A cell nanoinjector based on carbon nanotubes

Abstract

Technologies for introducing molecules into living cells are vital for probing the physical properties and biochemical interactions that govern the cell's behavior. Here, we report the development of a nanoscale cell injection system (termed the nanoinjector) that uses carbon nanotubes to deliver cargo into cells. A single multiwalled carbon nanotube attached to an atomic force microscope (AFM) tip was functionalized with cargo via a disulfide-based linker. Penetration of cell membranes with this "nanoneedle" was controlled by the AFM. The following reductive cleavage of the disulfide bonds within the cell's interior resulted in the release of cargo inside the cells, after which the nanoneedle was retracted by AFM control. The capability of the nanoinjector was demonstrated by injection of protein-coated quantum dots into live human cells. Single-particle tracking was used to characterize the diffusion dynamics of injected quantum dots in the cytosol. This technique causes no discernible membrane or cell damage, and can deliver a discrete number of molecules to the cell's interior without the requirement of a carrier solvent.

Fig. 1.

Schematic of the nanoinjection procedure. A MWNT-AFM tip with cargo attached to the MWNT surface via a disulfide linker penetrates a cell membrane. After disulfide reduction within the cell's cytosol, the cargo is released and the nanoneedle is retracted.

Fig. 2.

Characterization of nanoneedles before and after loading the cargo. (A) SEM image of a MWNT-AFM tip. (B) TEM image of the tip region of A. (C) TEM image of a MWNT-AFM tip coated with linker 1 and conjugated with QDot streptavidin.

Fig. 3.

Functionalization of MWNT-AFM tips. (A) QDot streptavidin was attached to the MWNT surface although linker 1 containing a disulfide bond: (i) 1, MeOH; (ii) QDot streptavidin, borate buffer. (B) QDot streptavidin was attached to the MWNT surface although linker 2 containing no disulfide bond: (iii) 2, MeOH; (iv) QDot streptavidin, borate buffer.

Fig. 4.

Nanoinjection of QDot streptavidin conjugates into a target HeLa cell. (A) Fluorescence image of the cells before nanoinjection. (B) Combined bright-field and fluorescence image of the cells before nanoinjection. The inserted arrow indicates the target cell. The dark shape in the lower left corner is the AFM cantilever. (C) Fluorescence image of the cells after the nanoinjection, showing fluorescent QDot streptavidin conjugates released inside the target cell. (D) Combined bright-field and fluorescence image of the cells after the nanoinjection. The QDot streptavidin conjugates are shown in red. The dark shape in the upper left corner is the retracted AFM cantilever. (E) Combined bright-field and fluorescence image of another four examples of HeLa cells after nanoinjection of QDot streptavidin. In all cases, fluorescence images were acquired with λ_ex = 415 nm and data collection with a 655-nm filter. Images are 70 × 70 μm in A–D and 30 × 30 μm in E.