Protein biosensing with track-etched single conical nanopores in polyimide

Currently, researchers are deeply involved in the field of nanopore technology due to its wide range of applications. Nanopores have been used for the detection and analysis of a variety of analytes, proteins, small organic molecules and metal ions [1-2]. So it becomes crucial to modify the chemistry of the nanopore walls to achieve desired interactions with molecules of interest. Single conical nanopores in 12 μm thick polyimide (PI), having carboxyl groups on the surface, were produced by an asymmetric track-etching process [3]. These pores have already been used successfully for the detection of DNA molecules [2] and possess the advantage of a very stable ion current signal. We have recently reported the modification of these pores by EDC/NHS activation [4]. The unmodified and modified pores were characterized by asymmetric I-V curves, which originate from their charged surfaces [3]. Here, we demonstrate the attachment of an aminoterminated biological ligand (biotin) to the inner surface of these pores by EDC/PFP coupling chemistry [5]. As a difference to the method reported before, the reactive intermediate pentafluorophenol (PFP) ester is ~10 times more active than the corresponding Nhydroxysuccinimide (NHS) ester, which leads to the higher efficiency of the modification reaction. The carboxyl groups were first activated by an ethanol solution of EDC (0.1 M) and PFP (0.2 M). The reactive intermediate PFP ester (I) was subsequently reacted with either ethylenediamine (EDA) or biotin-amine, leading to an amide bond formation having terminal –NH2 groups (II) and biotin moieties (III) respectively, as shown in the reaction scheme below