A high precision method for length-based separation of carbon nanotubes using bio-conjugation, SDS-PAGE and silver staining

Abstract

Parametric separation of carbon nanotubes, especially based on their length is a challenge for a number of nano-tech researchers. We demonstrate a method to combine bio-conjugation, SDS-PAGE, and silver staining in order to separate carbon nanotubes on the basis of length. Egg-white lysozyme, conjugated covalently onto the single-walled carbon nanotubes surfaces using carbodiimide method. The proposed conjugation of a biomolecule onto the carbon nanotubes surfaces is a novel idea and a significant step forward for creating an indicator for length-based carbon nanotubes separation. The conjugation step was followed by SDS-PAGE and the nanotube fragments were precisely visualized using silver staining. This high precision, inexpensive, rapid and simple separation method obviates the need for centrifugation, additional chemical analyses, and expensive spectroscopic techniques such as Raman spectroscopy to visualize carbon nanotube bands. In this method, we measured the length of nanotubes using different image analysis techniques which is based on a simplified hydrodynamic model. The method has high precision and resolution and is effective in separating the nanotubes by length which would be a valuable quality control tool for the manufacture of carbon nanotubes of specific lengths in bulk quantities. To this end, we were also able to measure the carbon nanotubes of different length, produced from different sonication time intervals.

Fig 1

X-ray diffraction (XRD) patterns of A) SWCNTs, B) lysozyme, and C) Conjugated lysozyme-SWCNTs.

Fig 2. FTIR spectra showing amide bonds in the conjugated lysozyme-SWCNTs.

FTIR spectrum for free lysozyme (red), the SWCNTs (green) and conjugated lysozyme-SWCNT blue).

Fig 3

A) SEM image of SWCNTs before conjugation B) SEM image of conjugated lysozyme-SWCNTs at a) a magnification 1.50K, Diameter of the SWCNT bundle ≈ 592 nm, b) a magnification of 10 K, c) a magnification 30.0K, diameter of conjugated lysozyme-SWCNT ≈ 89.5 nm.

Fig 4. The kinetics of bacterial cell lysis was monitored as represented by a plot of O.D. at 450 nm vs. time (Lysozyme (blue) and lysozyme-SWCNTs (red)).

Fig 5. SDS-PAGE electrophoresis of lysozyme, SWCNTs, and conjugated lysozyme with coomassie blue staining (left), and silver staining (right).

Lane 1 is lysozyme, lane 2 is conjugated lysozyme-SWCNT, and lane 3 is SWCNT.

Fig 6. SDS-PAGE and silver staining of sonicated samples of lysozyme-SWCNTs, lanes 1 and 4 are free lysozyme, and lane 3 is SWCNTs, lane 2, 9, and 10 are sonicated conjugated lysozyme-SWCNT samples for 3, 7, and 10 min, respectively.

It is clear the conjugated SWCNTs are separated based on their lengths.

Fig 7. Scanning electron micrographs of conjugated lysozyme-SWCNTs.

Fig 8. Distribution and the lengths of SWCNT fragments in two methods (MATLAB, 8A through 8C & ImageJ, 8D through 8F) after sonication time at 3 min (8A & 8D), 7 min (8B & 8E), and 10 min (8C & 8F).

The length of CNTs are in concordance with two methods ranging between 43 to 63 microns when calculated using MATLAB and 47 to 63 microns when ImageJ is used.

Fig 9. Length distribution of the conjugated SWCNTs.

The intensity of the CNTs at each lane is plotted versus length of CNTs calculated from Eq (1).