Single Conical Ion-Track Nanochannels Modified via Horseradish Peroxidase Enzyme-Concanavalin A Protein Bioconjugation

Synthetic nanochannels have recently attracted enormous interest because their geometry and surface chemistry can be fully controlled. They also feature excellent mechanical robustness and compatibility with various electronic measurement systems. Synthetic nanochannels fabricated in polymer and silicon-based films as well as glass nanopipettes have already been applied for the detection of DNA and protein analytes. Incorporation of bio-recognizable elements and biomolecular conjugation in nanoscale architectures have been achieved by two types of surface modification techniques: (i) covalent attachment to the channel walls, and (ii) electrostatic self-assembly of polyelectrolytes on the inner walls by exploiting the existing charged chemical groups on the channel surface. Moreover, biospecific interactions such as sugar– lectin affinity could also be used for the bioconjugation of biomolecules on a channel surface bearing sugar residues. However, the implementation of sugar–lectin interactions to immobilize biomolecules in nanochannels remains a challenge which is addressed here by using the horseradish peroxidase (HRP) enzyme. It is known that HRP is a mannose-rich glycoprotein and the mannose residues are accessible to binding with lectin protein molecules. Concanavalin A (Con A) is a lectin protein that exists as a tetramer at neutral pH. Single nanochannels were fabricated in heavy-ion irradiated PET foils of 12 μm thickness by asymmetric chemical track etching using 9 M NaOH solution. This resulted in a conical channel (small aperture 16 nm) with carboxylate (-COO ) groups on the channel surface. The COOH groups were first activated into sulfo-NHSesters by using an aqueous solution containing N-(3dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysulfosuccinimide for 30 minutes. Then, the amine-reactive sulfo-NHS-ester molecules were covalently coupled to the amino group present on the HRP enzyme molecule. After HRP-immobilization, the Con A solution was introduced on both sides of the enzyme-modified single-channel membrane. The exposure for 3 hours at room temperature allowed the sugar residues of glycoenzyme to couple through biospecific interaction to the Con A. The functionalized membrane was mounted in a conductivity cell, and current-voltage (I-V) curves were measured at neutral pH using 0.1 M KCl as an electrolyte solution. The ionic current through the as-prepared channel, measured at +1 V, was +1.5 nA (Fig. 1). After HRP immobilization, the current decreased to 0.95 nA. Finally, the Con A complexation with HRP enzyme resulted in a further reduction of current to 0.55 nA (approximately 3 times lower than the original value). At neutral pH, both the HRP enzyme and the Con A molecules are negatively charged. Consequently, the channel displayed current rectification before and after the enzyme and Con A immobilization. The ion current decreases for both voltage polarities, consistent with the dependence of the ionic current on the effective cross-section area of the nanochannel.