Optimierte Immobilisierung von Redoxproteinen an funktionale Elektrodenoberflächen für die molekulare Bioelektronik

Ziel dieser Arbeit war es, durch Kombination unterschiedlicher Immobilisierungstechniken eine Strategie zur funktionalen Immobilisierung von Cytochrom c (Cyt c) auf zwei-terminalen nanoskopischen Elektroden Bauelementen zu entwickeln. Die Immobilisierung von Redoxproteinen auf nanoskopischen Elektroden ist für die molekulare Bioelektronik von besonderem Interesse, um die für den Ladungstransfer verantwortlichen Prozesse an Einzelmolekülen aufklären zu können und so ein tieferes Verständnis der molekularen Mechanismen beim Elektronentransfer durch Biomoleküle zu bekommen. Redoxproteine sind aufgrund ihrer besonderen Eigenschaften vielversprechende Kandidaten für neuartige bioelektronische Bauelemente. Cyt c ist ein sehr gut charakterisiertes und stabiles Redoxprotein, das aufgrund seines Modellcharakters in Bezug auf Elektronentransferreaktionen in dieser Arbeit als Untersuchungsobjekt gewählt wurde. Für Cyt c werden unterschiedlichste Immobilisierungsstrategien für die Beschichtung von Elektrodenoberflächen beschrieben. Untersuchungen an makroskopischen 2D Elektroden sollten zeigen, welche der konventionellen Strategien sich besonders gut für die Immobilisierung von Cyt c eignen. Das Linkermolekül, über das die Immobilisierung des Proteins erfolgen sollte, musste dabei zwei wesentliche Funktionen erfüllen. So war erforderlich, dass das Linkermolekül, neben einer funktionalen Anbindung des Proteins an die Oberfläche, ohne es dabei zu denaturieren, auch zu einer guten elektronischen Verknüpfung mit dem Protein führte. Langkettige Moleküle, wie etwa Polymere oder Proteinkomplexe schieden somit als Linkermoleküle aus. Um eine gerichtete bifunktionale Immobilisierung einzelner Cyt c Moleküle zwischen zwei nanoskopischen Elektroden (z.B. Crossbarelektroden oder Nanoelektroden in einem Bruchkontakt) zu ermöglichen, sollte eine zweite komplementäre Immobilisierungsstrategie zum Einsatz kommen. Im Gegensatz zur unspezifisch gerichteten Bindung bei physikalischen und größtenteils auch chemischen Immobilisierungsstrategien, erfolgt die Immobilisierung von Proteinen über Affinitätstags spezifisch und sehr effizient. Über die Beschaffenheit und Position des Tags, lässt sich die Orientierung der Proteine an der Oberfläche beeinflussen. Mit Hilfe rekombinanter DNA-Techniken, sollten His$_{6}$ Tags und alternativ ein speziell entwickelter Cystein Tag an N- bzw. C-terminaler Position von Pferde Cyt c angehängt werden. Die Funktionalität der exprimierten rekombinanten Proteine sollte überprüft, das bifunktionale Immobilisierungsverhalten auf Goldoberflächen untersucht und die Redoxaktivität in Abhängigkeit der [...

Direct electrochemistry of novel affinity-tag immobilized recombinant horse heart cytochrome c

During the last decade protein electrochemistry at miniaturized electrodes has become important not only for functional studies of the charge transfer properties of redox proteins but also for fostering the development of sensitive biosensor and bioelectronic devices. One of the major challenges in this field is the directed coupling between electronic and biologically active components. A prerequisite for a fast and reversible electron transfer between electrode and protein is that the protein can be bound to the electrode in a favourable orientation. We examined electrostatic and bioaffinity-tag binding strategies for the directed immobilization of horse heart cytochrome c (cyt. c) on gold electrode surfaces to achieve this goal. Horse heart cyt. c was expressed in E. coli either as non-modified or genetically modified, i.e. histidine (his)-tag containing protein. The his-tags were introduced at defined positions at the N- or C-terminus of the polypeptide. It was our aim to generate tagged-versions of cyt. c that facilitate strong electronic coupling between protein and electrode and, at the same time, retain their catalytic and regulatory properties. The combination of different immobilization strategies, e.g. his-tag and electrostatic immobilization also opens new avenues for bivalent immobilization of proteins. This is of interest for molecular bioelectronic and biosensing applications where the proteins are immobilized between two crossing electrodes

A Novel Method for Antibiotic Detection in Milk Based on Competitive Magnetic Immunodetection

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

Bacterial Lighthouses—Real-Time Detection of <i>Yersinia enterocolitica</i> by Quorum Sensing

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

Brief Communication: Magnetic Immuno-Detection of SARS-CoV-2 specific Antibodies

SARS-CoV-2 causes ongoing infections worldwide, and identifying people with immunity is becoming increasingly important. Available point-of-care diagnostic systems as lateral flow assays have high potential for fast and easy on-site antibody testing but are lacking specificity, sensitivity or possibility for quantitative measurements. Here, a new point-of-care approach for SARS-CoV-2 specific antibody detection in human serum based on magnetic immuno-detection is described and compared to standard ELISA. For magnetic immuno-detection, immunofiltration columns were coated with a SARS-CoV-2 spike protein peptide. SARS-CoV-2 peptide reactive antibodies, spiked at different concentrations into PBS and human serum, were rinsed through immunofiltration columns. Specific antibodies were retained within the IFC and labelled with an isotype specific biotinylated antibody. Streptavidin-functionalized magnetic nanoparticles were applied to label the secondary antibodies. Enriched magnetic nanoparticles were then detected by means of frequency magnetic mixing detection technology, using a portable magnetic read-out device. Measuring signals corresponded to the amount of SARS-CoV-2 specific antibodies in the sample. Our preliminary magnetic immuno-detection setup resulted in a higher sensitivity and broader detection range and was four times faster than ELISA. Further optimizations could reduce assay times to that of a typical lateral flow assay, enabling a fast and easy approach, well suited for point-of-care measurements without expensive lab equipment

Sensitive Aflatoxin B1 Detection Using Nanoparticle-Based Competitive Magnetic Immunodetection

Food and crop contaminations with mycotoxins are a severe health risk for consumers and cause high economic losses worldwide. Currently, different chromatographic- and immunobased 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 thecMID is a promising method reducing the risks of processing contaminated commodities

Magnetic Nanoprobe Based Competitive Immunodetection for Fast and Quantitative Detection of Antibiotic Residues in Milk

Ensuring high quality standards for food is of global importance. In order to ensure this, suitable rapid test methods are needed. Especially reliable and quantitative detection of low molecular weight compounds like e.g. antibiotic residues is challenging and often not possible with existing rapid test methods. Here we present a novel rapid test format based on competitive magnetic immunodetection (CMID) employing magnetic nanoprobes in combination with a competitive immunofiltration assay and frequency magnetic mixing technology (FMMT) based detection. CMID was demonstrated for quantitative detection of penicillin G and other antibiotics in whole fat milk (WFM). We employed special immunofiltration columns (IFC) with a porous matrix coated with antibiotics conjugate. WFM was spiked with different concentrations of antibiotics and incubated with biotinylated monoclonal antibodies with binding specificity for the respective antibiotics. Subsequently pre-incubated samples were applied to the IFC. While passing the matrix in gravity flow, antibodies were able to compete for binding either to the antibiotics within the milk sample or to the coated antibiotics conjugate. Then magnetic nanoparticles were applied to the IFC to specifically label enriched antibodies. After a final washing step with PBS, IFC were placed in the detection head of the mobile magnetic reader device and readout signals were determined based on FMMT. Magnetic signals correlate with the amount of enriched antibodies and were thus inversely proportional to the antibiotics concentration within the milk samples. Based on this assay principle, calibration curves were recorded for penicillin G and other antibiotics enabling detection limits below 1 ng mL-1 which is below the regulatory limits. Additional spiked WFM samples were analysed using cMID, revealing highly accurate recovery rates and assay precision. We were able to demonstrate that competitive magnetic immunodetection (cMID) is a fast, sensitive and reliable method to quantitatively detect antibiotic residues in milk. Furthermore, the assay can be easily adapted for the detection of other low molecular weight contaminants as e.g. mycotoxins

3D Printed Modular Immunofiltration Columns for Frequency Mixing-Based Multiplex Magnetic Immunodetection

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

Sensitive and rapid detection of cholera toxin subunit B using magnetic frequency mixing detection

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