Flow injection analysis biosensor for urea analysis in adulterated milk using enzyme thermistor.

Urea in adulterated milk is one of the major health concern, it is especially harmful to pregnant women, children, and the sick. A sophisticated and reliable detection system is needed to replace current diagnostic tools for the urea in the milk. In this work, we report a flow injection analysis-enzyme thermistor (FIA-ET) bio-sensing system for monitoring of urea in adulterated milk. This biosensor was made of the covalently immobilized enzyme urease (Jack bean) on controlled pore glass (CPG) and packed into a column inside thermistor, which selectively hydrolysed the urea present in the sample. The specific heat registered from the hydrolysis of urea was found proportional to the concentration of urea present in the milk sample. The biosensor showed a linear range 1-200 mM, with % R.S.D. 0.96 for urea in 100 mM phosphate buffer, pH 7.2. Good recoveries were obtained (97.56-108.7%) for urea up to 200 mM in the spiked milk samples with % R.S.D. 0.95. In the adulterated milk, a simple filtration strategy and matrix matching technique was used to analyse urea. The response time of the sensor was evaluated for urea, which was 2 min, and it gives satisfactory output. A good comparison was observed between the urea concentrations measured through FIA-ET and the colorimetric method. These results indicate that utilizing this system could be very effective to detect low and high level of urea in adulterated milk. The immobilized urease column exhibited a good operational stability up to 180 days when used continuously at room temperature.

[1]  A. O. Rangel,et al.  Enzymatic determination of urea in milk by sequential injection with spectrophotometric and conductometric detection. , 2004, Journal of Agricultural and Food Chemistry.

[2]  B. Danielsson,et al.  Fructose-selective calorimetric biosensor in flow injection analysis. , 2010, Analytica chimica acta.

[3]  B Danielsson,et al.  An enzyme thermistor-based assay for total and free cholesterol. , 1999, Clinica chimica acta; international journal of clinical chemistry.

[4]  M. Delwiche,et al.  Manometric biosensor for on-line measurement of milk urea. , 2002, Biosensors & bioelectronics.

[5]  Bo Mattiasson,et al.  Determination of Serum Urea with an Enzyme Thermistor Using Immobilized Urease , 1976 .

[6]  B. Danielsson,et al.  Hydroxyapatite as a novel reversible in situ adsorption matrix for enzyme thermistor-based FIA , 2008 .

[7]  Bo Mattiasson,et al.  Enzyme thermistor analysis in clinical chemistry and biotechnology , 1979 .

[8]  J H Luong,et al.  The potential role of biosensors in the food and drink industries. , 1991, Biosensors & bioelectronics.

[9]  David K. Daniel,et al.  Enzyme Based Sensor for Detection of Urea in Milk , 2005 .

[10]  P. Johansson,et al.  A novel thermal biosensor: evaluation for determination of urea in serum , 1998 .

[11]  D. Mandler,et al.  A new approach for measuring the redox state and redox capacity in milk. , 2009, Analytical methods : advancing methods and applications.

[12]  Miguel Valcárcel,et al.  Sequential determination of glucose and fructose in foods by flow-injection analysis with immobilized enzymes , 1987 .

[13]  Rajan Sharma,et al.  A method for estimation of urea using ammonia electrode and its applicability to milk samples , 2008, Journal of Dairy Research.

[14]  Howard H. Weetall,et al.  [10] Covalent coupling methods for inorganic support materials , 1976 .

[15]  N. Verma,et al.  A disposable microbial based biosensor for quality control in milk. , 2003, Biosensors & bioelectronics.

[16]  C. J. Panchal,et al.  Potentiometric biosensor for urea determination in milk , 2009 .

[17]  M. Luzzana,et al.  Urea determination in milk by a differential pH technique , 1999 .

[18]  R. Kohn,et al.  Using milk urea nitrogen to predict nitrogen excretion and utilization efficiency in lactating dairy cows. , 1998, Journal of dairy science.