Graphene Biosensor-Based Field-Effect Biosensing to Detect Protein-Protein Interactions

Protein-protein interactions, PPIs, are critical in regulating most biological processes.

To detect PPIs using field-effect biosensing, begin with a graphene field-effect transistor chip. The chip comprises a carboxyl-functionalized graphene surface as the semiconductive channel between the source and drain electrodes.

Incubate the chip with a fresh solution of carbodiimide and N-hydroxysulfosuccinimide, sulfo-NHS, in a zwitterionic buffer. This buffer creates a suitable pH, enabling carbodiimide and sulfo-NHS to activate the chip's functionalized carboxyl groups, forming amine-reactive esters.

Add the target protein solution. The primary amines react with the amine-reactive esters, immobilizing the proteins on the chip through amide bond formation. Add amine-containing quenching solution to block unbound amine-reactive esters, preventing non-specific binding.

Insert the chip in the electronic reader and calibrate it with a buffer. The reader applies a constant voltage between the electrodes. The resulting electrical current flow between the electrodes in the analyte-unbound condition is measured.

Add the lowest analyte concentration. The interaction between analyte proteins and the targets immobilized on the chip alters the local charge distribution of the graphene surface, recorded as I-response — the change in current flow between the electrodes.

Post-recording, wash with a buffer to dissociate the bound analytes. Repeat the analysis with increasing analyte concentrations.

A gradual increase in I-response with increasing analyte concentrations indicates strong PPIs between the target and analyte.

Begin by mixing equal volumes of EDC and sulfo-NHS solution by pipetting up and down. Place the biosensor chips supplied by the company in a glass Petri dish with a fitted lid. All the functionalization steps involved in chip activation are suggested to be done within the Petri dish. Apply 50 microliters of 1 molar MES buffer to the biosensor chip. Incubate for 1 minute at room temperature.

Then, aspirate the buffer and apply 50 microliters of EDC/sulfo-NHS solution immediately to the sensor chip. Cover the Petri dish and incubate for 15 minutes at room temperature. Aspirate the EDC/sulfo-NHS solution from the chip, and apply 50 microliters of 1 molar MES buffer.

Aspirate MES buffer and rinse the chip twice with 50 microliters of 1x PBS. Aspirate the PBS from the chip, and add the target molecule Hsp90. Cover the glass Petri dish, and incubate for 30 minutes at room temperature. Then, aspirate the solution containing the target molecule, and rinse three times with 50 microliters of 1x PBS.

Aspirate the 1x PBS solution from the chip, and add 50 microliters of the Quench 1 solution. Cover the glass Petri dish, and incubate for 15 minutes at room temperature. Aspirate the Quench 1 solution from the chip, and add 50 microliters of the Quench 2 solution. Cover the glass Petri dish and incubate for 15 minutes at room temperature. After that, aspirate the Quench 2 solution from the chip, and rinse the chip five times using 50 microliters of 1X PBS leaving the last PBS droplet on the sensor.

Prepare the analyte dilution series for Cdc37 in the desired concentration range. Design the experiment to include at least eight different analyte concentrations to obtain a reliable dissociation constant or K_d value, and prepare these different dilutions in the same buffer used for calibration and target protein.

Place the chip carefully into the instrument. Make sure the software is on and the LED light turns green. Next, press the "Run Experiment" module on the automated software and choose "10 Points With Regeneration" or any other desired protocol. Fill in the details, such as operator name, experiment name, date, regeneration buffer, immobilized target, and the analyte in solution. Press the "Begin Experiment" button displayed on the software, and follow the instructions shown by the automated software.

To perform the instrument calibration, aspirate the remaining PBS solution from the chip and apply 50 microliters of the calibration buffer. Press the 'Continue' button and wait for five minutes until the calibration step is finished. The software displays the end point determined for the calibration step with a warning alarm to follow up.

Next, perform an analyte association by aspirating the calibration buffer from the chip and applying 50 microliters of the lowest analyte concentration. Press the 'Continue' button and wait for five minutes until the association step is finished. The software displays the end point for the association step with a warning alarm to proceed.

To perform an analyte dissociation, aspirate the analyte solution from the chip and apply 50 microliters of the dissociation buffer. Press the 'Continue' button. After the dissociation step duration, the software displays the end point for the dissociation step with a warning alarm.

Next, perform chip regeneration by aspirating the dissociation solution from the chip, and applying 50 microliters of the regeneration buffer. Then, press the 'Continue' button. The regeneration step typically takes approximately 30 seconds to finish. After that, the software displays the end point for the regeneration step with the warning alarm to follow up.

Finally, to wash the chip aspirate the regeneration solution from the chip, and apply 50 microliters of the wash buffer. Aspirate the solution from the chip and repeat this five times. Leave the last drop of the wash buffer on the chip. Then, press the 'Continue' button and wait for 30 seconds until the wash step duration is finished in the software display.

Repeat the steps for each analyte concentration used. The five steps of the calibration, analyte association, dissociation, regeneration, and washing five times constitute one cycle.