Abstract
New acrylic microspheres were synthesised by photopolymerisation where the succinimide functional group was incorporated during the microsphere preparation. An optical biosensor for urea based on reflectance transduction with a large linear response range to urea was successfully developed using this material. The biosensor utilized succinimide-modified acrylic microspheres immobilized with a Nile blue chromoionophore (ETH 5294) for optical detection and urease enzyme was immobilized on the surface of the microspheres via the succinimide groups. No leaching of the enzyme or chromoionophore was observed. Hydrolysis of the urea by urease changes the pH and leads to a color change of the immobilized chromoionophore. When the color change was monitored by reflectance spectrophotometry, the linear response range of the biosensor to urea was from 0.01 to 1,000 mM (R2 = 0.97) with a limit of detection of 9.97 μM. The biosensor response showed good reproducibility (relative standard deviation = 1.43%, n = 5) with no interference by major cations such as Na+, K+, NH4+ and Mg2+. The use of reflectance as a transduction method led to a large linear response range that is better than that of many urea biosensors based on other optical transduction methods.
Highlights
Urea biosensors have been reported for urea determinations in biomedical, clinical and food industry applications [1,2,3], e.g., for monitoring of urea levels in human urine and blood for diagnosis of kidney function and health [4,5,6], as well as for the determination of urea levels in milk [2]
Many urease enzyme immobilization techniques for biosensor construction have involved the incorporation of the enzyme into a polymer membrane or polymer backbone such as tetrafluoroethylene and poly(tetrafluoroethylene) [1], chitosan membranes [12], sol-gel-derived thick-films [3,6] and poly(N-vinylcarbazole)/stearic acid [5]
A mixture of 450 μL hexanadiol diacrylate (HDDA), 0.1 g DMPP, 6 mg NAS and 15 mL H2O with various amounts of n-butyl acrylate (n-BA) and Sodium dodecyl sulfate (SDS) was sonicated for 10 min, after which it was subjected to photopolymerisation for 600 s with UV light (350 nm) under nitrogen gas flow
Summary
Urea biosensors have been reported for urea determinations in biomedical, clinical and food industry applications [1,2,3], e.g., for monitoring of urea levels in human urine and blood for diagnosis of kidney function and health [4,5,6], as well as for the determination of urea levels in milk [2] These numerous applications have motivated the development of urea biosensors. N-Acryloxysuccinimide was used to modify acrylic microspheres to produce functional groups as a linker to immobilize enzyme urease via covalent bonds The efficiency of this bonding has be proven as reported [22,23]. The acrylic microspheres have the advantages of small size and providing a large surface area to volume ratio for enzymatic reactions to occur on the surface, in addition to preventing any barriers to diffusion of reactants and products This should improve biosensor performance in terms of response time and linear response range. Microspheres made from n-butyl acrylate possess good adhesion properties that allow these spheres to be coated directly on a plastic substrate for optical biosensor fabrication
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