Development of amperometric α-ketoglutarate biosensor based on ruthenium–rhodium modified carbon fiber enzyme microelectrode

Abstract

A rapid and highly sensitive miniaturized amperometric biosensor for the detection of α-ketoglutarate (α-KG) based on a carbon fiber electrode (CFE) is presented. The biosensor is constructed by immobilizing the enzyme, glutamate dehydrogenase (GLUD) on the surface of single carbon fiber modified by co-deposition of ruthenium (Ru) and rhodium (Rh) nanoparticles. SEM and EDX shed useful insights into the morphology and composition of the modified microelectrode. The mixed Ru/Rh coating offers a greatly enhanced electrocatalytic activity towards the detection of β-nicotinamide adenine dinucleotide (NADH), with a substantial decrease in overpotential of ∼400 mV compared to the unmodified CFE. It also imparts higher stability with minimal surface fouling, common to NADH oxidation. Further modification with the enzyme, GLUD leads to effective amperometric biosensing of α-KG through monitoring of the NADH consumption. A very rapid response to dynamic changes in the α-KG concentrations is observed with a response time of 6 s. The current response is linear between 100 and 600 μM with a sensitivity of 42 μA M⁻¹ and a detection limit of 20 μM. This proof of concept study indicates that the GLUD-Ru/Rh-CFE biosensor holds great promise for real-time electrochemical measurements of α-KG.

Introduction

α-Ketoglutarate (α-KG) is a key intermediate in Krebs cycle, and is one of the major precursors for the synthesis of most biochemical substances. It is produced from the oxidative decarboxylation of isocitrate, catalyzed by the enzyme, isocitrate dehydrogenase 1 (IDH1). It has been recently demonstrated that IDH1 is frequently mutated in low grade gliomas, a common form of brain tumor in humans to form single amino acid substitution at Arg¹³² (R132) (Ichimura et al., 2009). These monoallelic mutations (IDH1-R132) inactivate the enzyme and inhibit the production of α-KG. This in result increases the Hypoxia Inducible Factor 1, an angiogenic factor which is responsible for the growth of tumor cells (Zhao et al., 2009). α-KG has been emphasized to possess exciting angiogenesis suppressor activity (Schaur et al., 1979). Moreover, micronutrient application of α-KG has been shown to have beneficial effects on several malignant tumors (Wagner et al., 2010). Hence, the ability to follow the kinetics of α-KG formation in such gliomas would be useful in analyzing cellular activities. To our knowledge there are no reports on electrochemical biosensors for the measurement of α-KG. A flow injection system with a bi-enzymes (glutamate dehydrogenase and glutamate oxidase) reactor and a downstream electrochemical detector was developed for fermentation monitoring of α-KG (Collins et al., 2001).

This proof of concept study describes the development, optimization and characterization of an amperometric microsensor for α-KG based on a GLUD-Ru/Rh modified single carbon fiber electrode. The biocatalytic detection of α-KG at the modified CFE transducer involves the following reaction:

$$\alpha \text{-Ketoglutarate}+\text{N}{\text{H}}_3+\text{NADH}\stackrel{\text{GLUD}}{⟶}\text{<mi mathsize="small" is="true">L</mi>}\text{-Glutamate}+{\text{NAD}}^{+}+{\text{H}}_2\text{O}$$

where the enzyme, GLUD catalyzes the conversion of α-KG into l-glutamate in the presence of ammonia and NADH. The quantitation of α-KG is achieved through a low-potential anodic detection of the depleted NADH at the GLUD-Ru/Rh-CFE. CFE transducers have been widely used for exploring microscopic domains and measurements of local concentration profiles in living cells (Huffman and Venton, 2009). Moreover, these microelectrodes are widely used in biomedical sciences owing to their well-resolved spatially and temporally response in biological media for in vivo applications. However, like most carbonaceous electrodes, CFE requires high overvoltage for the oxidation of NADH. In addition, a serious problem associated with amperometric detection of NADH at carbon electrodes is the fouling of the electrode surface by the adsorption of reaction intermediates. Significant efforts have been directed towards developing new or modified electrode materials that lower the overpotential for NADH oxidation and minimize surface passivation effects. Mediator compounds that include polyazine dyes (Karyakin et al., 1999), catechol moieties (Jaegfeldt et al., 1981), metal complexes (Wu et al., 1996), or conducting polymer (Bartlett et al., 1997), have been used to modify the electrode surface to address these overpotential and passivation effects. One such effort is the modification of electrode by metal particles owing to their remarkable electrocatalytic activity (Wang, 2005). In the following sections, we demonstrate that the co-deposition of Ru and Rh metal nanoparticles onto the single CFE offers an effective electrocatalytic detection of NADH (compared to the individual metals) along with a highly stable response. This was followed by the immobilization of the GLUD enzyme and a low-potential anodic detection of α-KG via the NADH consumption (Eq. (1)).