Carbon nanotubes are novel carbon-based materials made up of “tubes” of graphene sheets. They have a range of fascinating properties that have made them the focal point of a huge amount of research. In particular, bioconjugates of carbon nanotubes are of interest because of their useful biomedical applications. 

Methods for the bioconjugation of carbon nanotubes commonly include aldehyde bioconjugation, amine-based techniques, cysteine bioconjugation, and conjugates that utilize tyrosine residues. 

What are Carbon Nanotubes? 

Carbon nanotubes, sometimes shortened to CNTs, are fascinating chemical molecules that were discovered in 1993. CNTs are made up of “tubes” of carbon which are effectively rolled-up sheets of single-layer carbon atoms. Their basic unit is made up of graphene, a simple single layer of carbon atoms arranged in a honeycomb lattice. Just like graphene, CNTs have very strong carbon-carbon bonds because of their sp2 hybridization. 

Carbon nanotubes are made up of sheets of carbon atoms in a hexagonal arrangement that are rolled up into cylindrical tubes. They can be single-walled (SWCNT) or multiwalled (MWCNT). 

CNTs can be formed of single-walled carbon nanotubes (SWCNTs) which have a diameter of less than 1 nm or multi-walled carbon nanotubes (MWCNTs) which contain many linked structures with much larger diameters of over 100 nm.

Bioconjugation of Carbon nanotubes can be easily accomplished using aldehyde and amine based methods
Carbon nanotubes are made up of rolled-up sheets of graphene into tubes. In the example above, the nanotube is an SWCNT (source).

What are the Benefits of Carbon Nanotubes?

The benefits of carbon nanotubes include variable electrical conductivity, high mechanical strength, lightweight, high stability, and more which allows them to be used in a range of applications such as electronics, sensors, batteries, light sources, and even for drug delivery. 

Just like their building block graphene, CNTs have many fascinating properties that have made them a target for research into potential applications over the last three decades. The most interesting of these properties is their conductivity. CNTs can be conducting, semiconducting, or non-conducting depending on their structure. Beyond that, they have high mechanical strength, are very lightweight, have high thermal conductivity, and are highly stable. 

Thanks to this incredible combination of properties, CNTs are excellent candidates for being used in electronic devices, sensors (chemical, electrochemical, and biological), batteries, field emitters, light sources, hydrogen storage, and more. There is even significant research into their potential applications for drug delivery, with new and efficient systems based on CNT nanomaterials.

Due to their unique structure and electrical conductivity, carbon nanotubes have a variety of applications in electronics. Source: RSC

What are Nano Bioconjugates of Carbon Nanotubes?

Bioconjugate chemistry is the study of linking molecules to biomolecules. This could mean linking one biomolecule to another or linking a biomolecule to a non-biological molecule such as a simple organic molecule, a metal, a particle, or a surface. The study of bioconjugation is a huge field and includes a range of biomolecules, applications, and possibilities. 

Nano bioconjugates of carbon nanotubes include two molecules attached together: a carbon nanotube structure and some other biomolecule like a protein or carbohydrate.

Bioconjugated CNTs have received a lot of attention in recent years, particularly for their biomedical applications. The potential applications of bioconjugated CNTs include biosensors, drug delivery systems, and biological/medical imaging

Functionalization of Carbon Nanotubes

The functionalization of CNTs has been studied in detail in the last three decades, but even now the majority of popular methods produce samples with mixtures of various diameters and chiralities of the nanotubes. To overcome that, successful methods often use post-synthesis processing to capture the desired CNT size and chirality. 

Carbon nanotubes can be functionalized using covalent functionalization or non-covalent functionalization. One example of covalent conjugation of carbon nanotubes involves creating amino-CNTs from fluorinated CNTs and then attaching them to the carboxyl group of a protein.

Covalent Functionalization of Carbon Nanotubes

CNTs can be functionalized by the covalent bonding of functional groups onto the carbon of CNTs. This can be performed at the end caps of the nanotubes or on the side walls depending on the desired functionality. However, sidewall functionalization changes the hybridization of the carbon bonds from sp2 to sp3 which changes the conjugation structure of the carbon layer.

Fluorinated CNTs have been one of the most popular methods of CNT functionalization since the discovery of CNTs. Fluorinated compounds are often highly reactive and are strong enough to react with the highly stable sp2 carbon-carbon bonds in the CNT structure. Furthermore, fluorinated CNTs have weaker C-F bonds than expected in a typical alkyl fluoride which makes them more ideal for replacement with other functional groups such as amino, alkyl, and hydroxyl groups which can then be used for bioconjugation reactions. 

Example method: Amino CNTs from fluorinated CNTs

The authors of this paper managed to produce amino-functionalized SWCNTs from fluorinated CNTS. They expected them to be potential synthetic precursors to bind amino acids, DNA, or even polymers to sidewalls of SWCNTs. To see how these amino-CNTs can be used to create a bioconjugate, see the section on “Amine Bioconjugation with Carbon Nanotubes” in the article!

Step 1. Suspend your fluorinated CNTs in your amine solution. 

The authors suspended a small quantity of the CNTs (10-20 mg) in 5 mL of a terminal alkylidene diamine and sonicated it for 3 minutes.

Step 2. Add pyridine to catalyze the reaction. 

The authors then added 5 drops of pyridine to act as a catalyst to start the reaction.

Step 3. Heat and stir the reaction under a nitrogen atmosphere for 3 hours at ~150-170°C.

The reaction must be performed under nitrogen to ensure you don’t form any unwanted side products. 

Step 4. Collect your amino CNT by filtration and wash with ethanol.

The authors used a 0.2 um pore size Teflon membrane to collect the amino CNT and washed it with large amounts of ethanol.

Step 5. Dry your amino CNT in a vacuum oven overnight. 

Once dried overnight, you’ve successfully made your amino-functionalized carbon nanotubes. 

Non-Covalent Functionalization of Carbon Nanotubes

Non-covalently functionalized CNTs have the great advantage of not destroying the conjugated system of the CNTs sidewalls which means you can maintain your desired functional properties. However, the downside to non-covalent methods is that they are less stable. CNTs can be wrapped with polymers or surfactants using non-covalent interactions, and aromatic small molecules can be absorbed onto the surface walls of the CNT by ππ stacking interactions

Example method: Surfactant Functionalized CNTs

The authors of this paper used a sodium dodecylbenzene sulfonate surfactant to successfully solubilize high weight SWCNTs in water. SWCNTs of high weight, unfortunately, have strong interactions and like to aggregate in water. This makes them difficult to suspend in water which is essential for making further modifications. 

Step 1. Add your CNT to water. 

Your CNTs are likely to have poor solubility and begin to aggregate at the bottom of the water.

Step 2. Add your surfactant to the water and sonicate. 

Sonication will begin the interaction between the surfactant and your CNTs. 

Step 3. Give your CNTs time to interact with the surfactant. 

The authors of the paper gave their CNTs 24 hours to interact with the surfactant. If your CNTs reaggregate, you may need to adjust the amount of surfactant or choose a different surfactant. 

Bioconjugation of Carbon Nanotubes

There are countless types of bioconjugate linkers that can be utilized with carbon nanotubes, too many to list here. Instead, here some common examples: 

Aldehyde Bioconjugation with Carbon Nanotubes

Aldehyde functional groups on biomolecules can be bioconjugated to carbon nanotubes to produce new biomolecular nano molecules. The authors of this work produced a label-free electrochemical immunosensor based on cyclodextrin functionalized CNTs with ionic liquid containing ferrocene and aldehyde groups. Here’s an outline of the steps they took:

Step 1. The CNTs were linked to cyclodextrin to produce a CD-CNT.

CNTs and cyclodextrin were stirred in ultrapure water for 12 hours resulting in the CD-CNT 

Step 2. Ferrocene was reacted with the CD-CNT to produce a ferrocene-CD-CNT with aldehyde groups.

This reaction occurred by mixing the two reagents (CD-CNT and CHO-ferrocene) in DCM for 6 hours. The product was collected via centrifugation. 

Step 3. The CHO-ferrocene-CD-CNT was added to a gold electrode.

The CNT in a homogenous solution was added to the surface of a gold electrode. The excess solution was washed away with water.

Step 4. Aldehyde groups on the electrode reacted with an antibody to create the electrochemical immunosensor.

The antibody in solution was dropped onto the surface of the electrode and allowed to incubate for 60 minutes in air. Excess antibody was washed away with water.

Amine Bioconjugation with Carbon Nanotubes

Amines are commonly used in bioconjugation reactions and that is no different when producing carbon nanotube bioconjugates. The authors of this paper managed to successfully conjugate biomolecules including proteins and DNA to amino-functionalized MWCNTs. The protein they used was bovine serum albumin. The conjugate links were formed via amide formation. Here’s an outline of how they did it:

Step 1. Prepare a carboxy-CNT.

The authors outlined their method 

Step 2. React your carboxy-CNT with ethylenediamine.

The authors refluxed their carboxyl-CNT with ethylenediamine to create their amino-CNTs through amide formation. 

Step 3. Finally, react your amino-CNT with your desired biomolecule (e.g. DNA). 

DNA was added to the amino-CNTs and found to coordinate with the amino-CNTs via non-covalent interactions. 

Cysteine Bioconjugation with Carbon Nanotubes

Cysteine is one of the essential amino acids and is found in most proteins. CNTs can be bioconjugated to cysteine for several reasons. The authors of this work managed to conjugate cysteine to CNTs during the development of an electrochemical sensor for Cd(II). The authors reacted carboxylic groups of an oxidized SWCNT with amino groups of S-triphenylmethyl cysteine using coupling chemistry. Here’s an outline of how they did it: 

Step 1. Purified SWCNTs were oxidized in an H2SO4/HNO3 mixture.

This was achieved by reflux for 3 hours followed by filtering and rinsing. 

Step 2. The oxidized SWCNTs were then activated with HBTU.

HBTU was added under an argon atmosphere and the mixture was stirred for 45 mins while cooled.

Step 3. S-triphenyl methyl cysteine was added to the mixture dropwise over 24 hours.

After adding dropwise, the reaction was allowed to continue over 48 hours under argon.

Step 4. Filter your cysteine bioconjugated CNT.

The product of the reaction was filtered with a 0.1 um pore size Teflon membrane and washed with DMF, ethanol, and acetone. 

Bioconjugation to Tyrosine Residues on a Protein

It is often difficult to selectively bioconjugate a specific protein functional group. This is no different for tyrosine residues on a protein, but these authors reported successful adsorption of tyrosine residues of silk sericin (a protein from the silkworm carcoon). The interactions between the CNTs and the tyrosine residues were non-covalent interactions. The authors noted the importance of this stable non-covalent bonding in the production of electrochemical sensors. Here’s how they did it:

Step 1. Purchase or extract silk sericin.

The authors explore the process in the paper but also mention it can be purchased from some suppliers. However, they preferred their method because it had a higher purity. 

Step 2. Dissolve your sericin in water, and then add your CNTs.

The authors used MWCNTs for this experiment. 

Step 3. Sonicate the mixture for 30 minutes.

The authors estimated the power of their sonicator at 250 W.

Step 4. Centrifuge the mixture at 2500 rpm twice to remove any unbound MWCNTs. 

The authors did this twice to ensure they removed all of the unbound MWCNTs. They were left with a sericin-MWCNT dissolved in solution. 

Bioconjugation Kits and Services for Carbon Nanotubes

There are many suppliers of carbon nanotubes for different purposes. Typically, you can buy carbon nanotubes with specific functionalization that you can go on to use for further purposes. 

Carbon nanotubes for bioconjugation can be bought from specialist suppliers such as Creative Diagnostics and Cheaptubes or major suppliers such as Sigma Aldrich.

Kit and Supplier How it works Price
Creative Diagnostics Single, double, or multi-walled CNTs CNTs can be purchased with a specific number of walls, diameter, and functionality for bioconjugation Prices vary based on quantity and product. 
Cheaptubes Functionalized CNTs CNTs can be purchased with O+, COOH, NH2, N2, and F groups. Groups can be tailored to meet needs.  Prices vary based on quantity and product.
Sigma Aldrich CNTs CNTs can be purchased as single, double, and multi-walled with a variety of functional groups and conductivity.  Prices vary based on quantity and product. 

By Karthik Raman, PhD

I am a PhD Bioengineer specialized in utilizing heparan sulfate and heparin for drug delivery to brain tumors. My expertise lies in the interface between polymer chemistry, protein biochemistry, and cellular biology.

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