Standard Protocols
Advanced Techniques
Under development
General Advice
Inspiration
Standard Protocols
Advanced Techniques
Under development
General Advice
Inspiration
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).
Pipettes:
Solutions:
It is recommended to use concentrated stock solution to prepare the solutions listed above directly before performing the experiment.
Protocol:
Condensed Protocol:
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).
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).
Pipettes:
Solutions:
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:
Condensed Protocol:
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.