ELECTROCHEMICAL MICROCELL

In recent years, enzymatic electrochemical biosensors have become increasingly popular in the field of rapid determination of analytes of industrial and environmental interest. The employment of such biosensors permits to avoid or limit the use of analytical methods that involve more complex and time-consuming techniques. One of the main goals in the last years is the development of reagentless electrochemical biosensors, i.e., ready-to-use devices which are directly usable by analysts regardless of their experience. In a reagentless electrochemical biosensor all the reagents which are necessary to perform the electrochemical measurement (i.e. supporting electrolytes, buffers, enzymes, co-factors) are pre-loaded onto or near the working electrode surface, thus allowing the operator to perform the electrochemical measurement by putting a small volume (i.e. 10-100 µL) of the sample onto the sensor and without the addition of reagents to the sample. In this thesis work, a reagentless amperometric biosensor has been developed by using alkaline phosphatase (ALP) enzyme as the biological recognition element and a commercial Screen-Printed Carbon Electrode (SPCE) as a transducer. This biosensor was tested toward the detection of the enzymatic substrate “sodium ascorbil-phosphate (AAP)” as an analyte. AAP is a widely used antioxidant in the cosmetic industry and can be found in several formulations such as anti age serums or hydrating creams. In the presence of alkaline phosphatase, AAP is dephosphorylated generating ascorbic acid (AA) which can be electrooxidized at the working electrode surface. To obtain a reagentless device, small electrochemical microcells were fabricated by 3D-printing and were specifically designed to host the commercial SPCE electrodes used. The possibility of pre-loading all the reagents into these 3D printed microcells and subsequently inserting SPCE electrodes inside, allowed to fabricate ready-to-use electrochemical biosensors kits for the detection of target analytes. The use of such reagentless devices allowed to perform fast electrochemical measurements by using the ALP enzyme in solution (not immobilized on the WE surface) with a minimum volume of sample required and with a higher sensitivity in respect to a traditional ALP biosensor built by immobilizing the ALP enzyme on the working electrode surface. We will demonstrate the use of these reagentless biosensors toward the determination of AAP in cosmetic samples and toward the detection of ALP enzyme in food samples.