• Simon Baumgarte
4. semester, Nanobioteknologi (cand.polyt.), Kandidat (Kandidatuddannelse)
In the course of this master thesis PTEN as a potential new biomarker contained in extracellular vesicles of prostate cancer patients was evaluated concerning its detectability in lateral flow assays. To do so, different labelling and detection strategies were exploited to find the most sensitive detection strategy.
In this respect, the AlphaLISA assay format — similar to an ELISA as the standard methodology for biomarker quantification — showed the highest sensitivity for the target of interest. When this assay format was transferred to lateral flow strips, a decrease in sensitivity by 25× could be observed, lying in the same range as that of a visual read-out, based on gold nanostars. These nanoparticles were additionally modified with a Raman-active molecule, however the detection of SERS signals from these conjugates did not result in a substantial increase of the assay sensitivity. Hence, it was shown that a Raman-based detection strategy was comparable to both the visual read-out and a fluorescence-based detection scheme for the quantitative detection of PTEN as the target of interest.
Still, experiments with Raman-active gold nanostars offered only preliminary results, but gave an insight into the technique’s capabilities for further optimization. Together with the possibility to detect signals using a portable reader prototype, this technique was slightly inferior to Europium-doped AlphaLISA acceptor beads, which depended upon another dedicated strip scanning device. Still, the additional visual assessment of test results using gold nanostars allows for the detection of PTEN without further instrumentation at similar sensitivity, rendering gold nanostars a more versatile technique.
Furthermore, spike-and-recovery experiments showed a strong matrix effect for detection strategies using AlphaLISA acceptor beads on lateral flow strips. These findings stress the importance of a suitable calibration of the system, as well as of finding appropriate purification or enrichment strategies for the samples to be tested. In these experiments, the chemiluminescence-based standard format of the AlphaLISA assay again showed most precise results, while requiring most complex instrumentation.
Lastly, different conjugation chemistries and suitable characterization techniques for nanoparticles to be conjugated with antibodies were exploited throughout this thesis, allowing to assess the success of each conjugation protocol.
Overall, a starting point for the formation of diverse conjugates to be used in lateral flow assays and an evaluation of their performance is reported, giving a first characterization of the most promising detection schemes. For future experiments, it remains to validate the performance of Raman-based detection strategies as the most versatile label found in this work. Furthermore, the transition from in vitro experiments to clinical samples presents another challenge for the assay format, requiring further optimization and calibration of the lateral flow assay reported here. However, the obtained data showed promising results, making an implementation of the assay worthwhile in the future.
Udgivelsesdato31 aug. 2019
Antal sider144
Ekstern samarbejdspartnerFraunhofer Institute for Cell Therapy and Immunology
Ana Leonor Heitor Lopes ana-leonor.lopes@izi.fraunhofer.de
ID: 310090566