Polymer/enzyme-modified HF-etched carbon nanoelectrodes for single-cell analysis

Highlights

• Carbon nanoelectrodes etched in aqueous HF were modified with polymer/enzyme films.

• Reliable fabrication of conical electrodes with a tip diameter of a few hundred nm.

• Glucose oxidase was electrically wired by means of a redox polymer.

• Polymer/enzyme modified electrodes could be used for intracellular measurements.

• The biosensors showed a stable current output even after intracellular measurement.

Abstract

Carbon-based nanoelectrodes fabricated by means of pyrolysis of an alkane precursor gas purged through a glass capillary and subsequently etched with HF were modified with redox polymer/enzyme films for the detection of glucose at the single-cell level. Glucose oxidase (GOx) was immobilized and electrically wired by means of an Os-complex-modified redox polymer in a sequential dip coating process. For the synthesis of the redox polymer matrix, a poly(1-vinylimidazole-co-acrylamide)-based backbone was used that was first modified with the electron transfer mediator [Os(bpy)₂Cl]⁺ (bpy = 2,2′-bipyridine) followed by the conversion of the amide groups within the acrylamide monomer into hydrazide groups in a polymer-analogue reaction. The hydrazide groups react readily with bifunctional epoxide-based crosslinkers ensuring high film stability. Insertion of the nanometre-sized polymer/enzyme modified electrodes into adherently growing single NG108-15 cells resulted in a positive current response correlating with the intracellular glucose concentration. Moreover, the nanosensors showed a stable current output without significant loss in performance after intracellular measurements.

Introduction

Electrodes at the sub-micron- and nanometre-scale are gaining increasing attention as they ensure single-entity analyses that are usually masked by bulk properties such as the addressability of individual cells [1], [2]. Advantages of micro- and nanoelectrodes in comparison to macroelectrodes include an improved signal-to-noise ratio and a high temporal resolution [1], [2]. Moreover, they enable electrochemical experiments in small confined volumes like the interior of cells [3] or synaptic clefts [4]. In addition, micro- and nanoelectrodes consume only minimal amounts of analyte and reveal small overall dimensions. Hence, probing becomes minimally invasive [1] and allows in-vivo measurements (e.g. in interstitial fluid in brain [5]) and the fabrication of implantable devices (e.g. for glucose measurements or drug-delivery monitoring [6]). In combination with scanning probe techniques they provide the opportunity for high resolution mapping of the substrate concentration [7].

Takahashi et al. described a protocol for the facile fabrication of nanometre-sized carbon electrodes based on pyrolysis of a gaseous carbon precursor that was purged through a laser-pulled glass capillary. This process results in the deposition of a thin carbon layer and thus the formation of carbon nanoelectrodes (CNEs) [8]. In particular, such electrodes were combined with scanning electrochemical (SECM) and scanning ion-conductance microscopy (SICM) as a tool for nanoscale imaging of single cells [8]. Furthermore, CNEs from pre-pulled capillaries have been successfully applied for the detection of reactive species in murine macrophages [9], [10], for dopamine detection in mouse brain slices [11], and for the fabrication of spear-head shaped field-effect transistors that were inserted into single cardiomyocytes [12].

Electrochemistry in complex matrices like the cytoplasm of cells does not only require small electrodes, but also a high specificity of the sensing device for the analyte of interest due to the presence of several interferences under physiological conditions [6]. Owing to their high substrate specificity, redox enzymes have been extensively used as biorecognition element in amperometric biosensors [13], [14], [15], [16], e.g. for the selective detection of glucose in diabetes management with sugar converting enzymes [17].

A common strategy to electrically wire enzymes to electrode surfaces is their incorporation into redox hydrogels that are equipped with specific redox mediators such as Os-complexes [18], [19]. The redox potential of the hydrogel matrix can be adjusted by the incorporation of Os-complexes with specific ligands that are modified with electron donating or electron withdrawing groups [18], [19], [20]. This allows for an adjustment of the operating potential of a corresponding sensor, and minimizes artefacts based on the oxidation/reduction of electrochemically active interferences like ascorbic or uric acid [20] or O₂ [20], [21]. Additionally, the formation of a 3D-structured immobilization matrix enables high biocatalyst loading that leads to a high sensitivity and a pronounced current response of the sensor [18], [19]. In addition, redox polymers could already be successfully employed for the fabrication of polymer/enzyme microelectrodes [22].

Recently we described miniaturised amperometric second generation biosensors fabricated from nanometre-sized electrodes [23]. Redox polymers and glucose converting enzymes were co-deposited into electrochemically etched cavities at the electrode tip. However, transmission electron microscopy (TEM) analysis revealed that the harsh conditions applied during the electrochemical etching process, i.e. application of extreme potentials to induce water splitting at the electrode induced the formation of holes in the glass sheath of electrochemically etched CNEs. These inhomogeneities at the electrode-tip led to irreproducible diffusion profiles and strong variations in the detected absolute currents. Moreover, during the etching process the electrode-tips tend to deteriorate which limits the usage of this electrochemically etched CNEs for a controlled and reliable sensor fabrication. Consequently, the fabrication of reproducible nanosensors remains challenging.

Wet-chemical etching of CNEs in aqueous HF enables the formation of highly defined electrode architectures of a conical shape and tip diameters of a few hundred nanometres [24], [25]. In addition, the process is highly reliable and can be conducted in a controlled manner. Absolute steady-state currents that reflect the electroactive surface area of these electrodes measured by means of [Ru(NH₃)₆]Cl₃ voltammetry experiments nicely correlate with the etching time [24].

Herein, we report the development and characterization of glucose sensors based on HF-etched CNEs [24]. Due to the improved fabrication process and stability in comparison to the previously described electrodes [23], sensors with reliable performance and reproducibility could be developed. The robust and stable polymer/enzyme-modified HF-etched CNEs enabled intracellular measurements inside single NG108-15 cells.