1. IntroductionThe determination of ammonia (NH3) in the environmental samples has become increasingly important. Usually, ammonia exist either in form of gases as ammonia or in water as ammonium (
) ions [1]. Ammonia is part of the natural nitrogen cycle and it is released to the environment by natural processes such as the decomposition of organic matter, human and animal excrements, or by volcanic eruptions [2]. It can also be distributed to the environment by activities like extensive use of fertilizers, spillage or leakage from wastewater treatment plants [3]. Ammonia is often found at low level in natural water however, elevated concentration of this compound can occur usually due to effluent discharges from sewage treatment plants, industrial processes or farming activeties [2,3].
Various approaches have been developed to detect dissolved ammonia in aqueous environment employing either electrochemical or optical methods [4-6]. These methods are time consuming and often require skill personnel to operate the sample preparation. An interesting alternative method for determination of dissolved ammonia involves the development of biosensors, which can offer simple, rapid, sensitive, specific and portable system [2,3,7].
Several papers have been published on the determination of dissolved ammonia employing enzyme system. The reaction involved the use of enzyme glutamate dehydrogenase (GLDH), which requires the cofactor β- nicotinamide adenine dinucleotide (NADH) and ammonium (
) in the enzymatic conversion of 2-oxoglutarate to L-glutamate [8-10]. During the reaction NADH is oxidized to NAD+, thereby making possible the indirect monitoring of ammonium by measuring the consumption of NADH either amperometrically at a potential of +0.76 V or optically at a wavelength of 340 nm. There are some intricacy present in the amperometric detection of NADH like the involvement high overpotentials and the formation of by-products that caused the adsorption of (NAD)2 dimers which foul the electrode surface [11,12]. Optical sensors have attracted the attention of many researches because of their small size, ease of operation and freedom from electrical interference [13].
Here, we demonstrate a colorimetric based biosensor employing stacked film immobilization of glutamate dehydrogenase (GLDH) and diaphorase (Dph) in combination with redox indicator thiazolyl blue tetrazolium bromide (MTT) for the determination of ammonium ion in aqueous environment. The biosensor offer several advantages including the ability of the sensors to give a quick indication on the presence of analyte of interest based on the color changes by using dual enzymes.
2. Experimental2.1. ReagentsDph, NADH, MTT, copper chloride, ferrous sulphate, zinc chloride, silver nitrate, mercury chloride, calcium chloride, potassium chloride and sodium nitrate were purchased from SIGMA. GLDH and α-ketoglutaric acid were obtained from FLUKA. Chitosan and sodium nitroprusside were supplied by Chito-Chem (M) Sdn. Bhd. and Merck, respectively. Thymol was acquired from BDH Chemicals. Sodium hypochlorite 10% was acquired from Systerm and ammonium chloride was purchased from R & M Marketing. All chemicals were of analytical grades and used without further purification.
2.2. Preparation of BiosensorChitosan solution (2%) was prepared by dissolving 2.0 g chitosan powder in 100 mL acetic acid (1%, v/v). The viscous solution was stirred overnight at room temperature. GLDH (40 mg/mL) and Dph (40 mg/mL) stock solution were prepared by dissolving 0.012 g of respective GLDH powder and Dph powder in 300 mL of 50 mM phosphate buffer solution pH 7. These solutions were then divided into 20 mL aliquots and kept at –20˚C for later use. A homogeneous stock solution of GLDH/ chitosan mixture was prepared by mixing GLDH solution (40 mg/mL) and chitosan solution (2%) at a volume ratio of 0.25 to 1.0 (v/v). Dph/chitosan mixture was prepared by using the same ratio as the latter.
Figure 1(a) illustrates the enzyme immobilization process: 10 mL of the Dph/chitosan mixture was deposited onto a glass slide in an area of 9 mm × 10 mm. Then it was spun at 2000 rpm for 3 s. The biosensor was kept at 4˚C for drying. A volume of 10 mL of the GLDH/chitosan mixture was then pipetted onto the dried film of Dph/chitosan on a glass slide and coated gently over an area of 9 mm × 10 mm. Again, it was spun at 2000 rpm for 3 s and dried at 4˚C.