Biosensors and Bioelectronics 26 (2010) 1560–1564 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios Flow injection analysis biosensor for urea analysis in adulterated milk using enzyme thermistor Geetesh K. Mishra, Rupesh K. Mishra, Sunil Bhand ∗ Biosensor Lab., Chemistry Group, Birla Institute of Technology & Science, Pilani–KK Birla Goa Campus, NH17B Bypass, Zuari Nagar, Goa 403726, India article info Article history: Received 14 April 2010 Received in revised form 28 July 2010 Accepted 29 July 2010 Available online 4 August 2010 Keywords: Flow injection analysis Enzyme thermistor Adulterated milk Urea Immobilized urease Control pore glass abstract 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 diag- nostic 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 col- umn 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 phos- phate 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 concentra- tions 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. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Adulteration of natural milk with synthetic chemicals is a seri- ous concern for human health. Milk is an excellent source of energy, protein, minerals and vitamins (Noyhouzer et al., 2009). In India, Urea [CO (NH 2 ) 2 ] is commonly used as an adulterant for milk. Adul- teration of milk with urea decreases the nutritive value of the milk. Urea is also a normal constituent of milk. The typical concentration of urea in milk is 18–40 mg/dL (Jonker et al., 1989). A cut-off limit for urea concentration in milk is normally accepted at 70 mg/dL. It also forms a major part (55%) of the nonprotein nitrogen of milk (Sharma et al., 2008). Urea being relatively cheap and rich in nitro- gen is an economical choice to adulterate the milk. The effect of urea above cut-off limit in milk may cause indigestion, acidity, ulcers, cancers, malfunctions of kidney, etc. (Trivedi et al., 2009). Hence urea estimation in milk is of great significance. Several conventional methods have been reported in recent years for urea analysis in milk, i.e. spectrophotometric and conduc- tometric detection (Reis Lima et al., 2004), differential pH technique (Luzzana and Giardino, 1999). The conventional techniques as well ∗ Corresponding author. Tel.: +91 832 2580332; fax: +91 832 2557033. E-mail addresses: sunil17 bhand@yahoo.com, sgbhand@gmail.com (S. Bhand). as current wet chemistry and analytical practices are time con- suming and may require highly skilled workers and expensive equipment (Loung et al., 1991). The food and dairy industries need rapid, reliable and affordable techniques for quality control. The application of thermal biosensors in food analysis is a growing field with increasing demand for reliable sensors (Bhand et al., 2010). Numerous biosensors have been reported in recent years for milk urea analysis, such as manometric biosensor (Jenkins and Delwiche, 2002; Renny et al., 2005) and potentiometric biosensor (Verma and Singh, 2003; Trivedi et al., 2009). Some of these reported biosen- sors have very short detection limit and low operational stability, which renders them unfit for routine analysis. There is a need for economical, reliable, robust and reproducible biosensors specifi- cally for urea analysis in milk. Thus, thermal biosensors are good alternative devices owing to their simplicity of operation and long term stability (Pirvutoiu et al., 2002). Thermal biosensors are based on the measurement of heat, produced when a biological reaction takes place. The amount of reacted substrate is related to the heat produced through the specific enthalpy, H r , of the reaction. An advantage of this princi- ple as compared to other analytical tools like spectrophotometric and electrochemical methods is the universal detection principle which is combined with the specificity of the biological reactions (Bjarnason et al., 1998). Thermometric detection usually employs a 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.07.113