DNA-Guided Single-Molecule Modifications of Carbon Nanotubes by Magnetic Beads-Assisted Purification

Single-walled carbon nanotubes (SWCNTs) offer significant potential for probing molecular interactions at single-molecule resolution, owing to their unique optical/electronic properties and their one-dimensional structure. However, achieving precise SWCNT surface modifications has been challenging, primarily due to the difficulty in manipulating chemical reactions at the single-molecule scale. In this study, we propose a facile, magnetic beads-assisted sorting strategy to achieve single-molecule modifications of SWCNTs.<br/><br/>Our methodology encompasses a two-step process: 1- Synthesizing SWCNT dispersion with a mixture of unmodified ssDNA and biotin conjugated ssDNA (ssDNA-Bt). With a sufficiently low ssDNA-Bt/ssDNA ratio in the starting material, we predict SWCNTs that have ssDNA-Bt, will only have one ssDNA-Bt at a distinct probability; thus, a single biotinylation on a SWCNT. 2- These singly-biotinylated SWCNTs are selectively isolated using streptavidin-coated magnetic beads. To overcome the strong biotin-streptavidin binding during recovery, we employ desthiobiotin (dtBt), a biotin analogue with reduced affinity for streptavidin, facilitating the release of SWCNTs through an affinity-based exchange. Finally, a single biomolecule can be conjugated selectively to a SWCNT through the ssDNA-dtBt bond either by using a streptavidin-assisted conjugation or by including conjugation chemistry on a ssDNA-dtBt strand.<br/><br/>As a proof-of-concept, we prepared (GT)₁₅-dtBt/(GT)₁₅-SWCNT with a starting mixing ratio of as low as 1/1000. The synthesized SWCNT sample was then incubated with streptavidin-coated magnetic beads for 1 h, followed by the addition of biotin to recover the captured biotinylated SWCNTs. The recovery of SWCNT was characterized by near-infrared (NIR) fluorescence microscopy. Accordingly, 80% of the dtBt-functionalized SWCNTs were selectively recovered. Notably, recovery from the control samples [(GT)₁₅-Bt/(GT)₁₅-SWCNT or (GT)₁₅-SWCNT] was negligible, highlighting the ability of our strategy to enable single molecule SWCNT functionalization with high efficiency. We further quantified the functionalization sites by atomic force microscopy and an enzymatic activity assay. Thus, we herein developed a facile strategy of single-molecule functionalization of SWCNTs, a method which may be generalized for broader applications in SWCNT based sensing and sorting, including chiral separations and single-defect engineering.