Abstract
The determination of trypsin in the human real sample is a routine medical investigation to assess the pancreatic disease. Herein, we fabricated an interferometric reflectance spectroscopy based biosensor for the determination trypsin. For this purpose, urease and fluorescein 5(6)-isothiocyanate (FLITC) were immobilized on the nanoporous anodic alumina (NAA). The operation principle of the proposed biosensor is based on the change in the pH of the solution during the reaction of urease and urea and therefore change in the light-absorbing ability of FLITC in the presence of trypsin. The reaction of the urease enzyme with urea increased the pH of the solution because of producing ammonia. This increase in the pH of solution increased the light-absorbing ability of the immobilized FLITC on NAA and therefore the intensity of the reflected light from the NAA to the charge-coupled device detector decreased. In the presence of trypsin, the catalytic activity of immobilized urease on NAA decreased. This decrease in the activity of urease enzyme consequent on the decrease in the amount of the generated ammonia. Therefore, the immobilized FLITC on the NAA did not absorb more light and consciously, the intensity of the light reflected light into the detector increased. The proposed biosensor exhibited a good response to the concentration of trypsin in the range of 0.25–20 μg.mL−1 with the limit of detection of 0.06 μg.mL−1.
Highlights
The determination of trypsin in the human real sample is a routine medical investigation to assess the pancreatic disease
The morphological characterization of the nanoporous anodic alumina (NAA) was examined by Scanning electron microscopy (SEM) (Fig. 2A–F)
The band in the range of 1140 cm−1 are attributed to the aromatic –C–H in-plane bend, a band at 1250 cm−1 due to the –C–O–C stretching mode and a band at 1414 cm−1 due to the –C–C stretching of quinone and symmetric carboxylic (–COO−) stretch mode from fluorescein 5(6)-isothiocyanate (FLITC) and a band at 1600 cm−1 due to the amide II stretching, and a band at 1723 cm−1 due to amide I of urease enzyme[53,54]. These results demonstrate that FLITC and urease enzyme was immobilized on the NAA, successfully
Summary
The determination of trypsin in the human real sample is a routine medical investigation to assess the pancreatic disease. We fabricated an interferometric reflectance spectroscopy based biosensor for the determination trypsin For this purpose, urease and fluorescein 5(6)-isothiocyanate (FLITC) were immobilized on the nanoporous anodic alumina (NAA). Several optical biosensors have been reported by using urease enzyme to detect the urea in the sample[42,43] In these kinds of the optical-based biosensor, a pH-sensitive optical probe molecule such as fluorescein was used to study the generated ammonia from urea. To fabricate the IRS sensor, NAA, urease enzyme and fluorescein 5(6)-isothiocyanate (FLITC) were used as a reflective thin film, an enzyme that its activity can be changed by to protease proteins and an optical probe molecule, respectively. Compare with the fluorescence biosensor based on quantum dots, the proposed IRS based biosensor exhibited high analytical performance in terms of selectivity, sensitivity, stability, linear range and limit of detection in nearly every case
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