TY - JOUR

T1 - Aptamer-based Depletion of Small Molecular Contaminants

T2 - A case study using ochratoxin A

AU - Schax, Emilia

AU - Lönne, Maren

AU - Scheper, Thomas

AU - Belkin, Shimshon

AU - Walter, Johanna Gabriela

N1 - Funding Information: Parts of this study were funded by grants from the German Research Foundation (DFG) to the REBIRTH Cluster of Excellence (EXC 62/2) and by Europäische Fonds für regionale Entwicklung (EFRE). We thank Professor Birgit Glasmacher and Lutz Dreyer, Institut für Mehrphasenprozesse, Leibniz Universität Hannover for their support with the confocal microscopy. Furthermore, we want to thank Dr. Astrid Sitte (NanoTemper Technology GmbH, Munich) for helpful discussion of the MST experiments.

PY - 2016/1/19

Y1 - 2016/1/19

N2 - Based on the increasing demand for detection and depletion of small molecules like mycotoxins or pesticides in food, water, or pharmaceuticals, aptamers are gaining more importance as sensitive, specific-depletion molecules. Here, we present an aptamer-based method for depletion of ochratoxin A (OTA) as a model system and show the advantages and the limitations of aptamers in the depletion of small molecular contaminants. OTA is a mycotoxin produced by various Penicillium and Aspergillus strains and is often found in grain and grain derivatives. We immobilized a well-described DNA aptamer against OTA on an agarose gel and used the column as a clean-up system. The aptamer shows a high specificity and sensitivity for OTA: Ochratoxin B, a molecule similar to OTA, was not bound by the aptamer; and a control oligonucleotide was not able to bind OTA. After optimizing the process for better economic feasibility, the column could be used for several times without loss of aptamer activity. We investigated the location of immobilized aptamer within the gel using fluorescent-labeled aptamers. Furthermore, beer samples spiked with OTA were used to investigate aptamer activity in complex samples. Using these complex samples we have observed a significant loss of aptamer activity. We have further investigated this limitation by performing microscale thermophoresis experiments to determine the KD values of the aptamer in different complex samples like beer, coffee, juice and wine. Our results indicate that the applicability of aptamers to real processes is currently restricted by the selection buffer used during its selection process (SELEX). We therefore suggest using conditions closer to those of the later application of the aptamer during future SELEX experiments.

AB - Based on the increasing demand for detection and depletion of small molecules like mycotoxins or pesticides in food, water, or pharmaceuticals, aptamers are gaining more importance as sensitive, specific-depletion molecules. Here, we present an aptamer-based method for depletion of ochratoxin A (OTA) as a model system and show the advantages and the limitations of aptamers in the depletion of small molecular contaminants. OTA is a mycotoxin produced by various Penicillium and Aspergillus strains and is often found in grain and grain derivatives. We immobilized a well-described DNA aptamer against OTA on an agarose gel and used the column as a clean-up system. The aptamer shows a high specificity and sensitivity for OTA: Ochratoxin B, a molecule similar to OTA, was not bound by the aptamer; and a control oligonucleotide was not able to bind OTA. After optimizing the process for better economic feasibility, the column could be used for several times without loss of aptamer activity. We investigated the location of immobilized aptamer within the gel using fluorescent-labeled aptamers. Furthermore, beer samples spiked with OTA were used to investigate aptamer activity in complex samples. Using these complex samples we have observed a significant loss of aptamer activity. We have further investigated this limitation by performing microscale thermophoresis experiments to determine the KD values of the aptamer in different complex samples like beer, coffee, juice and wine. Our results indicate that the applicability of aptamers to real processes is currently restricted by the selection buffer used during its selection process (SELEX). We therefore suggest using conditions closer to those of the later application of the aptamer during future SELEX experiments.

KW - affinity depletion

KW - affinity separation

KW - aptamer

KW - downstream processing

KW - microscale thermophoresis

KW - Ochratoxin A

UR - http://www.scopus.com/inward/record.url?scp=84954501326&partnerID=8YFLogxK

U2 - 10.1007/s12257-015-0486-1

DO - 10.1007/s12257-015-0486-1

M3 - Article

AN - SCOPUS:84954501326

VL - 20

SP - 1016

EP - 1025

JO - Biotechnology and Bioprocess Engineering

JF - Biotechnology and Bioprocess Engineering

SN - 1226-8372

IS - 6

ER -