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2.2. Reagents

Chloramphenicol (Mw = 323.13 g·mol−1) was purchased from Sigma (Diegem, Belgium). Tris buffer containing 100 × 10−3 mol·L−1 NaCl, 20 × 10−3 mol·L−1 Tris HCl, 2 × 10−3 mol·L−1 MgCl2, 5 × 10−3 mol·L−1 KCl, and 1 × 10−3 mol·L−1 CaCl2 (pH = 7.6) was obtained from VWR (Leuven, Belgium) and used as a binding buffer solution. CAP binding aptamer sequence (5'-SH-(CH2)6-AGC-AGC-ACA-GAG-GTC-AGA-TGA-CTG-AGG-GCA-CGG-ACA-GGA-GGG-CAT-GGA-GAG-ATG-GCG-3') selected and designed at the SPHERE laboratory of the University of Antwerp [,] was purchased from Eurogentec (Seraing, Belgium). Type B gelatine (GelB, IEP = 5, Bloom strength = 257) isolated from bovine skin by the alkaline process and Type A gelatin (GelA, IEP = 8.8, Bloom strength = 202) isolated from porcine skin by the acid treatment were provided by Tessenderlo Chemie (Brussels, Belgium).

2.3. Procedure

In first step of procedure, the electrochemical pretreatment and biomodification of SPE were done. Prior to immobilization of thiolated DNA aptamer, a multiple-pulse amperometric pretreatment on the gold surface is carried out in a stirred 0.5 mol·L−1 H2SO4, 10 mmol·L−1 KCl solution with following triple-potential pulse sequence: −0.3 V for 3.0 s; 0.0 V for 3.0 s and +1.0 V for 1.5 s (15 cycles) [,]. The gold working electrode surface of SPE was then exposed to the mixture of aptamer (5 µM) containing GelB solution (5 w/v%; solved in Tris buffer at 40 °C). The percentage of the incorporation was 70:30 v/v% for aptamer:Gel solution in Tris buffer (pH 7.6). Their interaction was allowed to proceed for around 4 h, while the electrodes were stored in a wet chamber for protection from evaporation. Four hours was the best time for the immobilization of aptamer; because the minimum time for formation of a self-assembled monolayer of thiolated groups on the gold surface was 3 h [,,] and the drop of gelatin was completely liquid in the wet chamber (≈40 °C) during this period. After that, the wet chamber was switched off and cooled down gradually to reach room temperature. At room temperature, the drop of apt/GelB became rigid.

3. Results and Discussion

depicts the diagnostic performance of the aptasensor. To study the role of gelB as matrix in function of the biosensor, the CAP reduction signal was investigated on the aptamer/GelB-modified SPE. It can be seen that the peak current increases with increasing CAP concentration (0.10 to 12.0 nmol·L−1) with two different slopes [,,]. The peak current shows a linear relationship with the concentration of CAP in the range from 0.30 to 2.0 nmol·L−1 (inset). The calculated detection limit is 1.83 × 10−10 mol·L−1 based on three times standard deviation of the blank divided by the slope of the calibration curve (3σ/m).