Periodic Reporting for period 2 - DeDNAed (Cluster decorated recognition elements on DNA origami for enhanced raman spectroscopic detection methods)
Reporting period: 2022-03-01 to 2024-08-31
Biosensor development has made great progress in recent years in terms of selectivity and sensitivity. Nevertheless, there are areas of application in which very specific requirements are placed for monmitoring that cannot be fully met by available sensors to date. For example, detecting small variations in interleukin-6 (IL-6) concentrations is crucial to prevent cytokine storms during cancer therapy or SARS-CoV-2 infection, which can lead to autoimmune shock in vulnerable patients. Similarly, early detection of Aflatoxin-B1 (AF-B1), a carcinogen, is essential for food quality control and preventing contamination. The COVID-19 pandemic also highlighted the importance of early pathogen detection and maintaining hygiene in hospitals.
The objective of this project is to develop a flexible and adaptable biosensor platform that exhibits high selectivity, sensitivity, and rapid detection capabilities. The platform will utilise DNA origami, a folded DNA strand, in order to combine the advantages of biomolecular sensors with surface-enhanced Raman spectroscopy (SERS) for the purpose of precise optical measurements. Individual, exchangeable bio-recognition elements (e.g. DNA aptamer, antibodies; bio-RE) with integrated nano clusters (NC) can be integrated into nanoparticle (NP) arrangements, in order to achieve a fast, sensitive and selective detection of the respective target during binding to the bio-RE through near-field coupling. The integration in higher-order sensor arrays will be done on both solid silicon-based substrates and flexible surfaces (polymers, textiles, ect.).
For the development of such a biosensor the following objectives are essential:
(1) Establishment of DNA origami as “nano breadboard” for the bio-RE
(2) Proof of signal enhancement through spatial alignment of bio-Re and NP
(3) Demonstration of detection of food containments and bio markers on novel sensor platform
(4) Transfer of the sensor platform to a flexible substrate
The objective of this project is to develop a flexible and adaptable biosensor platform that exhibits high selectivity, sensitivity, and rapid detection capabilities. The platform will utilise DNA origami, a folded DNA strand, in order to combine the advantages of biomolecular sensors with surface-enhanced Raman spectroscopy (SERS) for the purpose of precise optical measurements. Individual, exchangeable bio-recognition elements (e.g. DNA aptamer, antibodies; bio-RE) with integrated nano clusters (NC) can be integrated into nanoparticle (NP) arrangements, in order to achieve a fast, sensitive and selective detection of the respective target during binding to the bio-RE through near-field coupling. The integration in higher-order sensor arrays will be done on both solid silicon-based substrates and flexible surfaces (polymers, textiles, ect.).
For the development of such a biosensor the following objectives are essential:
(1) Establishment of DNA origami as “nano breadboard” for the bio-RE
(2) Proof of signal enhancement through spatial alignment of bio-Re and NP
(3) Demonstration of detection of food containments and bio markers on novel sensor platform
(4) Transfer of the sensor platform to a flexible substrate
The major scientific and technical outcomes during the project duration and by the end of the project (M42):
- Synthesized and verified two multifunctional DNA oligo sequence (origami attachment, bioRE, fluorescent AgNC coordination) for the analytes AF-B1 and Ochratoxin A
- Synthesized NCs functionalized antibodies probes and oriented functionalization for complementary bioRE attachment sequence DNA strand conjugation
- Three DNA probes were designed and validated for N-gene of SARS-CoV-II as bio-RE
- Tri-peptide structure as bioRE was introduced as further bio-RE
- Binding rate analysis of the bio-RE with switchSENSE technology were performed
- Synthesized monodisperse AuNPs, oligo-functionalised for DNA origami attachment
- Optimized design, synthesis and purification parameters for DNA origami templates with different NP spacing (2-15 nm)
- Optimized DNA origami templates for array formation and surface integration on lipids
- Synthesized and characterised of DNA origami hybrids based on 3 of the available bio-RE types (DNA aptamer, antibody, DNA probe)
- Microtechnology processing of suitable immobilisation systems on Si and glass (SiO2/Parylene-F; SiO2/PMMA/APTES) with nanostructuring of binding point arrays for selective immobilization
- Optimized parameters for SiO2/Parylene-F and APTES based immobilization process
- Selective immobilized DNA origami hybrids structures (AFB1-Aptamer-Hybrid, Antibody-Hybrid, Probe A-Hybrid)
- Validation of aptamer based detection of AF-B1 by antibody-gold-nanoparticle conjugate
- Validation of the DNA origami hybrid sensor concept by benzophenone detection
The major achievements in terms of Exploitation and Innovation management by the end of the project (M42) are:
- Realization and updating (2 times) of an Exploitation plan and identification of 4 KER:
- Exploitation activities are underway with a spin-off from a partner institute that is interested in exploiting two of the KERs
- Further development of one KER to higher the TRL by serving evidence, illustration of technology and upscaling are in progress based on a national funded project in Germany
- New funding opportunities to further develop the DeDNAed sensor platform have been identified, in particular EIC Transition programme is considered to be the most relevant to the continuation of DeDNAed-generated biosensor
- The major output of IPR management is the recommendation of considering patenting or extension of existing patents to Europe and the USA (in progress)the generated IP as well as a considering licensing methods for the IPR
Activities undertaken to disseminate the results obtained during the project period up to the end of the project (M42):
- Defined and implemented comprehensive C&D strategy (PEDR) to involve different target groups
- Organized 2 dissemination workshops
- Coordinated participation in 36 conferences, 6 workshops, 1 pitch event, 5 other events
- Published 5 articles in peer reviewed, open access scientific journals
- Published 3 press releases and 11 non scientific articles
- Maintained consistent social media presence (LinkedIn, Youtube, Twitter)
- Construction and maintenance of the public website with Key facts of the project, consistently updated news and events and list of publications, posters and public deliverables and provision of link
- Synthesized and verified two multifunctional DNA oligo sequence (origami attachment, bioRE, fluorescent AgNC coordination) for the analytes AF-B1 and Ochratoxin A
- Synthesized NCs functionalized antibodies probes and oriented functionalization for complementary bioRE attachment sequence DNA strand conjugation
- Three DNA probes were designed and validated for N-gene of SARS-CoV-II as bio-RE
- Tri-peptide structure as bioRE was introduced as further bio-RE
- Binding rate analysis of the bio-RE with switchSENSE technology were performed
- Synthesized monodisperse AuNPs, oligo-functionalised for DNA origami attachment
- Optimized design, synthesis and purification parameters for DNA origami templates with different NP spacing (2-15 nm)
- Optimized DNA origami templates for array formation and surface integration on lipids
- Synthesized and characterised of DNA origami hybrids based on 3 of the available bio-RE types (DNA aptamer, antibody, DNA probe)
- Microtechnology processing of suitable immobilisation systems on Si and glass (SiO2/Parylene-F; SiO2/PMMA/APTES) with nanostructuring of binding point arrays for selective immobilization
- Optimized parameters for SiO2/Parylene-F and APTES based immobilization process
- Selective immobilized DNA origami hybrids structures (AFB1-Aptamer-Hybrid, Antibody-Hybrid, Probe A-Hybrid)
- Validation of aptamer based detection of AF-B1 by antibody-gold-nanoparticle conjugate
- Validation of the DNA origami hybrid sensor concept by benzophenone detection
The major achievements in terms of Exploitation and Innovation management by the end of the project (M42) are:
- Realization and updating (2 times) of an Exploitation plan and identification of 4 KER:
- Exploitation activities are underway with a spin-off from a partner institute that is interested in exploiting two of the KERs
- Further development of one KER to higher the TRL by serving evidence, illustration of technology and upscaling are in progress based on a national funded project in Germany
- New funding opportunities to further develop the DeDNAed sensor platform have been identified, in particular EIC Transition programme is considered to be the most relevant to the continuation of DeDNAed-generated biosensor
- The major output of IPR management is the recommendation of considering patenting or extension of existing patents to Europe and the USA (in progress)the generated IP as well as a considering licensing methods for the IPR
Activities undertaken to disseminate the results obtained during the project period up to the end of the project (M42):
- Defined and implemented comprehensive C&D strategy (PEDR) to involve different target groups
- Organized 2 dissemination workshops
- Coordinated participation in 36 conferences, 6 workshops, 1 pitch event, 5 other events
- Published 5 articles in peer reviewed, open access scientific journals
- Published 3 press releases and 11 non scientific articles
- Maintained consistent social media presence (LinkedIn, Youtube, Twitter)
- Construction and maintenance of the public website with Key facts of the project, consistently updated news and events and list of publications, posters and public deliverables and provision of link
This project aims to combine the advantages of SERS and DNA origami to develop a new generation of biosensors, enabling both qualitative and quantitative detection directly from a supernatant solution. This will allow a direct correlation between analyte concentration and the SERS signal. The combination of a highly ordered SERS array and a customizable bioRE as a biosensor has not been previously described and offers significant innovation potential in terms of selectivity, sensitivity, speed, and adaptability. The variability of DNA origami as a binding matrix for different bioRE and NP allows for the creation of tailored structures and arrays that can meet specific measurement requirements. Additionally, the planned application of DNA origami-based biosensors on flexible substrates opens new possibilities for wipe tests or wearable devices, a concept not yet explored in the literature.
The direct detection of the analyte from the solution and its transfer to application-related surfaces will bring a market launch into the existing biosensor technology significantly closer. The demonstration of the sensor principle in the fields of healthcare and food safety was therfore intended to provide a positive example of the performance of the technology. The sensor platform is to be validated for its potential in point-of-care diagnosis through the verification of its performance with a number of model analytes of high research relevance, including IL-6, AF-B1, SARS-Cov-2 and influenza. However, as has been demonstrated within the project in the field of biosensors, the recognition element can be readily exchanged in order to adapt to new applications or measurement regimes. Nevertheless, the potential applications of the DeDNAed approach for biosensor technology are not the only area of interest. Nanotechnology also presents a number of challenges, particularly in the context of DNA origami technology. The initial efforts to adapt the technology to the field of nanotechnology are currently being realised in a funded EU project on optical nanotechnology and in a national project on telecommunications.
The direct detection of the analyte from the solution and its transfer to application-related surfaces will bring a market launch into the existing biosensor technology significantly closer. The demonstration of the sensor principle in the fields of healthcare and food safety was therfore intended to provide a positive example of the performance of the technology. The sensor platform is to be validated for its potential in point-of-care diagnosis through the verification of its performance with a number of model analytes of high research relevance, including IL-6, AF-B1, SARS-Cov-2 and influenza. However, as has been demonstrated within the project in the field of biosensors, the recognition element can be readily exchanged in order to adapt to new applications or measurement regimes. Nevertheless, the potential applications of the DeDNAed approach for biosensor technology are not the only area of interest. Nanotechnology also presents a number of challenges, particularly in the context of DNA origami technology. The initial efforts to adapt the technology to the field of nanotechnology are currently being realised in a funded EU project on optical nanotechnology and in a national project on telecommunications.