Nanotube-Protein conjugation using N-(1-Pyrene)maleimide linker

This protocol details the use of pyrene-linker N-(1-Pyrene)maleimide to attach a protein with a thiol (-SH) group found on the amino acid residue Cysteine to a carbon nanotube. For this reaction to function correctly, the protein must have an exposed thiol group for the linker to bind to. The linker covalently bonds to the protein and non-covalently binds to the carbon nanotube through pi-pi stacking. Preparation and execution of this protocol should take approximately 4 hours.

Preparation: Gather all the glassware needed beforehand and make sure all tools are thoroughly washed and rinsed with DI water. Proteins are very sensitive to their environment and must be handled carefully. Recommended glassware and plastics are listed below with amounts varying depending on the number of chips being processed (minimum shown).

  • 100×50 Bath Dish (x4) - For various baths
  • 1.5 mL Microtube (x2) - For pyrene-linker and protein soaks
  • 50 mL Plastic Falcon Tubes (x3) - For mixing solutions
  • 100 mL Beaker (x2) - For holding Ethanol and diH2O

Pipettes:

  • 1000 uL Pipette with corresponding tips
  • 100 uL Pipette with corresponding tips

Solutions:

  • 20mM Na2HPO4 (Sodium Phosphate) (100 mL minimum)
  • 0.1% Tween-20 in Ethanol (150 mL minimum)
  • 1mM Pyrene Maleimide in Ethanol (1 mL minimum)
  • 10mM KCl (Potassium Chloride) (50 mL minimum)
  • diH2O (250mL minimum)

It is recommended to use concentrated stock solution to prepare the solutions listed above directly before performing the experiment.

Protocol:

  1. Pipette 1 mL of 1mM Pyrene Maleimide into a 1.5 mL Microtube. Make sure the pyrene maleimide solution is vortexed before pipetting as the pyrene maleimide will fall out of solution. Place a dry chip in the pyrene maleimide solution and close the top (shaking the microtube to ensure the pyrene maleimide is still in solution before adding the chip). Make sure the face of the chip is not stuck to the side wall of the Microtube. Let the chip soak in solution for 30 minutes.
  2. While the chip is soaking in the pyrene maleimide, prepare a 100 mL solution of 0.1% Tween-20 in Ethanol in a 100×50 Bath Dish. It is recommended that a 50 mL Falcon Tube is used to mix a stock solution of Tween with Ethanol. Once the chip has soaked in pyrene maleimide for 30 minutes, quickly transfer the chip to the 0.1% Tween+ethanol bath. Place the bath on the shaker and set at 110 RPM for 30 min. The chip will sit in this wash phase for 30 minutes total.
  3. While the chip is shaking in the 0.1% Tween+ethanol, prepare a 100 mL solution of 0.05% Tween+ethanol and 10mM Na2HPO4. It is recommended to mix a 0.1% Tween+ethanol solution in a 50 mL Falcon Tube and a 20mM Na2HPO4 solution in another 50 mL Falcon Tube and add both solutions to the 100×50 Bath Dish directly before the solution is needed. Once the chip has been washed in the 0.1% Tween+ethanol solution for 30min, quickly transfer it to the 0.05% Tween+ethanol, 20mM Na2HPO4 solution and set on the shaker for 10 minutes at 110 RPM. It is also good to take the protein out of the freezer at this time to begin thawing. The protein should be gradually warmed up to room temperature (do not use a hot plate or warm water bath to quickly warm the protein).
  4. While the chip is shaking in the 0.05% Tween+ethanol, 20mM Na2HPO4 solution, prepare a 100 mL diH2O water bath in a 100×50 Bath Dish. After soaking for 10 minutes in the 0.05% Tween+ethanol, 20mM Na2HPO4 solution, transfer the chip to the water bath and place on the shaker for 5 minutes at 110 RPM.
  5. While the chip is shaking in the water bath, prepare a 0.5 mL - 1.0 mL protein solution (making sure to keep it appropriately buffered, 20mM Na2HPO4 works for a variety of proteins) of the desired concentration in a 1.5 mL Microtube. The 2012 Science paper reports using a 50uM concentration, however lower concentrations (~100 nM) are more typical for protein experiments. After 5 minutes in the shaking water bath, the chip should be quickly transferred to the Microtube and sealed in. Make sure the face of the chip is orientated up and that the chip is completely submerged in the protein solution. The chip should soak in the protein solution at room temperature for 1 hour.
  6. While the chip is soaking in the protein solution, prepare a 100 mL solution of 5mM KCl, 10mM Na2HPO4, 0.05% Tween+diH2O. It is recommended that a 10mM KCl, 20mM Na2HPO4 solution is prepared in a 50 mL Falcon Tube and a 0.01% Tween+diH2O solution is prepared in another 50 mL Falcon Tube and that both are added to the 100×50 Bath Dish right before use. After soaking for 1 hour in the protein solution, quickly transfer the chip to the 100 mL solution of 5mM KCl, 10mM Na2HPO4, 0.05% Tween+diH2O and set on the shaker at 110 RPM for 30 minutes.
  7. While the chip is shaking in the 100 mL solution of 5mM KCl, 10mM Na2HPO4, 0.05% Tween+ethanol, prepare a 100 mL diH2O bath. After shaking for 30 minutes in the 100 mL solution of 5mM KCl, 10mM Na2HPO4, 0.05% Tween+ethanol, quickly transfer the chip to the water bath and set on the shaker at 110 RPM for 5 minutes.
  8. After shaking in the water bath for 5 minutes, remove the chip and carefully blow dry with nitrogen. The chip has now undergone the full protocol and should have protein bound to the nanotube through a pyrene maleimide linker molecule.

Condensed Protocol:

  1. 30 min soak in 1mM pyrene linker in ethanol, no agitation
  2. 30 min shake in 0.1% Tween-20 in ethanol, 110 RPM
  3. 10 min shake in a 50-50 ratio of 10mM Na2HPO4 and 0.05% Tween-20 in ethanol, 110 RPM
  4. 5 min shake in diH2O, 110 RPM
  5. 60 min protein soak, no agitation
  6. 30 min shake in a 50-50 ratio of 5mM KCl+10mM Na2HPO4 in diH2O and 0.05% Tween-20 in diH2O, 110 RPM
  7. 5 min shake in diH2O, 110 RPM
  8. Blow dry with nitrogen

Notes: Pyrene Maleimide is relatively stable and longer protein soaks are possible if the binding efficiency is low (binding efficiency is relative to the amount and accessibility of thiol group on the protein). If preparing the device for eventual electrical measurements in solution, instead of performing the final diH2O wash and drying the chip, store it in buffer (MOPS, Na2HPO4, ect). After starting the protocol, a chip should never dry out until the entire experiment is completed. Some proteins may require additional buffers to perform correctly, these should be introduced in appropriate concentrations after the protein soak stage (but not be included in the diH2O wash if the chip is getting dried immediately after the protocol is finished).

Nanotube-protein conjugation using pyrenebutanoic acid, succinimidyl ester (Pyrene-NHS) linker

This protpcol details the use of pyrene-linker pyrenebutanoic acid, succinimidyl ester (now referred to as pyrene-NHS) to attach a protein non-covalently to a carbon nanotube. The interaction is a combination of a covalent bond formed between pyrene-NHS and the target protein, and a non-covalent bond, specifically pi-pi stacking, between the pyrene-NHS molecule and the carbon nanotube. Preparation and execution of this protocol should take approximately 16-24 hours which includes an “overnight” waiting period and a 6 hour waiting period.

Preparation: Gather all the glassware needed beforehand and make sure all tools are thoroughly washed and rinsed with DI water. Proteins are very sensitive to their environment and must be handled carefully. Recommended glassware, plastics, and solutions are listed below with amounts varying depending on the number of chips being processed (minimum shown).

  • 100×50 Bath Dish (x1) - For diH2O bath
  • 1.5 mL Microtube (x2) - For pyrene-NHS and protein soaks
  • 50 mL or 100 mL Beaker (x1) - For holding DMF

Pipettes:

  • 1000 uL Pupette with corresponding tips
  • 100 uL Pipette with corresponding tips

Solutions:

  • 2.3 mg/mL Pyrene-NHS in DMF (1 mL)
  • 30 mL DMF
  • 1 mL of protein at desired concentration (50uM can be a good place to start)
  • diH2O (100mL minimum) - For washing

Note: This protocol may change depending on the solubility of various proteins. If a protein is not soluble in H2O, a different washing solution is needed.

It is recommended to use concentrated stock solution to prepare the solutions listed above directly before performing the experiment.

Protocol:

  1. Pipette 1 mL of 2.3 mg/mL Pyrene-NHS+DMF in to a 1.5 mL Microtube. Place a dry chip in the solution and close the lid. Make sure the face of the chip is completely covered in liquid and is not stuck to the side of the Microtube. Put the Microtube sideways (still ensuring the entire chip is covered) on the shaker and set to 150 RPM for 2 hours.
  2. Roughly 30 minutes before the chip is done shaking, start preparing the protein solution in a 1.5 mL Microtube. The protein should be in a buffer that keeps the protein in a stable configuration and does not allow unfolding.
  3. Once the chip has been shaking for 2 hours, pull the chip out, quickly dip it in a DMF bath (30 mL or greater), then transfer it to the protein solution. Make sure the chip is completely submerged in the protein solution and let it sit overnight (6+ hours) without agitation.
  4. After the chip as been sitting in the protein solution for a number of hours, transfer it to a 100 mL wash solution in a 100×50 Bath Dish. The was solution is dependent on the solubility of the protein. If the protein is water soluble, a bath of 0.05% Tween in diH2O can be used. The chip should be transferred straight from the protein solution to the wash bath. The bath should be placed on the shaker and set at 110 RPM for 6 hours.
  5. After shaking 6 hours, place the chip in a 100mL diH2O bath for 5 minutes then carefully blow dry with nitrogen. The chip should now have protein bound to the nanotube through a pyrene-NHS linker molecule.

Condensed Protocol:

  1. 2 hour shake in 2.3 mg/mL Pyrene-NHS+DMF, 150 RPM
  2. Quick dip in DMF bath
  3. Overnight soak in protein solution
  4. 6 hour shake in wash buffer
  5. 5 min shake in diH2O bath
  6. Blow dry with nitrogen

Notes: Pyrene-NHS is relatively reactive and degrades quickly so it is recommended that a fresh solution be prepared for every experiment. If preparing the device for eventual electrical measurements in solution, directly after the final diH2O wash, store the chip in a buffered solution that conserves the electronic integrity and protein integrity. After starting the protocol, a chip should never dry out until the entire experiment is completed.


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