Impedimetric Biosensor for the Assessment of the Clotting Activity of Rennet Maria A. Panagopoulou, † Dimitrios V. Stergiou, † Ioannis G. Roussis, ‡ and Mamas I. Prodromidis* ,† Laboratories of Analytical Chemistry and Food Chemistry, Department of Chemistry, University of Ioannina, 451 10 Ioannina, Greece 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 hexacy- anoferrate(II)/(III) couple as a redox probe. Gold elec- trodes were modified with self-assembled monolayers of different thiols (thioctic acid, dithiobis-N-succinimidyl propionate, and cysteamine), and (artificial) casein mi- celles 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 per- formed before and after the interaction of the im- mobilized micelles with rennet. Finally, the proposed biosensors were successfully tried for various com- mercial samples. The casein content of milk represents about 80% of milk proteins. The principle casein fractions are R s1 -, R s2 -, -, and κappa- casein (κ-CN), and their distribution in cow’s milk is 38, 10, 40 and 12%, respectively. 1 In combination with appreciable quanti- ties of colloidal calcium phosphate (CCP) nanoclusters, they appear in the form of not-quite spherical colloids, 50-500 nm in diameter, called casein micelles. 2 The distinguishing property of all caseins is their low solubility at pH 4.6. Rennet is a natural complex of enzymes produced in any mammalian stomach, and it is widely used in the cheese making industry as the major milk coagulant. 3 The milk-clotting activity of rennet relies on its ability to degrade casein micelles, and this action depends on the chymosin (pI ≈ 4.6) content of the complex. Chymosin proportion may differ by source (cow, lamb, goat, chicken, camel, or nonanimal sources) and age, accompanied by a concomitant alteration of pepsin content. 4 The proportions normally present in commercial rennets are 70% chymosin and 30% pepsin. 5 Rennet coagulation of milk may be divided into primary (enzymic hydrolysis) and secondary (aggregation) stages, al- though these stages normally overlap to some extent during cheese making. During the primary stage, chymosin hydrolyses the outer “hairy layer” of κ-CN (Figure 1A). More specifically, κ-CN is cleaved by rennet at the Phe105-Met106 bond, producing the soluble glycomacropeptide (GMP, residue 106-169), which diffuses away from the micelle into the serum phase, and the positively charged (pI > 7) 6 insoluble para-κ-casein molecules, which remain attached to the casein micelle. 1-3 This results in a reduction of the net negative charge of micelles, as well as of the electrostatic repulsion among them. As a consequence, rennet- altered micelles become susceptible to aggregation 7 (Figure 1B). The nature of the attractive forces during the aggregation of casein micelles is still not completely clear, although calcium bridges, van der Waals forces, and hydrophobic interactions appear to be involved. 3,8 The evaluation of the clotting power of rennet has always been a parameter of great importance from scientific, technological, and commercial viewpoints. The importance of this parameter has raised the scientific interest since the end of the 19th century when the first method, based on visual observation of the formation of a clot, was published. 9 Since then, numerous methods, which also depend upon visual observations of the coagulation point of the milk, have been used to measure rennet activity. 10-12 Even though they are still in routine use, they suffer from the subjective nature of the observation, especially when * To whom correspondence should be addressed. E-mail: mprodrom@ cc.uoi.gr. † Laboratory of Analytical Chemistry. ‡ Laboratory of Food Chemistry. (1) Qi, P. X. Lait 2007, 87, 363–383. (2) Fox, P. F. In Advanced Dairy Chemistry; Fox, P. F., McSweeney, P. L. H., Eds.; Springer: New York, 2003; Vol. 1: Proteins, Part A, pp 1-48. (3) Lucey, J. A. In Encyclopedia of Dairy Sciences; Roginski, H., Fox, P. F., Fuquay, J. W., Eds.; Academic Press: San Diego, 2002; pp 286-293. (4) Guinee, T. P.; Wilkinson, M. G. J. Soc. Dairy Technol. 1992, 45, 94–104. (5) Irigoyen, A.; Izco, J. M.; Ibanez, F. C.; Torre, P. Food Chem. 2001, 72, 137–144. (6) Larsson, K. I.; Andre ´n, A. Int. Dairy J. 1999, 9, 381–382. (7) Walstra, P. J. Dairy Sci. 1990, 73, 1965–1979. (8) Andren, A. In Encyclopedia of Dairy Sciences; Roginski, H., Fox, P. F., Fuquay, J. W., Eds.; Academic Press: San Diego, 2002; pp 281-286. (9) Soxhlet, F. Milch-Zeitung 1877, 6, 497–501. (10) Sommer, H. H.; Matsen, H. J. Dairy Sci. 1935, 18, 741–749. (11) Kunitz, M. J. Gen. Physiol. 1935, 18, 459–466. (12) Berridge, N. J. Analyst 1952, 77, 57–62. Anal. Chem. 2010, 82, 8629–8636 10.1021/ac1017925  2010 American Chemical Society 8629 Analytical Chemistry, Vol. 82, No. 20, October 15, 2010 Published on Web 09/16/2010