New SPR-based methods for analysis of allergenic agents used in wine treatment

The use of allergenic agents in wines needs to be monitored by both producers and supervisory authorities for the protection of sensitive individuals. Currently established as the gold standard is the ELISA-technique which relies on the affinity and specificity of antibodies. Since antibodies are produced in animals and current legislative developments demand the reduction of the use of animals for scientific purposes, the biosensor technology could be interesting for the quantification of allergens. By the combination of a technical device, the surface plasmon resonance, and a specific antibody it was possible to develop an antibody-based analytical method which is capable to detect lysozyme in quantities below 0.25 ppm. Since the biosensor is reusable multiple times, this approach can contribute to reduce the antibody consumption and therefore the use of animals for analysis. Furthermore, first insights in the use of new molecular receptors, aptamers were gained

<i>Just in Time</i>-Selection: A Rapid Semiautomated SELEX of DNA Aptamers Using Magnetic Separation and BEAMing

A semiautomated two-step method for in vitro selection of DNA aptamers using magnetic separation and solid-phase emulsion polymerase chain reaction has been developed. The application of a magnetic separator allows the simultaneous processing of up to 12 SELEXs (systematic evolution of ligands by exponential enrichment) with different targets or buffer conditions. Using a magnetic separator and covalent target immobilization on magnetic beads, the selection process was simplified and the substeps of aptamer/target incubation, washing, and elution of the aptamers were merged into one automated procedure called “FISHing”. Without further processing the resulting FISHing eluates are suitable for BEAMing (beads, emulsion, amplification, and magnetics), which includes the amplification by emPCR (emulsion polymerase chain reaction) and strand separation by the implementation of covalently immobilized reverse primers on magnetic beads. The novel selection process has been proved and validated by selecting and characterization of aptamers to the wine fining agent lysozyme

Food Sensing: Selection and Characterization of DNA Aptamers to <i>Alicyclobacillus</i> Spores for Trapping and Detection from Orange Juice

The quality of the beverage industry’s products has to be constantly monitored to fulfill consumers’ high expectations. The thermo-acidophilic Gram-positive <i>Alicyclobacillus</i> spp. are not pathogenic, but their heat-resistant endospores can survive juice-processing conditions and have become a major economic concern for the fruit juice industry. Current detection methods rely on cultivation, isolation, and organism identification, which can take up to a week, resulting in economic loss. This work presents the selection and identification of DNA aptamers targeting <i>Alicyclobacillus</i> spores by spore-SELEX (systematic evolution of ligands by exponential enrichment) in orange-juice-simulating buffer. The selection process was verified by various techniques, including flow cytometric binding assays, radioactive binding assays, and agarose gel electrophoresis. The subsequent aptamer characterization included the determination of dissociations constants and selectivity by different techniques, such as surface plasmon resonance spectroscopy and fluorescence microscopy. In summary, 10 different aptamers with an affinity to <i>Alicyclobacillus</i> spp. have been developed, analyzed, and characterized in terms of affinity and specificity

Food Targeting: A Real-Time PCR Assay Targeting 16S rDNA for Direct Quantification of Alicyclobacillus spp. Spores after Aptamer-Based Enrichment

Spore-forming Alicyclobacillus spp. are able to form metabolites that induce even in small amounts an antiseptical or medicinal off-flavor in fruit juices. Microbial contaminations could occur by endospores, which overcame the pasteurization process. The current detection method for Alicyclobacillus spp. can take up to 1 week because of microbiological enrichment. In a previous study, DNA aptamers were selected and characterized for an aptamer-driven rapid enrichment of Alicyclobacillus spp. spores from orange juice by magnetic separation. In the present work, a direct quantification assay for Alicyclobacillus spp. spores was developed to complete the two-step approach of enrichment and detection. After mechanical treatment of the spores, the isolated DNA was quantified in a real-time PCR-assay targeting 16S rDNA. The assay was evaluated by the performance requirements of the European Network of Genetically Modified Organisms Laboratories (ENGL). Hence, the presented method is applicable for direct spore detection from orange juice in connection with an enrichment step