Precise and Rapid Hydrolases Activity Determination Applying Nanoparticles Cloud

Roumen Zlatev, Engineering Institute, Autonomous University of Baja California, Mexicali, Mexico
Margarita Stoytcheva, Engineering Institute, Autonomous University of Baja California, Mexicali, Mexico
Silvia Ahumada, Engineering Institute, Autonomous University of Baja California, Mexicali, Mexico
Ana Luisa Reyes, Engineering Institute, Autonomous University of Baja California, Mexicali, Mexico

The nanostructured disposable spectrophotometric sensor developed by the autors earlier for rapid hydrolases activity determination was improved avoiding its main drawback, the insufficient reproducibility of the results obtained by different sticks of the sensor. It was found that this irreproducibility is due to the non-homogeneous substrate deposition on the transparent stick traversed by the light beam. Thicker deposit zones cause decreased sensibility compared with the finer. The improvement of the deposition process would annul one of the main advantages of this type of sensors – the cost efficiency. To keep this advantage a different approach was applied: the spectrophotometer beam was directed to pass parallel to the stick surface covert by the substrate instead to penetrate it.

This way the substrate during its degradation by the enzyme liberate the nanoparticles forming a cloud around the sensor stick absorbing the light beam sliding near its surface. The registered curve in coordinates: light absorbance vs. time has the shape of wave which slope of the razing part is proportional to the hydrolases activity.

The sensor was applied to the enzymes trypsin and lipase using gelatin and olive oil as substrates respectively charged with Fe₂O₃ nanoparticles.