Development of aptamer-based biosensors for mycotoxin determination

In this thesis, the development of aptamer-based electrochemical biosensors for the rapid, reliable, and selective detection of patulin (PAT), a mycotoxin that poses a significant risk to food safety, was investigated. Mycotoxins, especially those found in fruit and fruit-based products, threaten both human and animal health. The frequent detection of PAT above regulatory limits in apple juice and similar foods highlights the need for novel and effective analytical tools. In this context, biosensors utilizing synthetic biorecognition elements such as aptamers offer clear advantages in specificity and sensitivity over conventional analytical techniques. The study presents two distinct biosensor platforms: a structural-switching, methylene blue-labeled aptamer-based sensor using square wave voltammetry (SWV), and an impedimetric sensor based on thiol-modified aptamers (EIS). Gold-coated electrode surfaces were functionalized and optimized for aptamer immobilization, with experimental parameters statistically designed via response surface methodology (RSM). Analytical performance of both sensors was rigorously evaluated in buffer and real samples such as apple juice. The SWV-based structural-switching sensor exhibited a linear range of 0.5–5 ng/mL with a calibration equation of i=0.783x+1.649 (R²=0.99). The limit of detection (LOD) was calculated as 0.33 ng/mL and the limit of quantification (LOQ) as 1.0 ng/mL, with a sensitivity of 0.783 kΩ/(ng/mL). These results are comparable to, or exceed, many previously reported aptasensors. For the impedimetric sensor, a linear range of 0.5–10 ng/mL was achieved, with an LOD of 0.45 ng/mL and an LOQ of 1.35 ng/mL, maintaining a high degree of linearity (R²>0.98). Selectivity tests revealed cross-reactivity with structurally similar mycotoxins, notably zearalenone (ZEA) and ochratoxin A (OTA-A). On the EIS platform, positive signal shifts of 26.7% ± 11.7 for ZEA and 16.1% ± 6.9 for OTA were observed, while SWV-based sensors displayed lower cross-reactivity. These findings underscore the impact of aptamer sequence and terminal modification on the degree of interference among different mycotoxins. Application in real samples resulted in high recoveries (91–104%) and low relative standard deviations (RSD<8%), with both sensors maintaining over 90% performance after two weeks of storage (shelf-life testing). A key outcome of this research is the demonstration that different electrochemical techniques (SWV vs. EIS) and aptamer modifications (MB vs. SH) impart unique advantages and limitations in terms of signal amplification and selectivity. The methylene blue-labeled structural-switching sensor offered superior signal enhancement and lower LODs, while the impedimetric platform was distinguished by its high selectivity and stability. Both approaches provide rapid, low-cost alternatives suitable for field analysis in food safety applications. Future directions may include multiplexed detection platforms, advanced nanomaterial modifications, and in silico aptamer sequence optimization for even higher performance. Overall, this study highlights both the innovative and practical aspects of aptamer-based biosensors in mycotoxin detection, contributing valuable insights to both fundamental and applied analytical sciences.