A novel reagentless glutamate microband biosensor for real-time cell toxicity monitoring

A reagentless glutamate biosensor was applied to the determination of glutamate released from liver hepatocellular carcinoma cells (HepG2) in response to toxic challenge from various concentrations of paracetamol. A screen printed carbon electrode (SPCE) containing the electrocatalyst Meldola's Blue (MB-SPCE) served as the electron mediator for the oxidation of NADH.A mixture of the enzyme glutamate dehydrogenase (GLDH), cofactor nicotinamide adenine dinucleotide (NAD+) and the biopolymer chitosan (CHIT) were drop-coated onto the surface of the transducer (MB-SPCE) in a simple one step fabrication process.The reagentless biosensor was used with amperometry in stirred solution at an applied potential of +0.1 V (vs. Ag/AgCl). All experiments were carried out at the following conditions: pH 7, temperature 37 °C, atmosphere 5% CO2.The linear range of the device was found to be 25–125 μM in phosphate buffer (75 mM, containing 0.05 M NaCl) and 25–150 μM in cell culture medium. The limits of detection (LOD) were found to be 1.2 μM and 4.2 μM based on three times signal to noise, using PBS and culture medium respectively. The sensitivity was calculated to be 106 nA μM−1 cm−2 and 210 nA μM−1 cm−2 in PBS and cell medium respectively. The response time was ∼60 s in an agitated solution.HepG2 cells were exposed to various concentrations of paracetamol (1 mM, 5 mM and 10 mM) in order to investigate the drug-induced release of glutamate into the culture medium in real time. Two toxicity studies were investigated using different methods of exposure and analysis.The first method consisted of a single measurement of the glutamate concentration, using the method of standard addition, after 24 h incubation. The concentrations of glutamate were found to be 52 μM, 93 μM and 177 μM, released on exposure to 1 mM, 5 mM and 10 mM paracetamol respectively.The second method involved the continuous monitoring of glutamate released from HepG2 cells upon exposure to paracetamol over 8 h. The concentrations of glutamate released in the presence of 1 mM, 5 mM and 10 mM paracetamol, increased in proportion to the drug concentration, ie: 16 μM, 28 μM and 62 μM respectively. This result demonstrates the feasibility of using this approach to monitor early metabolic changes after exposure to a model toxic compound

Development of a novel reagentless, screen-printed amperometric biosensor based on glutamate dehydrogenase and NAD+, integrated with multi-walled carbon nanotubes for the determination of glutamate in food and clinical applications

© 2015 Elsevier B.V. Abstract A screen printed carbon electrode (SPCE) containing the electrocatalyst Meldola's Blue (MB) has been investigated as the base transducer for a reagentless glutamate biosensor. The biopolymer chitosan (CHIT) and multiwalled carbon nanotubes (MWCNTs) were used to encapsulate the enzyme glutamate dehydrogenase (GLDH) and the co-factor nicotinamide adenine dinucleotide (NAD+). The biosensor was fabricated by sequentially depositing the components on the surface of the transducer (MB-SPCE) in a layer-by-layer process, details of which are included in the paper. Each layer was optimised to construct the reagentless device. The biosensor was used in conjunction with amperometry in stirred solution using an applied potential of +0.1 V (vs. Ag/AgCl). Optimum conditions for the analysis of glutamate were found to be: temperature, 35°C; phosphate buffer, pH 7 (0.75 mM, containing 0.05 M NaCl). The linear range of the reagentless biosensor was found to be 7.5-105 μM, and limit of detection was found to be 3 μM (based on n = 5, CV: 8.5% based on three times signal to noise) and the sensitivity was 0.39 nA/μM (±0.025, coefficient of variation (CV) of 6.37%, n = 5). The response time of the biosensor was 20-30 s. A food sample was analysed for monosodium glutamate (MSG). The endogenous content of MSG was 90.56 mg/g with a CV of 7.52%. The reagentless biosensor was also used to measure glutamate in serum. The endogenous concentration of glutamate was found to be 1.44 mM (n = 5), CV: 8.54%. The recovery of glutamate in fortified serum was 104% (n = 5), CV of 2.91%

The design, development and application of electrochemical glutamate biosensors

The development of biosensors for the determination of glutamate has been of great research interest for the past 25 years due to its importance in biomedical and food studies. This review focusses on the various strategies used to fabricate glutamate biosensors as well as their performance characteristics. A brief comparison of the enzyme immobilisation method employed and the performance characteristics of a range of glutamate biosensors are described in tabular form and then described in detail throughout the review: some selected examples have been included to demonstrate the various applications of these biosensors to real samples

Droplets- and digital-based methods

Liquid droplets may be manipulated within microfluidic channel structures, or on planar surfaces to provide experimental platforms where each droplet is a discrete experimental entity. Sometimes described as digital methodology, this provides a unique approach within the fields of chemistry and biochemistry. Subsequent splitting, combining and sorting processes with the droplets offer a limitless opportunity for complex experimental design that includes high sample numbers with high-throughput capability. Distinct advantages include nanolitre, or lower sample volumes, rapid mixing and short diffusion distances. When placed within an automated microfluidic environment, droplets may be driven sequentially or in parallel through a pre-set pattern of unit operations. Many applications have been reported that include the broad fields of synthesis and sample assay. Synthetic applications have been reported for organic synthesis, biochemical synthesis and materials synthesis. The majority of assay-based applications have been reported for bio-assays and biological assays with single cells. The distinct advantages of low sample volumes and high-throughput have been the primary drivers for the development of droplet and digital-based methods

FIA determination of CrIII in tap water with chemiluminescence detection

CrIII has been determined by measuring the light emitted as a result of the CrIII-catalysed reaction between luminol and hydrogen peroxide. Increase in the sensitivity of the determination was achieved by introducing bromide into the reaction mixture while the selectivity was improved by the addition of EDTA. All the operations were performed online in an automated FIA system. The calibration curve for CrIII was linear in the range 0-500 μg 1-1 and the limit of detection was 0.5 μg 1-1. The method was applied to the determination of CrIII in tap water; the CrIII concentration varied between 10 and 20 μg 1-1 with a typical coefficient of variation of 2% (n = 5)

Data smoothing in stripping voltammetry by simplex function fitting

Stripping voltammetry (SV), in common with all trace voltammetric analytical techniques, suffers from poor signal-to-noise (S/N) ratios due to external interference pick up and inherent generation of noise in the electrochemical system. In addition to other methods developed to smooth noisy data (such as time and frequency domain filtering), optimisation Simplex fitting is that the distortion of the signal due to 'leakage' (associated with digital filters)2 is avoided. In addition, this procedure, in contrast to most conventional filtering techniques, eliminates completely the noisy components and is capable of calculating the peak parameters automatically. The proposed method can be applicable to other instrumental techniques producing a peak shaped response such as mass spectrometry, atomic absorption spectrometry and chromatography

Comparison of data smoothing techniques as applied to electrothermal atomic absorption spectrometry

Detection of trace metals by electrothermal atomic absorption spectrometry ETAAS at low concentrations is limited by the noise in the measurement system. In this work, three noise reduction techniques, namely digital filtering (recursive time domain and non recursive time domain), modified simplex function fitting and adaptive Wiener filtering, were applied to the determination of lead by ETAAS at low concentration levels. The three methods are based on different formal and operational principles to realise the same goal of noise reduction. The results obtained from the application of these three techniques are compared, the factors that must be considered in each case are discussed and the relative advantages and drawbacks of each technique are pointed out