Bio-Layer Interferometry-Based SELEX and Label-Free Detection of Patulin Using Generated Aptamer.

ABSTRACT

This study reports a novel bio-layer interferometry (BLI)-based SELEX for generation of high affinity aptamers against patulin. Unlike conventional SELEX, the present method enabled real-time monitoring of increasing affinity of the oligonucleotides to the toxin. After seven rounds of selection cycles, the enriched pool of aptamers was characterized by cloning and sequencing and clustered into two families based on similarity. Two sequences, PAT C3 and PAT C4, each belonging to different clades, were further evaluated for their binding affinity. SPR studies determined the dissociation constants (KD) of 8.2 × 10-8 and 1.9 × 10-7 M for aptamer PAT C3 and PAT C4, respectively. The highest affinity PAT C3 aptamer was used to develop a patulin BLI aptasensor, which indicated a linear detection range from 0.045 to 100 ng/mL [limit of detection (LOD) = 0.173 ng/mL; limit of quantification (LOQ) = 0.526 ng/mL]. The aptasensor displayed no cross-reactivity with its structural analogue isopatulin or any of the other mycotoxin groups tested. Spiking studies in simulated apple juice samples showed recoveries in the range of 82.11 to 100.23%, indicating good sensor performance. The study is the first report of BLI-based SELEX for a non-protein toxin, which resulted in the generation of high affinity aptamers and development of an aptasensor which can have wide application in the food industry for high throughput screening of samples for patulin contamination within a short span of time.