Ultra-empfindlicher Nachweis von Analyten durch kinetische Korrekturlesung
Biochemie
Zusammenfassung der Projektergebnisse
The aim of this project was developing a novel platform technology for the reliable, rapid, and affordable early detection of protein biomarkers. Such an assay would open up the door for better diagnostics of e.g., bloodstream pathogens causing severe infectious diseases. Amongst these the Lyme disease poses a major health risk since more than 14% of the world’s population likely has, or has had encountered the tick-born pathogen. Whilst the illness can be successfully treated with antibiotics, better diagnostics are needed to guide the clinician towards each patient’s complete recovery. The early, direct detection of Lyme disease is especially difficult because of the transient presence of the pathogen Borrelia burgdorferi in the blood stream. The pathogen is only present during the first few days of exposure, before disseminating to the joints, tendons, or bursae. PCR-based methods targeting the detection of B. burgdorferi in synovial fluid only have 50-60% sensitivity and are not a reliable method to determine the eradication of the pathogen after antibiotic therapy due to residual genomic material. Indirect detection on the other hand is hindered by the delayed expression of IgM or IgG antibodies which only gradually increase over time4. Measurable levels for classic serological test like standard Elisa are only reached two to three weeks post infection. All in all, with current immunoassay-based detection techniques only one-third to one-half of Lyme disease patients can be reliably diagnosed. In the course of this one-year funded project I designed a nanoscale DNA sensor that was crafted using DNA origami design principles. The sensor comprises an amplification domain, a target recognition domain, and a site-specific DNA vulnerability for sensor destruction. In its folded form, the sensor seeds the polymerization of single-stranded DNA slats into macroscopic structures as demonstrated in previous work. This amplification step from nano meter sized sensors to micrometre sized aggregates allows easy read out by agarose gel electrophoresis or other spectrophotometric or microscopic techniques (set aim I). Zero background detection should be achieved through destroying the sensors, which then lose its ability to seed polymerization. The destruction is implemented by toehold mediated strand displacement when so called invader strands are added to the sensors. After a certain time of proofreading, we also call it a stress test, target bound sensors will survive, whereas unbound ones are unfolded and thus destroyed by the invading strands (set aim II). We iterated different sensor designs using single stranded DNA as a target and selected the most promising design for testing streptavidin as a mock biomarker target.
