Remote biosensor for the determination of trypsin by using nanoporous anodic alumina as a three-dimensional nanostructured material

Abstract

tabrizi, Josep ferré-Borrull & Lluis f. Marsal * 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. Millions of people in the world suffer from some form of pancreatic diseases 1. According to world health statistics reports, pancreatic diseases id associated with a tremendous economic burden 2,3. Due to reduce the economic losses caused by this disease and also to improve the public health, the fabrication of a device to diagnose this disease is important for every government. The routine laboratory test for pancreatic disease diagnosis is the determination of trypsin the human real sample. Trypsin is a serine protease with a potential role in pancreatic cancer that can cleavage the peptides on the carbonyl side of arginine and lysine amino acids 4. In the acute pancreatitis patient that is a fatal disease, the concentration of trypsin reaches up to 84.4 µg.mL −1 in the urine sample that is so higher than its normal rage (115-350 ng.mL −1) 5. Therefore, it is necessary to develop a simple, selective, sensitive, and low-cost sensor for trypsin detection. However, most of them are complicated, low sensitivity, time-consuming, and expensive method 6-10. To overcome these problems, biosensors become more interested to detect trypsin 11,12. The biosensor is an analytical device which includes a transducer and a biomolecule (such as the enzymes, antibodies, and aptamers) 13-15. Among the various biosensors that have been reported for the sensing of bio-targets, the optical biosensors are more interested because of their high sensitivity, small and lightweight, remote sensing ability, immunity to electromagnetic interference, capability for monitoring a wide...

Recent Advances in Nanoporous Anodic Alumina: Principles, Engineering, and Applications

“…In addition, an aptamer padding proved to be a valuable solution [209]. Works of Tabrizi et al show vast versatility of such structures that are capable of the precise recognition of specific biomolecules and ions: determination of lead (II) ions [210], quantitative detection of trypsin [211], or Salmonella sensing through the recognition of specific DNA fragments [212] were reported. These systems are often based on simple principles, yet providing impressive effectivity-often employing precision and selectivity of biomolecules.…”

A Paper-Based Near-Infrared Optical Biosensor for Quantitative Detection of Protease Activity Using Peptide-Encapsulated SWCNTs

“…Although the urine affected both the baseline PL of SWCNTs and the extent of the fluorescence response, we have demonstrated that our biosensor can detect trypsin and differentiate between trypsin concentrations. Recent studies have reported the development of trypsin biosensors with a limit of detection of 8.6 ng/mL using a quartz crystal microbalance (QCM) [ 59 ] and 60 ng/mL using interferometric reflectance spectroscopy (IRS) [ 58 ] under the optimum conditions. Although the minimal tested trypsin concentration in this work was 1 μg/mL, we assume that after further careful optimization, the sensitivity could be significantly enhanced.…”

“…It was reported that trypsin levels in serum and urine are dramatically increased during acute pancreatitis. While the normal range of trypsin concentration in the serum of a healthy person is 0.25 ± 0.1 μg/mL [ 56 ] and in the urine is about 115 to 350 ng/mL, patients with pancreatitis show maximum levels of 1.4 ± 0.6 [ 57 ] and 84.4 µg/mL, respectively [ 58 ]. One of the most frequently used methods for quantitative detection of proteins is enzyme-linked immunosorbent assay (ELISA) [ 59 ].…”

Advances in Optical Biosensors and Sensors Using Nanoporous Anodic Alumina

“…The analysis of the results also demonstrated that the catalytic activity of the urease enzyme in the presence of trypsin as the protease enzyme would change [ 156 ]. In the presence of trypsin, the activity of urease decreased and the amount of the catalyzed urea to hydroxide ion and carbon dioxide decreased.…”