Tunable 3D nanofibrous and bio-functionalised PEDOT network explored as a conducting polymer-based biosensor

Highlights

• 3D Nano-PEDOT-COOH network with nanofibrous structure and carboxylic acid groups was realised simultaneously via soft-template.

• Nanofibrous structure was favourable for probe charge transfer and NADH transduction.

• High density of carboxylic acid groups for covalent immobilisation of NADH-dependent dehydrogenase to create Bio-Nano-PEDOT.

• Bio-Nano-PEDOT interface showed good analytical performance for NADH sensing and lactate biosensing.

Abstract

Conducting polymers that possess good electrochemical properties, nanostructured morphology and functionality for bioconjugation are essential to realise the concept of all-polymer-based biosensors that do not depend on traditional nanocatalysts such as carbon materials, metal, metal oxides or dyes. In this research, we demonstrated a facile approach for the simultaneous preparation of a bi-functional PEDOT interface with a tunable 3D nanofibrous network and carboxylic acid groups (i.e. Nano-PEDOT-COOH) via controlled co-polymerisation of EDOT and EDOT-COOH monomers, using tetrabutylammonium perchlorate as a soft-template. By tuning the ratio between EDOT and EDOT-COOH monomer, the nanofibrous structure and carboxylic acid functionalisation of Nano-PEDOT-COOH were varied over a fibre diameter range of 15.6 ± 3.7 to 70.0 ± 9.5 nm and a carboxylic acid group density from 0.03 to 0.18 μmol cm⁻². The nanofibres assembled into a three-dimensional network with a high specific surface area, which contributed to low charge transfer resistance and high transduction activity towards the co-enzyme NADH, delivering a wide linear range of 20–960 μM and a high sensitivity of 0.224 μA μM⁻¹ cm⁻² at the Nano-PEDOT-COOH50% interface. Furthermore, the carboxylic acid groups provide an anchoring site for the stable immobilisation of an NADH-dependent dehydrogenase (i.e. lactate dehydrogenase), via EDC/S–NHS chemistry, for the fabrication of a Bio-Nano-PEDOT-based biosensor for lactate detection which had a response time of less than 10 s over the range of 0.05–1.8 mM. Our developed bio-Nano-PEDOT interface shows future potential for coupling with multi-biorecognition molecules via carboxylic acid groups for the development of a range of advanced all-polymer biosensors.

Introduction

Poly(3,4-ethylenedioxythiophene) (PEDOT), an emerging conducting polymer (CP) with extraordinary redox reversibility, biocompatibility and electrochemical properties, shows considerable potential as an interfacing material in biosensing to bridge between the organic electronics world and bioelectronics, since PEDOT possess sufficient similarities in its organic chemical nature with biological molecules to facilitate the concept of all-polymer biosensors (Inal et al., 2018). One key to merge organic electronics and bioelectronics is to establish an effective signal transduction interface between biomolecules and PEDOT. However, the lack of reactive functional groups for bioconjugation and the relatively low electrochemical catalytic activity of pristine PEDOT, has hindered the development of a direct PEDOT-based electrochemical biosensors with bioelectrochemical signal transduction between the catalytic enzyme and PEDOT. In most of the reported PEDOT sensors and biosensors, nanomaterials were used as dopants to prepare PEDOT-composites, including carbon materials (carbon nanotubes, graphene), metal or metal oxides (Au, TiO₂) and dyes (Prussian blue) (Lin et al., 2010; Meng et al., 2019; Wang et al., 2018). The PEDOT mainly served as a conducting matrix while the bioelectrochemical signal transduction still relied on the doped carbon, metal or dye nanocatalyst. Thus, pure PEDOT interfaces possessing functionality for both bioconjugation and good electrochemical activity are still largely absent while remaining critical for future advances in biosensing.

Functionalisation endows conducting polymers (CPs) with functional groups that facilitate effective immobilisation of biorecognition molecules for biosensing applications. In recent years, much effort has been devoted to design functionalised CPs with active chemical groups, such as carboxylic acid (Luo et al., 2009), amine (Godeau et al., 2016), phosphorylcholine (Goda et al., 2015), sialic acid (Hai et al., 2017) etc. Of these, the functionalisation of CPs with carboxylic acid groups delivers more profound value compared to the others because of the variety of biological molecules, such as nucleotides and proteins (antibodies and enzymes), that can be covalently immobilised on the CP transducer interface, via ethyl(dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysulfosuccinimide (NHS) chemistry. The introduction of carboxylic acid groups can be achieved either by using carboxylic acid containing molecules as a dopant during polymerisation or by using their derivatives (e.g. EDOT-COOH) as monomers. Carboxylic acid containing molecules, such as citrate (Meng et al., 2019a) and hyaluronic acid (Mantione et al., 2016) can be incorporated into the CP matrix as a dopant to compensate for the backbone charge, thus endowing the resulting CP interface with a high –COOH density. However, this strategy suffers from several drawbacks including potential leakage of dopant, difficulty in controlling the amount of dopant and destruction of the CP's bulk properties and conductivity. Alternatively, CP derivatives bearing carboxylic acid groups have been used for the preparation of a close-structured planar functionalised PEPOT film coupled with DNA and or antibodies for indirect affinity biosensing (Luo et al., 2009; Sekine et al., 2011).

Advances in nanomaterials based on conventional carbon (CNTs, graphene) and metal nanomaterials (Au, Pt nanoparticles) used in bioelectronics (Jin et al. 2018a, 2018b) encourages the exploration of nanostructured CPs. Nanostructured CPs inherit prominent physical-chemical properties from their bulk polymer equivalents, while acquiring beneficial nanomaterial characteristics such as large active surface area to promote catalytic reactions. While bulk CP interfaces may suffer from hysteresis and sluggish electron/mass transfer, nanostructured CPs are capable of realising effective electrode interface kinetics and facilitate signal transfer due to their high surface area and porous structure, leading to faster response times and high sensitivity for the resulting sensors (Lu et al., 2019). Various CP structures, including nano and microspheres (Nguyen et al., 2015; Vagin et al., 2016), hollow-capsules (Wannapob et al., 2015), nanowires (Travas-Sejdic et al., 2014) and nanotubes (Lin et al., 2016), have been implemented via sacrificial hard-template (e.g. calcium carbonate and polystyrene), soft-template (e.g. surfactants) or template-free (dimer as seeds) fabrication. Although these techniques yield a controlled nanostructured CP interface, the demand for a high density of functional groups for biorecognition elements still remains unfulfilled.

In this study, we developed an innovative bioorganic/organic polymer bio-interface possessing functionality for bioconjugation and good electrochemical properties for signal transduction towards an all-polymer-based biosensor. By combining functionalisation and nanostructure formation strategies, we demonstrate herein a facile approach for the simultaneous preparation of a bi-functional PEDOT interface with a tunable 3D nanofibrous network and carboxylic acid groups (i.e. Nano-PEDOT-COOH) via controlled co-polymerisation of EDOT and EDOT-COOH monomers using tetrabutylammonium perchlorate (TBAP) as a soft-template (Scheme 1a). The ratio between EDOT and EDOT-COOH was evaluated to modulate the resulting nanostructure morphologies and density of the carboxylic acid groups. The 3D nanofibrous network endowed the Nano-PEDOT-COOH interface with enhanced charged transfer and catalytic activity towards the electrochemical oxidation of NADH, which may be ascribed to the relatively large 3D active surface area and porous structure. Furthermore, the carboxylic acid functionality provided anchoring sites for the stable immobilisation of a NADH-dependent dehydrogenase (i.e. lactate dehydrogenase) via EDC/S–NHS chemistry for the fabrication of a Bio-Nano-PEDOT-based biosensor for lactate detection that delivers a fast response time and high sensitivity (Scheme 1b). This strategy also provides a generic design for electrocatalytic devices based on the many available NADH-dependent dehydrogenase enzymes.