created in MetaMorph® 4.6.5 (Molecular Devices, Sunnyvale, CA, USA). Adirondack Green Q-dots can be excited at a broad range of wavelengths, so Q-dot-labeled SWNTs deposited on a glass slide were imaged using FITC-Alexa Fluor-488 excitation and emission filters.