Introduction 1.1 General Points

1. Surfactant Solvent Methanol Triton X-100 Ethanol Acetone SDS Toluene SDBS DMF H2O Time (h) Time (d) 3+ 30 3+ 2.5 22 3+ 3+ days 2.5 2 Tab.1 Average time length for CNTs to settle down from even suspension (surfactant factor) Tab.2 Average time length for CNTs to settle down from even suspension (solvent factor) Fig.1 TEM (transmission electron microscopy) images of CNTs Left: The parallel lines are the cross-sections of the sidewalls of MWNT concentric cylinders. Right: A bundle of SWNTs. Each circle represents the cross-section of a SWNT. Fig.3 TEM images of CNTs before and after air oxidation Left: AR (as received) CNT sample (purity approx. 5-10%). Right: A sample of CNTs after oxidation at 673 K in air for 40 min (purity approx. 10-20%). • The data listed here was based on the results collected from 3 batches each. The samples were sonicated for 4 h. The organic solvent used was methanol. • SDS- sodium dodecyl sulfate; SDBS- sodium dodecyl benzene sulfonate. Synthesis Purification Raw sample Air oxidation Acid refluxing • The data listed here was based on the results collected from 3 batches each. The samples were sonicated for 4 h. The surfactant used was SDBS. • DMF- dimethylformamide. Surfactant-aided sonication for CNT dispersion Immobilisation Protein immobilisation to the processed CNTs Annealing Filtration Fig.7 Comparison of annealed samples treated with and without sonication Left: the sample was oxidized, refluxed, and followed by annealing at 1273 K for 4 h, without sonication; there were still many particles in the final product. Right: the sample was oxidized, refluxed and sonicated with SDBS as surfactant in methanol for 4 h, followed by annealing at 1273 K for 4 h; there were only few particles left (purity approx. 90%). Fig.8 Raman graphs of samples before (left) and after (right) purification Purification and Protein Immobilisation of Carbon Nanotubes Hua Gao Supervisor: Professor NKH Slater Cambridge Unit for Responsive Biopolymers (CURB) & Cambridge Unit for Bioscience Engineering (CUBE) • Introduction • 1.1 General Points • Carbon nanotube (CNT) is a one-dimension nanotube which offers lots of intriguing properties for applications. • There are two forms of CNTs: single-walled carbon nanotube (SWNT) and multi-walled carbon nanotube (MWNT) (Fig.1). • CNT synthesis methods include arc-discharge, laser ablation, and chemical vapor deposition (CVD) etc.. • Purification is carried out to remove the impurities in CNTs. Oxidation, filtration, and annealing are general CNT purification processes. • Purified CNTs are promising materials for applications in many areas. • 1.2 About This Project Fig.6 Comparison of samples treated with HCl and HNO3 A: a CNT sample refluxed with HCl. The carbon particle near the tube was damaged while the tube was intact. B: a CNT sample refluxed with nitric acid. The tube was severely eroded after treatment. Fig.5 A sample of CNTs after refluxing in HCl for 6 hours, with a purity of around 30% 3.3 Surfactant-Aided Sonication, Filtration and Annealing • CNTs are usually synthesized with a lot of impurities. Possible impurities include amorphous carbon, graphite, fullerenes, particulate carbon, metal catalysts (e.g. Fe, Ni, Y, Co, etc.). This project aims to find a method for CNT purification, with high purity, low loss, and low cost. • CNTs tend to entangle together, making it difficult for purification and applications. Surfactant-aided sonication will be used to solve this problem. • Among many intriguing applications of CNTs, the idea of using CNTs as biosensors and drug delivery systems will be studied. The electronic properties of CNTs coupled with the specific recognition properties of the immobilized biosystems would make for an ideal miniaturized sensor. Additionally the cylindric structure would be useful as a drug delivery vector. Immobilisation of biomolecules on CNTs will be carried out. • After acid refluxing, the CNT sample was purer. However, tubes were entangled together, trapping most of the impurities, such as carbon particles and catalyst particles, which made it difficult to remove the impurities by filtration. So surfactant-aided sonication was carried out. The proper processing surfactant and organic solvent were to be determined. • Based on the experimental data listed in Tab 1 and 2, SDBS-aided sonication with ethanol (or methanol) as organic solvent was chosen. Because it took the longest time for CNTs to settle down, indicating an even suspension state was achieved. • The sample was then filtered with an ultrafiltration unit (MWCO 300,000) and annealed at 1273 K in N2 for 4 h. Annealing was effective in optimizing the CNT structures, as shown in Fig 7 (the TEM photo on the right is the sample after annealing). • Effects of the sonication process can be seen from Fig. 7. It proved the surfactant-aided sonication was effective to untangle CNTs, thus to free the particulate impurities embedded in the entanglement. • As compared with the initial sample, Raman graphs showed that the amorphous carbon and other non-ordered particles had been removed (Fig.8). 2.Experimental Work • 2.1 Synthesis • Raw CNT samples were either synthesized by arc-discharge method in ICL, Oxford, or received for free from commercial supplier, with approx. 10% in purity. • 2.2 Purification • Many purification processes have been used to purify CNTs. These include air oxidation, refluxing, filtration, and annealing. • Each purification process was tested to examine its effects on the properties of the samples produced. Based on the results obtained, the ideal method was identified. This consists of a combination of processes that will give CNTs with desired properties and high purity. • Time and temperature of air oxidation were tested at different levels and a suitable condition was obtained. • Experiments were carried out to evaluate which one of HNO3, HCl, and H2SO4 was the most suitable refluxing reagent based on their influences on purity and yield of CNT samples. • It is difficult to remove the majority of nano-particulate impurities from CNT sample via filtration, because of CNTs’ tendency to entangle together. Surfactant-aided sonication can help to untangle CNTs. A suitable dispersion condition, including surfactant, solvent, and sonication time length was investigated. • After CNT samples were properly dispersed, filtration was carried out. • Annealing was a necessary step to optimize the final CNT structures, thus made them ready for further applications. • 2.3 Immobilisation • A bifunctional molecule, 1-pyrenebutanoic acid, succinimidyl ester (1) has a pyrene group, which was reported to be easy to interact with the basal plane of graphite via π-stacking. So it is believed to interact with CNT walls, the pyrene segment immobilized to the CNT sidewalls (Fig.2). • Its succinimidyl ester group is highly reactive with nucleophilic substitution via primary and secondary amines, which are commonly found on the surface of most proteins (Fig.2). • Most proteins are theoretically able to immobilize at CNTs walls via this pyrene-succinimidyl, or more precisely, π-stacking nucleophilic-substitution bridge. • The protein used for this research was streptavidin labeled with 10 nm gold. • 3.4 Protein Immobilisation • With CNT samples of high purity, protein attachment could be carried out. The micrograph of the beginning CNT sample is shown in Fig.9. The sample was incubated in a 1-pyrenebutanoic acid, succinimidyl ester solution, then in a streptavidin solution. The TEM images indicated that the streptavidin molecules were attached to CNTs. The micrographs of the resulting sample is shown in Fig.10. Fig.2 An illustration of protein immobilisation to CNTs 1: 1-pyrenebutanoic acid, succinimidyl ester Fig.10 The micrograph of the CNT sample after protein immobilisation The black dots were the Au particles labeled to streptavidins, indicating immobilisation of streptavidins onto CNT surfaces. Fig.9 A micrograph of the CNT sample before streptavidin was attached 3.Results and Discussions • 3.1 Air Oxidation • The AR (as-received) carbon nanotubes were low in purity, the average purity was about 5-10%, as seen in Fig. 3. So purification was needed before attachment of proteins onto CNTs. • Air oxidation was useful in reducing the amount of amorphous carbon and damaging the carbon passivation coating covering the metal catalyst particles (Ni, Y). This enables the maximum exposure of the catalyst particles while maintaining low losses of SWNTs. • Time and temperature of air oxidation were determined based on TGA (thermo-gravimetric analysis) results. Optimal oxidation condition was found to be at 673 K for 40 min (Fig.3shows the images of CNTs before and after oxidation). 4.Conclusions and Future Work • Raw CNT samples were purified from 10% to 90 % in purity; the sequence of purification processes was decided; parameters for every stage of purification were determined. • A surfactant-aided sonication for CNT dispersion was added to the method to help further purification and applications. • Streptavidin was immobilized to CNTs via 1-pyrenebutanoic acid, succinimidyl ester. • Other kinds of proteins will be tried for CNT immobilisation. Biotin will be a major research object, as it can bind specifically with avidin, thus avoiding non-specific targeting. • Protein nanoparticles and polymer nanoparticles used as drug delivery vectors will also be examined. 5.References Dai H (2002), Surface Science, 500, 218-241. Chen R.J, Zhang Y, Wang D, Dai H (2001), Journal of American Chemical Society, 123, 3838-3839. • 3.2 Acid Refluxing • Refluxing the sample in strong acid was effective in reducing the amount of metal particles and amorphous carbon. This is demonstrated in Fig. 5. • The effectiveness of HCl, HNO3 and H2SO4 were studied. Under similar conditions, H2SO4 was found to require longer time to achieve the same level of purity as HCl and HNO3. Whilst HNO3 was found to damage the CNT structure to a greater extent as compared to HCl (Fig. 6). Therefore, HCl was identified to be the ideal refluxing acid. 6.Acknowledgements Many thanks to Prof Nigel Slater, Dr Sharon Williams,Dr Mark Eccleston, and all our group members; to ICL at Oxford University and MSM at Cambridge University; to The Gates Cambridge Trust for the Gates scholarship and Universities UK for the ORS award.