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Novel Mechanism for Attachment of Peptides to Functionalized Carbon Nanotubes

Background:

 

This project was originally started as a part of my nanochecmistry research project at COSMOS at UCSC in Summer, 2013. The project was supervised by Dr. Shaowei Chen from UCSC and Dr. Roger Terrill from SJSU. The following is the my presentation I gave at COSMOS UCSC.

I conducted lab experients under the supervison of Dr. Roger Terrill at SJSU to find if there is a possible mechanism for the direct attachment of peptides to carbon nanotubes so that the biological functionalization of carbon nanotubes is made more simple and effective.

 

Objectives:

1. To discover a novel reaction for the attachment of proteins to the sidewalls of carbon nanotubes

2. To explore the properties of the modified carbon nanotubes to prove they have been functionalized

  
Lab Research

Chemicals:

1. SWNT – 99% (w/t) provided by CheapTubes

2. Dimethyl Foramide(DMF) – 99% anyhydrous

3. Paraformadelhyde – pure reagent

4. L-(+)-Cysteine Hydrochloride

5. Toluene

6. Distilled Water

7. Acetonitrile

Observations: 

Clear solution of carbon nanotubes, paraformaldehyde, and DMF turned yellow upon addition of cysteine in DMF. Reaction continued to darken in color, from pale yellow to a golden orange. Within one hour, the reaction had darkened into a deep red and after the addition of the second portion of cysteine in DMF, the color shifted to a red-brown. This color was maintained throughout the remainder of the reaction.

 

Centrifugation:

Supernatant was drawn off from the reaction vessel and centrifuged at 2000 rpm for 10 minutes. After centrifugation, black CNTs were observed at the bottom of the centrifuge tube. When shaken, the carbon nanotubes dissolved back into solution and were no longer observed at the bottom of the tube. Repeated rounds of centrifugation produced the same result.

 

Sonification:

Supernatant and carbon nanotubes drawn from reaction vessel were placed in a vial and sonificated. Semi-suspension was observed.

Procedure:

6mg of carbon nanotubes and 20mg of paraformaldehyde were suspended in 20mL of DMF and placed into round-bottom flask (the reaction vessel). The heterogenous mixture was stirred rapidly and heated to 130ºC to allow for a steady reflux. A solution of 20mg of cysteine in 2mL of DMF was added in 2 portions. The first was added to begin of the reaction and the second was added 2 hours later. The reflux was allowed to proceed for 24 hours. 2mL of supernatant was drawn off and centrifuged. The carbon nanotubess remaining at the bottom of the reaction vessel were placed into a glass vial and centrifuged. The supernatant was drawn off and the carbon nanotubes were washed with DMF. Solubility tests were completed with the modified carbon nanotubes in acetonitrile, water, DMF, and toulene. FTIR scans were also done with the modified carbon nanotubes, using toulene as a transfer solvent. The solvent was evaporated with a heat gun before scanning began.

Equipments:

1. Reflux setup consisting of round-bottom flask, condensor, rubber septum, machine for water control, magnetic stir bar, and heating mantle

2. Glassware, including beakers, pipets, vials, and graduated cylinders for the preparation and transfer of chemicals Metal spatula

3. Centrifuge machine and plastic centrifuge tubes

4. Syringe and Filter

5. Sonificator

 

Interpretation of Data:

From the data and observations, it was proven that the reaction was successful and a new mechanism for the functionalization of carbon nanotubes was discovered. Throughout the reaction, the color change indicated that some reaction was occurring, most likely involving the carbon nanotubes. carbon nanotubes tend to change color during reactions. In addition, when supernatant from the reaction was centrifuged, modified carbon nanotubes were observed to come out of solution and could be pushed back into solution by shaking the mixture several times. Also, the carbon nanotubes were observed to bundle differently than the pristine carbon nanotubes. When sonificated, the reacted carbon nanotubes, spread out and suspended in solution. This is not observed with unreacted carbon nanotubes, increasing evidence that the carbon nanotubes were modified. The solubility tests with the carbon nanotubes were the final confirmation that the carbon nanotubes had reacted. Pristine carbon nanotubes do not interact at all with organic or non- organic solvents. However, my modified tubes were observed to stay suspended in varying solvents. The carbon nanotubes seemed to prefer neither extremely polar or nonpolar solvents, but rather a middle between this range. This was observed in the full suspension of the carbon nanotubes in both acetonitrile and toulene.

Solubility Tests:

Reacted carbon nanotubes were collected and placed into centrifuge tubes. Acetonitrile, water, DMF, and toulene were added until the heterogeneous solution was 1mL. The mixtures were sonificated for 30 seconds and partial suspension for all solvents. The carbon nanotubes were especially soluble in acetonitrile and toulene. A white, milky suspension was observed in toulene, indicating the carbon nanotubes were completely suspended in solvent.

 

FTIR Scans: [citreation 57]

Abstract:

This project explores novel reaction for the direct attachment of proteins to the sidewalls of carbon nanotubes, eliminating the problems found in other methods of functionalization. This research also determines the properties of the modified carbon nanotubes to prove they have been functionalized. A 1,3 cycloaddition with a peptide and aldehyde was used to functionalize the carbon nanotubes. Cysteine was chosen as the peptide to attach to the carbon nanotubes because it could be further reacted with.Centrifugation, sonification, solubility tests, and FTIR spectroscopy were used to analyze the modified carbon nanotubes. Both the centrifugation and sonification of the reacted carbon nanotubes showed the nanoobjects had reacted. The carbon nanotubes were able to go in and out of solution through centrifugation. Sonification of the nanotubes resulted in semi-suspension of the nanotubes. The solubility tests showed that the nanotubes were able to fully suspend moderately polar solvents. The FTIR scans of modified carbon nanotubes showed remarkable differences from those of unreacted carbon nanotubes.This project has shown the successful attachment of peptides to carbon nanotubes and the hypothesis was proven correct. The modified carbon nanotubes are soluble in multiple solvents and this new mechanism allows proteins to be attached efficiently to carbon nanotubes under mild reaction conditions. After attachment of the first peptide, multiple peptides can be attached and a custom peptide chain can be created on the carbon nanotube, increases the versatility of carbon nanotubes in biological applications.This research has provided a novel reaction for the direct linkage of peptides to carbon nanotubes.

Acknowledgement:

 

Thanks to Dr. Roger Terrill, Associate Professor at SJSC, for his guidance in this project. I also thank him for allowing me to use his equipment to complete my research in his lab.

Thanks to CTY Cogito Research Awards for sponsoring my reseach.

Thanks to Dr. John D. Tovar, Associate Professor at Johns Hopkins University for being my mentor on this project.

Thanks to Cheap Tubes to provide me SWNTs for my research.

Conclusion:

This project has shown the successful attachment of peptides to carbon nanotubes and the hypothesis was proven correct. This research has built upon research done on 1,3 cycloaddition of azomethine ylides to fullerenes and illustrated a new method for sidewall functionalization of carbon nanotubes with peptides. The modified carbon nanotubes are soluble in multiple solvents and this new mechanism is a step towards the synthesis of peptide-based carbon nanotubes. This mechanism has the advantage of needing no intermediates between the carbon nanotubes and the peptides. Proteins can be attached directly to carbon nanotubes and this reaction increases the versatility of carbon nanotubes in biological applications. Carbon nanotubes containing peptides can now be made quickly and efficiently. After attachment of the first peptide, multiple peptides can be attached and a custom peptide chain can be created on the carbon nanotubes. The mild reaction conditions of this project also provide added benefits that the carbon nanotubes are not damages or changed in any way, other than the sidewall functionalizations. This research has provided a novel reaction for the direct linkage of peptides to carbon nanotubes. From gene delivery to vaccination, carbon nanotubes have a bright future in the biomedical fields and this research will make it allow the full potential of these nanoobjects to be fulfilled.

 

Future Research:

Future research can be done to improve this reaction. Firstly, this reaction was carried out with only single-walled carbon nanotubes (SWNTs) but not with multi-walled carbon nanotubes (MWNTs). Similar research (original) involving other cycloadditions has shown that MWNTs should react similarly to SWNTs. However, more experimentation is need to confirm this. Also, the ratios of reagents need to be made more efficient. it was not clear how much reactants were needed to react with the carbon nanotubes so an excess was added. Further research could result in an ideal formula for the reaction so that the process could be streamlined for laboratory and even industrial use. Finally, only one peptide, cysteine, was tried in this project. Other proteins should be tried to observe the effect of varying functional groups during the reaction. This project has paved way for a new process of carbon nanotube functionalization. With further research, the benefits of carbon nanotubes can be brought to the world.

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