A Reagentless, Screen-Printed Amperometric Biosensor for the Determination of Glutamate in Food and Clinical Applications

Abstract

A reagentless biosensor has been successfully developed to measure glutamate in food and clinical samples. The enzyme, glutamate dehydrogenase (GLDH) and the cofactor, nicotinamide adenine dinucleotide (NAD) are fully integrated onto the surface of a Meldola's Blue screen-printed carbon electrode (MB-SPCE). The biological components are immobilized by utilizing unpurified multi-walled carbon nanotubes (MWCNT's) mixed with the biopolymer chitosan (CHIT), which are drop-coated onto the surface of the MB-SPCE in a layer-by-layer fashion. Meldola's Blue mediator is also incorporated into the biosensor cocktail in order to increase and facilitate electron shuttling between the reaction layers and the surface of the electrode. The loadings of each component are optimized by using amperometry in stirred solution at a low fixed potential of +0.1 V. The optimum temperature and pH are also determined using this technique. Quantification of glutamate in real samples is performed using the method of standard addition. The method of standard addition involves the addition of a sample containing an unknown concentration of glutamate, followed by additions of known concentrations of glutamate to a buffered solution in the cell. The currents generated by each addition are then plotted and the resulting line is extrapolated in order to determine the concentration of glutamate in the sample (Pemberton et al., Biosens Bioelectron 24:1246-1252, 2009). This layer-by-layer approach holds promise as a generic platform for the fabrication of reagentless biosensors.

The design, development and application of electrochemical glutamate biosensors

“…The biosensor was successfully applied to the determination of glutamate in spiked serum. A recovery of 104% (n = 5, CoV: 2.91%) was determined, which compares favourably to previously discussed biosensors [19,23,26]. An interference study was conducted in both serum and food samples (stock cubes)…”

“…Screen-printing technology has offered the possibility of the mass production at low cost of glutamate biosensors; these have been successfully applied to the measurement of glutamate in serum and food samples [26]. As screen-printed devices are inexpensive to manufacture they can be considered disposable, in comparison to glassy carbon electrodes, which are expensive and are not considered disposable devices.…”

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

“…Both reagentless and non-reagentless conventional sized glutamate biosensors have previously been developed by drop-coating the required biomolecules onto the working electrode of a screen-printed carbon electrode [21][22][23]. We have recently reviewed electrochemical glutamate biosensor construction and their applications in food and clinical analysis [24].…”

Enzyme-Based Electrochemical Glutamate Biosensors

“…Also, the use of low-cost and disposable devices such as screen printed carbon electrodes (SPCE) is advantageous in the analysis of biological fluids where contamination may be a problem. Hughes et al described the development and optimization of a disposable screen-printed amperometric biosensor for glutamate based on GLDH [79]. The authors also developed a stable, reagentless amperometric glutamate biosensor by incorporating the GLDH biorecognition components using a layer-by-layer deposition involving chitosan and MWCNTs on SPCE [81].…”

“…In addition, the influence of experiment conditions such as pH, temperature, ionic strength, and stirring on the enzyme-catalyzed reaction can be minimized by optimizing and keeping these conditions constant throughout the biosensor use. For example, GLDH-based biocatalytic sensors have utilized pHs ranging from 7.0 to 9.0 during the detection step [12,67,79]. Also, GmOx-based biosensors appear to utilize pHs from 7.0 to 7.4 [40,68].…”