Mihaela Nistor

Label free capacitive immunosensor for detecting calpastatin — A meat tenderness biomarker

An immunological capacitive biosensor for calpastatin was developed, optimized and applied for the analysis of meat extract samples. Anti-calpastatin antibody was immobilized on a gold electrode modified with a self-assembled monolayer of mercaptoundecanoic acid and Protein A from Staphylococcus aureus, and the obtained immunosensor was inserted as the working electrode in an electrochemical cell of a flow injection system. The dynamic range of the sensor was 20 to 160 ng/mL calpastatin. The electrode could be regenerated and re-used for more than 7 days with minimal reduction in sensitivity. For the analysis of real samples, the target analyte was extracted from the Longissimus dorsi muscle from beef carcasses directly after slaughtering. The extract was analyzed both with the developed immunosensor and microtiter plate ELISA, and a good correlation was obtained. However the immunosensor offers advantages of speed, simplicity, sensitivity and possibility for miniaturization over conventional assays for calpastatin quantification.

Monitoring of glucose and glutamate using enzyme microstructures and scanning electrochemical microscopy.

Glucose oxidase and glutamate oxidase lines, with typical width of 100 microm, were patterned on gold surfaces using a micro-dispensing system, by shooting 100 pl droplets of the corresponding enzyme solutions. The probe of a scanning electrochemical microscope (SECM) was then carefully positioned in the close proximity of the enzyme microstructure and poised to +600 mV vs. Ag/AgCl, KCl 0.1 M. The H(2)O(2), generated by the enzyme lines at different concentrations of glucose and glutamate in the surrounding solution, was sequentially monitored. Reproducible calibration curves for glucose and glutamate were obtained in one single experiment, proving that the combination of enzyme microstructures with SECM can provide a new way of achieving multianalyte detection.

Indirect, non-competitive amperometric immunoassay for accurate quantification of calpastatin, a meat tenderness marker, in bovine muscle

A method is described for quantification of the beef tenderness marker, calpastatin, in meat samples by amperometric detection. Using a novel bovine recombinant partial calpastatin protein as standard antigen a low detection limit of 0.2 ng/mL was achieved. The influence of the complex matrix was minimised by heat pretreatment and dilution of the samples prior to detection of calpastatin. The relative error between the direct sample measurement and standard addition methods was 5.89%, confirming the accuracy of the developed amperometric immunoassay.

Design, development and application of a bioelectrochemical detection system for meat tenderness prediction.

The analytical method described, based on antibody-antigen bio-recognition and the measuring system for amperometric detection, was designed for accurate, easy to use and cost effective quantification of calpastatin, a meat tenderness biomarker. The novel assay for calpastatin quantification was integrated in a portable electrochemical device known as the Tendercheck system and was used to analyze meat samples collected from animals of different breeds and ages. The data obtained were correlated (R² = 0.62) with Warner Bratzler Shear Force (WBSF) measurements, a routinely used method for meat tenderness determination.

Comparison of two glutathione S -transferases used in capacitive biosensors for detection of heavy metals

This work describes the development of a heavy-metal biosensor based on either recombinant 6His-Tag glutathione S-transferase (GST-(His)6) or glutathione S-transferase Theta 2-2 (GST-theta 2-2), and a capacitive transducer. The dynamic range of the pure bovine liver GST-Theta 2-2 biosensor was 1 fM to 1 mM for Zn2+, and 10 pM to 1 mM for Cd2+. The GST-(His)6 biosensor was able to detect Zn2+ and Cd2+ in the range of 1 fM to 10 µM, and Hg2+ in the range of 1 fM to 10 mM. The bovine liver GST Theta 2-2 biosensor displays an increased selectivity and a wider dynamic range for Zn2+ compared with the GST-(His)6 biosensor. Therefore, by using different GST isozymes, it is possible to modulate important characteristics of capacitive biosensors for the detection of heavy metals.