Impedimetric Biosensor for the Assessment of the Clotting Activity of Rennet

Cheese production is relied upon the action of rennet (a mixture of chymosin and pepsin) onto casein micelles of milk. For the first time, the monitoring of this interaction with electrochemical impedance spectroscopy (EIS) was used to develop a faradic impedimetric biosensor for the assessment of t...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2010-10, Vol.82 (20), p.8629-8636
Main Authors: Panagopoulou, Maria A, Stergiou, Dimitrios V, Roussis, Ioannis G, Prodromidis, Mamas I
Format: Article
Language:English
Subjects:
Acids - analysis
Analytical chemistry
Biological and medical sciences
Biosensing Techniques - methods
Biosensors
Biotechnology
Charged particles
Chemical reactions
Chemistry
Chymosin - chemistry
Chymosin - ultrastructure
Electrocatalysis
Electrochemical methods
Electrodes
Exact sciences and technology
Fundamental and applied biological sciences. Psychology
Hydrogen-Ion Concentration
Methods. Procedures. Technologies
Micelles
Microscopy, Atomic Force
Milk
Proteins
Spectrometric and optical methods
Various methods and equipments
Volumetric analysis
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Summary:Cheese production is relied upon the action of rennet (a mixture of chymosin and pepsin) onto casein micelles of milk. For the first time, the monitoring of this interaction with electrochemical impedance spectroscopy (EIS) was used to develop a faradic impedimetric biosensor for the assessment of the clotting activity of rennet, using hexacyanoferrate(II)/(III) couple as a redox probe. Gold electrodes were modified with self-assembled monolayers of different thiols (thioctic acid, dithiobis-N-succinimidyl propionate, and cysteamine), and (artificial) casein micelles were immobilized on the modified gold surfaces. The proposed method is based on the measurement of charge-transfer resistance (R ct) changes attributed to the degradation of the negatively charged immobilized casein micelles by rennet to neutral biostructures. This action results in the increase of the flux of the redox probe, which exists in the bulk solution, to the surface of the electrode and, consequently, in the decrease of R ct. Experimental parameters such as the micelle loading, the reaction time, the concentration of rennet, and the working pH, were optimized. Besides EIS measurements, cyclic voltammetry, FT-IR, and atomic force microscopy (AFM) experiments were also performed before and after the interaction of the immobilized micelles with rennet. Finally, the proposed biosensors were successfully tried for various commercial samples.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac1017925