Abstract
Numerous studies have addressed the utilization of glutaraldehyde (GA) as a homobifunctional cross-linker. However, its applicability has been impeded due to several issues, including the tendency of GA molecules to undergo polymerization. Herein, a portable urea biosensor was developed for the real-time monitoring of the flow of physiological fluids; this was achieved by using disuccinimidyl cross-linker-based urease immobilization. Urease was immobilized on a porous polytetrafluoroethylene (PTFE) solid support using different disuccinimidyl cross-linkers, namely disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS) and bis-N-succinimidyl-(pentaethylene glycol) ester (BS(PEG)5). A urease activity test revealed that DSS exhibited the highest urease immobilizing efficiency, whereas FT-IR analysis confirmed that urease was immobilized on the PTFE membrane via DSS cross-linking. The membrane was inserted in a polydimethylsiloxane (PDMS) fluidic chamber that generated an electrochemical signal in the presence of a flowing fluid containing urea. Urea samples were allowed to flow into the urea biosensor (1.0 mL/min) and the signal was measured using chronoamperometry. The sensitivity of the DSS urea biosensor was the highest of all the trialed biosensors and was found to be superior to the more commonly used GA cross-linker. To simulate real-time monitoring in a human patient, flowing urea-spiked human serum was measured and the effective urease immobilization of the DSS urea biosensor was confirmed. The repeatability and interference of the urea biosensor were suitable for monitoring urea concentrations typically found in human patients.
Highlights
Biosensors are analytical devices that detect biomolecules, comprising three parts, namely a molecular recognition layer, a transducer and a signal generator [1]
Urease was covalently immobilized on a parylene-A-coated PTFE membrane with disuccinimidyl cross-linkers of various spacing, namely disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS) and BS(PEG)5, and compared to the immobilization of urease using GA
A portable urea biosensor (2 cm wide, 3.5 cm long and 1.5 cm high) was fabricated based on a urease-immobilized membrane that generated an electrochemical signal in the presence of a flowing fluid containing urea
Summary
Biosensors are analytical devices that detect biomolecules, comprising three parts, namely a molecular recognition layer, a transducer and a signal generator [1]. Molecular recognition layers have been developed for the specific capture of various biomolecules, including antibodies, aptamers, enzymes and receptors [2,3,4,5,6]. Enzyme-based biosensors are powerful analytical devices that use an enzyme to capture analytes and generate a measurable recognition signal [7,8]. Urease is an enzyme that hydrolyzes urea and is widely used for the detection of urea. Mammals, including humans, produce urea as the main end-product of protein metabolism, and its levels are directly related to the protein intake and nitrogen metabolism [9]. As urea is excreted by the kidneys, it is an important biomarker of kidney dysfunction [10,11]
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