Automated Enrichment of Sulfanilamide in Milk Matrices by Utilization of Aptamer-Linked Magnetic Particles

Abstract

The present work demonstrates the first automated enrichment approach for antibiotics in milk using specific DNA aptamers. First, aptamers toward the antibiotic sulfanilamide were selected and characterized regarding their dissociation constants and specificity toward relevant antibiotics via fluorescence assay and LC-MS/MS detection. The performed enrichment was automated using the KingFisherDuo and compared to a manual approach. Verifying the functionality, trapping was realized in different milk matrices: (i) 0.3% fat milk, (ii) 1.5% fat milk, (iii) 3.5% fat milk, and (iv) 0.3% fat cocoa milk drink. Enrichment factors up to 8-fold could be achieved. Furthermore, it could be shown that novel implementation of a magnetic separator increases the reproducibility and reduces the hands-on time from approximately half a day to 30 min.

Aptamer-based assays: strategies in the use of aptamers conjugated to magnetic micro- and nanobeads as recognition elements in food control

“…Despite the large number of aptamers selected for different targets, their reliable applications in MSPE are still in their infancy as well as limited to few small molecules. As for food safety and quality monitoring, published articles focus exclusively on mycotoxins or antibiotic residues [36][37][38][39][40][41].…”

“…In this regard, it would have been very interesting to also investigate the behavior of non-functionalized or functionalized magnetic adsorbents with scrambled oligonucleotide sequences. Aptamer-linked MNPs were exploited for the development of an automated enrichment approach for sulfanilamide in milk [ 41 ]; firstly, the selection of aptamer sequence towards the target analyte was carried out in a milk-simulating buffer having a pH value and ion conditions comparable to those of bovine milk. Despite a poor aptamer selectivity, especially towards chlortetracycline, manual and automated aptamer-based enrichment procedures were investigated in five matrices with different fat contents, i.e., milk-simulating buffer, skimmed milk, skim milk, full-cream milk, and cocoa milk drink, followed by spectrophotometric analysis.…”

Aptamer-based and immunosorbents

“…This example shows the need to study the effect of different cations to determine the optimal conditions for a strong binding of the target on the OS during the extraction step. Some parameters such as temperature (20)(21)(22)(23)(24), ionic strength and pH (20) also strongly affect the conformation of aptamers. These parameters must be controlled and adapted to ensure high extraction recoveries or favor the elution (25).…”

“…The use of these sorbents in parallel to IS/OS is particularly helpful for optimizing the washing step and then obtaining an optimal selectivity (48,57). At least the evaluation of the selectivity can be achieved by controlling the lack of retention of compounds often present in the studied matrices and not recognized by antibodies/aptamers (18,22,23,(25)(26)(27)(28)30,31,40,(45)(46)(47)(48)72,74,85).…”

Original oder Fälschung?

“…Analog zu Antikörper‐basierten Verfahren erfolgt der Einsatz von Aptameren in ELASA‐ ( Enzyme‐Linked Aptamer Sorbent Assay ) oder ELONA‐Verfahren ( Enzyme‐Linked Oligonucleotide Assay ) , welche teilweise bereits als kommerzielle Analysekits erhältlich sind. Darüber hinaus werden sie aber auch immobilisiert auf Teststreifen ( Lateral Flow Assays ) für einen schnellen und qualitativen Nachweis vor Ort verwendet, für einen schnellen Nachweis verschiedener Antibiotika in Milchprodukten .…”