2.7. Detection of Furaneol in a Real Sample and Validation with HPLC

Furaneol can naturally be found in many fruits, specifically in strawberries, with a concentration-dependent on the maturation stage. In this way, by measuring the concentration of furaneol in strawberry, it is possible to confirm the ripening stage of the fruit [29]. Therefore, to confirm the reliability of the constructed aptasensor, we grew strawberry plants (Elsanta strawberry) in a climate chamber under controlled temperature and humidity (T = 24 °C and RH = 70%). Fruits (n = 3) at different maturity stages were collected, and their content of furaneol was quantified using the developed aptasensor and high-performance liquid chromatography (HPLC), subsequentially comparing the results.

The fruit maturation stages were quantified in terms of color using a hand-held Spectrophotometer (Chroma Meter CR-400, Konica Minolta Corp., Osaka, Japan). For the color, L is the lightness (black (0) and white (100)), a is the color difference between green and red, and finally, b represents the color difference between yellow color and blue color. L, a, and b scores were the mean of five random measurements.

For the preparation of the real samples for HPLC and the aptasensor measurement, strawberries were cut into small pieces. Pieces from three different fruits from each maturation stage (unripe, during ripening, and ripe), were randomly sampled and ground with 5 mL of distilled water at room temperature. Whatman filter paper was first added on top of Ceramic Porcelain Buchner Chemistry Lab Filter (Haldenwanger, Erlangen, Germany), and then Celite 545 (10 g) was added on top of it. Finally, covered with another Whatman filter paper so that pouring the sample did not disturb the surface of the Celite 545. After that, the filtration system was wetted first with distilled water. The grounded, real sample was filtered, then washed three times with 10 mL of distilled water. Subsequentially, the resulting solution was filtered again first through a 0.45 µm and then through a 0.2 µm nylon membrane before HPLC analysis. Finally, the real sample was diluted, and then the measurement results were multiplied by the dilution factor.

Furaneol content was analyzed using 1525 Waters HPLC (Waters Corporation, MA, USA) equipped with a binary pump, an auto-sampler injection system, a Symmetry C18 Column (2.1 × 50 mm, 3.5 µm, Waters Corporation, MA, USA) and a photo-diode array detector (PDA 2998) set at 286 nm. Shimadzu Chem Station for Windows (Shimadzu Technologies) was used to control the system. The resultant from the sample preparation was subjected to HPLC analysis. The mobile phase consisted of methanol/0.5% formic acid solution (v/v), and the following methanol flow gradient: 15/85 for 2 min, 50/50 for 24 min, 100/0 for 27 min, 100/0 for 29 min, and 15/85 for 33 min [30]. The chromatographic separation was conducted out at a flow rate of 0.8 mL/min for 40-min, and injection volume of 20 μL. The furaneol standards (Sigma-Aldrich) was used to prepare the calibration curve (0.1–2.5 mM, R2 = 0.9964). The samples were injected in duplicate.