Encyclopedia of Experiments: Biological Techniques
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Transcript
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.
