Hydrogen peroxide sensing with horseradish peroxidase-modified polymer single conical nanochannels

Inspired from the funtioning and responsiveness of biological ion channels, researchers attempt to develop biosensing systems based on polymer and solid-state nanochannels. The applicability of these nanochannels for detection/sensing of any foreign analyte in the surrounding environment depends critically on the surface characteristics of the inner walls. Attaching recognition sites to the channel walls leads to the preparation of sensors targeted at a specific molecule. There are many nanochannel platforms for the detection of DNA and proteins, but only a few are capable of detecting small molecules. Here, we describe a nanochannel platform for the detection of hydrogen peroxide, H₂O₂, which is not only a toxic waste product in the cellular systems but also a key player in the redox signaling pathways. The sensor is based on single conical nanochannels fabricated in an ion tracked polymer membrane. The inner walls of the channel are decorated with horseradish peroxidase (HRP) enzyme using carbodiimide coupling chemistry. The success of the HRP immobilization on the channel surface is confirmed by measuring the pH-dependent current-voltage (I-V) curves of the system. The reported HRP-nanochannel system detects nanomolar concentrations of H ₂O₂ with 2,2'-azino-bis(3-ethylbenzothiazoline-6- sulfonate) (ABTS) as the substrate. The immobilized HRP enzyme is thus capable of inducing redox reactions in a subfemtoliter volume of single nanochannels. We demonstrate that functioning of the designed biosensor is reversible and can be used multiple times to detect H₂O₂ at various concentrations.