In this study, we have investigated a new method to determine the pI values of proteins compared to the conventional methods. We developed a new sensing platform and paradigm to succeed it. The track-etched poly(ethylene terephthalate) (PET) membranes were used to fabricate both single and multipore membranes. Two-step chemical etching method was prefered to fabricate single nanopore in a reproducible manner. EDC-coupling was used to modify and attach the proteins inside the nanopore surface. By the aid of ion current rectification, the surface charge characteristics of nanopores were identified with and without protein. A close approximation was successfully made to the literature values for the pI values of proteins using nanopore membranes respect.