Aptamer P-domain Binding in the Presence of Food Matrices

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preparations, aptamer target binding was only observed in presence of undiluted CP oyster-suspension. In presence of diluted oyster preparations, aptamer target binding was observed for all oyster preparations. However, compared to the Buf-2-P-domain binding in SB, binding intensity in the presence of oyster-preparations was reduced by about 50 %. The data suggest that aptamer-target binding was inhibited by the presence of undiluted oyster matrices. In addition to this observation, it was noted that aptamer-target-binding in CP oyster-suspension was facilitated, when binding in IQF oyster-suspension was not observed. The food matrix present during SELEX was also prepared with oysters from CP (with a different batch of oysters). It is possible, that the aptamer bound its target in presence of the CP oysters, as these were the same kind of oysters (harvested in the same geographical location), which were also present during SELEX. This could be an issue for aptamer applicability, as oyster composition exhibits seasonal variability.

Furthermore, oyster composition is depending on available feed and other environmental conditions. Therefore, variations in oyster composition between harvest areas can be expected.

The idea to conduct a comparative study assessing the influence of food matrices was inspired by the fact that SELEX allows the choice of the selection conditions based on inherent application conditions. However, if the inherent conditions are not defined, the choice of selection conditions cannot be defined either, making the design of an according SELEX experiment difficult. As this problem is not limited to oysters, but applies to a variety of food with additional variables, such as level of ripeness of fruit and vegetable, geographical differences in foods and the high variability of ready-to-eat foods, these challenges need to be addressed in further studies.

While completing FRA in the presence of food matrix preparations, the background signal for binding studies in presence of oyster matrices was as high as the signal detected for the entire P-domain dilution series. In addition, a slower flow-through rate was observed during the FRA experiment in presence of oyster matrices. This raises the question whether FRA is a good analytical method to assess the aptamer binding in food matrices, as specific binding cannot be detected if it is lower than the background signal. An alternative for FRA would be sensor applications, which have been vastly promoted. Indeed, aptamers have been used in sensor or extraction applications from food. The protocol for these applications usually involves sample preparation and matrix dilution prior to aptamer application. This is also the case for the well-studied aptamer for OTA, which has been used in a variety of food matrices. The binding of the OTA aptamer to its target has been predicted as an induced fit upon target binding²¹⁵. This could be advantageous over aptamers that bind their target due to an intramolecular-stabilized complex structure, which could attract more non-specific targets in food matrices. The application described for the OTA aptamer required pre-extraction as well as four fold dilution of a 10 %

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oyster-suspension in SB¹⁸⁴. The food matrices used in this study were partly pre-extracted as described in the experimental section. However, efforts were taken to omit a dilution step since extraction or screening methods for foodborne viruses require high sample quantities, as the number of viral particles compared to the number of bacteria found in foods can be low, due to the lack of virus replication outside of the host organism. It was shown that the P-domain was detected by FRA in presence of 10-fold diluted oyster matrices. This 10-fold dilution of a sample during virus extraction, would however translate in 10-fold increase of extract volume, to be able to process the entire sample. This would result in an unfeasible sample volume for downstream applications such as high throughput applications. This problem does not necessarily apply to extraction or detection methods for bacteria. This is due to the bacterial replication in the contaminated food, contributing to a much higher number of bacteria in foods compared to viruses. Moreover, most bacteria can be cultured after successful recovery from a food source, increasing the overall likelihood of detection. Still, problems have also been reported with aptamer application in food matrices for the detection of bacteria. Aptamers for Campylobacter detection in chicken juice among other food matrices have recently been described in a sensor application¹⁷². However, the limit of detection for bacteria detected in buffer (2.5 bacteria/mL) was considerably lower than in chicken juice (100 bacteria/ mL). That study stimulated a discussion about the usefulness of aptamers to distinguish viable from non-viable organisms, which would be of pronounced interest for the food industry. This is especially important in instances where viable organisms cannot be cultured or where the culture or propagation require high copy numbers, as is the case for NoV²¹⁷.

Despite the issue of the food matrix interfering with sample preparation and analytical assays in general it is noteworthy, that the aptamers for OTA and Salmonella have been used successfully in a multitude of food analytical assays (see section 1.2.4). As mentioned above, the OTA aptamer interacts with its target based on an induced fit mechanism. Further studies to explore the role of the target fit mechanism and whether the mechanism of target interaction determines an aptamers suitability in food matrices are yet to be completed. Aptamer Buf-2 binds the P-domain with high specificity and selectively, still the utility of the aptamer in undiluted food matrix samples could not be confirmed by FRA. Based on this context, the target specificity and its selective binding characteristics, do not determine, whether the aptamer can be used in food analytical application.

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