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Foodborne zoonotic pathogens have a severe impact on food safety. The demand for animal-based food products (meat, milk, and eggs) is increasing, and therefore faster methods are necessary to detect infected animals or contaminated food before products enter the market. However, conventional detection is based on time-consuming microbial cultivation methods. Here, the establishment of a quorum sensing-based method for detection of foodborne pathogens as Yersinia enterocolitica in a co-cultivation approach using a bacterial biosensor carrying a special sensor plasmid is described. We combined selective enrichment with the simultaneous detection of pathogens by recording autoinducer-1-induced bioluminescent response of the biosensor. This new approach enables real-time detection with a calculated sensitivity of one initial cell in a sample after 15.3 h of co-cultivation, while higher levels of initial contamination can be detected within less than half of the time. Our new method is substantially faster than conventional microbial cultivation and should be transferrable to other zoonotic foodborne pathogens. As we could demonstrate, quorum sensing is a promising platform for the development of sensitive assays in the area of food quality, safety, and hygiene.

The misuse of antibiotics as well as incorrect dosage or insufficient time for detoxification can result in the presence of pharmacologically active molecules in fresh milk. Hence, in many countries, commercially available milk has to be tested with immunological, chromatographic or microbiological analytical methods to avoid consumption of antibiotic residues. Here a novel, sensitive and portable assay setup for the detection and quantification of penicillin and kanamycin in whole fat milk (WFM) based on competitive magnetic immunodetection (cMID) is described and assay accuracy determined. For this, penicillin G and kanamycin-conjugates were generated and coated onto a matrix of immunofiltration columns (IFC). Biotinylated penicillin G or kanamycin-specific antibodies were pre-incubated with antibiotics-containing samples and subsequently applied onto IFC to determine the concentration of antibiotics through the competition of antibody-binding to the antibiotic-conjugate molecules. Bound antibodies were labeled with streptavidin-coated magnetic particles and quantified using frequency magnetic mixing technology. Based on calibration measurements in WFM with detection limits of 1.33 ng·mL−1 for penicillin G and 1.0 ng·mL−1 for kanamycin, spiked WFM samples were analyzed, revealing highly accurate recovery rates and assay precision. Our results demonstrate the suitability of cMID-based competition assay for reliable and easy on-site testing of milk.

Food and crop contaminations with mycotoxins are a severe health risk for consumers and cause high economic losses worldwide. Currently, different chromatographic- and immuno-based methods are used to detect mycotoxins within different sample matrices. There is a need for novel, highly sensitive detection technologies that avoid time-consuming procedures and expensive laboratory equipment but still provide sufficient sensitivity to achieve the mandated detection limit for mycotoxin content. Here we describe a novel, highly sensitive, and portable aflatoxin B1 detection approach using competitive magnetic immunodetection (cMID). As a reference method, a competitive ELISA optimized by checkerboard titration was established. For the novel cMID procedure, immunofiltration columns, coated with aflatoxin B1-BSA conjugate were used for competitive enrichment of biotinylated aflatoxin B1-specific antibodies. Subsequently, magnetic particles functionalized with streptavidin can be applied to magnetically label retained antibodies. By means of frequency mixing technology, particles were detected and quantified corresponding to the aflatoxin content in the sample. After the optimization of assay conditions, we successfully demonstrated the new competitive magnetic detection approach with a comparable detection limit of 1.1 ng aflatoxin B1 per mL sample to the cELISA reference method. Our results indicate that the cMID is a promising method reducing the risks of processing contaminated commodities.

Cholera is a life-threatening disease caused by the cholera toxin (CT) as produced by some Vibrio cholerae serogroups. In this research we present a method which directly detects the toxin's B subunit (CTB) in drinking water. For this purpose we performed a magnetic sandwich immunoassay inside a 3D immunofiltration column. We used two different commercially available antibodies to capture CTB and for binding to superparamagnetic beads. ELISA experiments were performed to select the antibody combination. The beads act as labels for the magnetic frequency mixing detection technique. We show that the limit of detection depends on the type of magnetic beads. A nonlinear Hill curve was fitted to the calibration measurements by means of a custom-written python software. We achieved a sensitive and rapid detection of CTB within a broad concentration range from 0.2 ng/ml to more than 700 ng/ml.

For performing point-of-care molecular diagnostics, magnetic immunoassays constitute a promising alternative to established enzyme-linked immunosorbent assays (ELISA) because they are fast, robust and sensitive. Simultaneous detection of multiple biomolecular targets from one body fluid sample is desired. The aim of this work is to show that multiplex magnetic immunodetection based on magnetic frequency mixing by means of modular immunofiltration columns prepared for different targets is feasible. By calculations of the magnetic response signal, the required spacing between the modules was determined. Immunofiltration columns were manufactured by 3D printing and antibody immobilization was performed in a batch approach. It was shown experimentally that two different target molecules in a sample solution could be individually detected in a single assaying step with magnetic measurements of the corresponding immobilization filters. The arrangement order of the filters and of a negative control did not influence the results. Thus, a simple and reliable approach to multi-target magnetic immunodetection was demonstrated.

I Toxins i was able to uphold its high standards for published papers due to the outstanding efforts of our reviewers. [Extracted from the article]

Cholera is a life-threatening disease caused by the cholera toxin (CT) as produced by some Vibrio cholerae serogroups. In this research we present a method which directly detects the toxin’s B subunit (CTB) in drinking water. For this purpose we performed a magnetic sandwich immunoassay inside a 3D immunofiltration column. We used two different commercially available antibodies to capture CTB and for binding to superparamagnetic beads. ELISA experiments were performed to select the antibody combination. The beads act as labels for the magnetic frequency mixing detection technique. We show that the limit of detection depends on the type of magnetic beads. A nonlinear Hill curve was fitted to the calibration measurements by means of a custom-written python software. We achieved a sensitive and rapid detection of CTB within a broad concentration range from 0.2 ng/ml to more than 700 ng/ml. [ABSTRACT FROM AUTHOR]

Reliable techniques to detect the presence and zygosity of transgenic events in plants are crucial for biotechnology research and plant breeding industry. Therefore, we have introduced a novel assay, which enables high-throughput genotyping of transgenic events in maize using duplex real-time PCR. We demonstrated that the presence and zygosity of two selected transgenic events could be detected with high reproducibility and that our method is suitable for rapid genotyping of a large number of samples. [ABSTRACT FROM AUTHOR]

Foodborne zoonotic pathogens have a severe impact on food safety. The demand for animal-based food products (meat, milk, and eggs) is increasing, and therefore faster methods are necessary to detect infected animals or contaminated food before products enter the market. However, conventional detection is based on time-consuming microbial cultivation methods. Here, the establishment of a quorum sensing-based method for detection of foodborne pathogens as Yersinia enterocolitica in a co-cultivation approach using a bacterial biosensor carrying a special sensor plasmid is described. We combined selective enrichment with the simultaneous detection of pathogens by recording autoinducer-1-induced bioluminescent response of the biosensor. This new approach enables real-time detection with a calculated sensitivity of one initial cell in a sample after 15.3 h of co-cultivation, while higher levels of initial contamination can be detected within less than half of the time. Our new method is substantially faster than conventional microbial cultivation and should be transferrable to other zoonotic foodborne pathogens. As we could demonstrate, quorum sensing is a promising platform for the development of sensitive assays in the area of food quality, safety, and hygiene. [ABSTRACT FROM AUTHOR]

The misuse of antibiotics as well as incorrect dosage or insufficient time for detoxification can result in the presence of pharmacologically active molecules in fresh milk. Hence, in many countries, commercially available milk has to be tested with immunological, chromatographic or microbiological analytical methods to avoid consumption of antibiotic residues. Here a novel, sensitive and portable assay setup for the detection and quantification of penicillin and kanamycin in whole fat milk (WFM) based on competitive magnetic immunodetection (cMID) is described and assay accuracy determined. For this, penicillin G and kanamycin-conjugates were generated and coated onto a matrix of immunofiltration columns (IFC). Biotinylated penicillin G or kanamycin-specific antibodies were pre-incubated with antibiotics-containing samples and subsequently applied onto IFC to determine the concentration of antibiotics through the competition of antibody-binding to the antibiotic-conjugate molecules. Bound antibodies were labeled with streptavidin-coated magnetic particles and quantified using frequency magnetic mixing technology. Based on calibration measurements in WFM with detection limits of 1.33 ng·mL−1 for penicillin G and 1.0 ng·mL−1 for kanamycin, spiked WFM samples were analyzed, revealing highly accurate recovery rates and assay precision. Our results demonstrate the suitability of cMID-based competition assay for reliable and easy on-site testing of milk. [ABSTRACT FROM AUTHOR]

Food and crop contaminations with mycotoxins are a severe health risk for consumers and cause high economic losses worldwide. Currently, different chromatographic- and immuno-based methods are used to detect mycotoxins within different sample matrices. There is a need for novel, highly sensitive detection technologies that avoid time-consuming procedures and expensive laboratory equipment but still provide sufficient sensitivity to achieve the mandated detection limit for mycotoxin content. Here we describe a novel, highly sensitive, and portable aflatoxin B1 detection approach using competitive magnetic immunodetection (cMID). As a reference method, a competitive ELISA optimized by checkerboard titration was established. For the novel cMID procedure, immunofiltration columns, coated with aflatoxin B1-BSA conjugate were used for competitive enrichment of biotinylated aflatoxin B1-specific antibodies. Subsequently, magnetic particles functionalized with streptavidin can be applied to magnetically label retained antibodies. By means of frequency mixing technology, particles were detected and quantified corresponding to the aflatoxin content in the sample. After the optimization of assay conditions, we successfully demonstrated the new competitive magnetic detection approach with a comparable detection limit of 1.1 ng aflatoxin B1 per mL sample to the cELISA reference method. Our results indicate that the cMID is a promising method reducing the risks of processing contaminated commodities. [ABSTRACT FROM AUTHOR]

In modern bioanalytical methods, it is often desired to detect several targets in one sample within one measurement. Immunological methods including those that use superparamagnetic beads are an important group of techniques for these applications. The goal of this work is to investigate the feasibility of simultaneously detecting different superparamagnetic beads acting as markers using the magnetic frequency mixing technique. The frequency of the magnetic excitation field is scanned while the lower driving frequency is kept constant. Due to the particles' nonlinear magnetization, mixing frequencies are generated. To record their amplitude and phase information, a direct digitization of the pickup-coil's signal with subsequent Fast Fourier Transformation is performed. By synchronizing both magnetic fields, a stable phase information is gained. In this research, it is shown that the amplitude of the dominant mixing component is proportional to the amount of superparamagnetic beads inside a sample. Additionally, it is shown that the phase does not show this behaviour. Excitation frequency scans of different bead types were performed, showing different phases, without correlation to their diverse amplitudes. Two commercially available beads were selected and a determination of their amount in a mixture is performed as a demonstration for multiplex measurements. [ABSTRACT FROM AUTHOR]

For performing point-of-care molecular diagnostics, magnetic immunoassays constitute a promising alternative to established enzyme-linked immunosorbent assays (ELISA) because they are fast, robust and sensitive. Simultaneous detection of multiple biomolecular targets from one body fluid sample is desired. The aim of this work is to show that multiplex magnetic immunodetection based on magnetic frequency mixing by means of modular immunofiltration columns prepared for different targets is feasible. By calculations of the magnetic response signal, the required spacing between the modules was determined. Immunofiltration columns were manufactured by 3D printing and antibody immobilization was performed in a batch approach. It was shown experimentally that two different target molecules in a sample solution could be individually detected in a single assaying step with magnetic measurements of the corresponding immobilization filters. The arrangement order of the filters and of a negative control did not influence the results. Thus, a simple and reliable approach to multi-target magnetic immunodetection was demonstrated. [ABSTRACT FROM AUTHOR]