Periodic Reporting for period 1 - DeDNAed (Cluster decorated recognition elements on DNA origami for enhanced raman spectroscopic detection methods)
Reporting period: 2021-03-01 to 2022-02-28
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, averting a cytokine storm, which can occur during cancer therapy or SARS-Cov-2 disease and is triggered by a sudden increase in the inflammatory marker interleukin-6 (IL-6), requires the fastest possible detection of even small variations in IL-6 concentration. Only in this way can countermeasures be initiated in time to prevent auto-immune shock in already weakened patients. The early and highly resolved detection of Aflatoxin-B1 (AF-B1), as one of the most carcinogenic substances produced is essential for quality assurance of our food. Already a dose of 10 μg/kg body weight leads to a strongly damaging effect on the liver. Several studies have also shown that there is a risk of toxin accumulation in animal muscle fibers, organs as well as milk. An highly sensitive detection of an AF-B1 primary contamination of plants guarantees timely segregation and leads to a reduced risk of secondary contamination or feeding of them. The emergence of the Corona pandemic/epidemic also showed once again how quickly the spread of pathogens can get out of control and how ensuring adequate hygiene in hospitals and care facilities as well as the early detection of pathogens is essential to contain it.
In our project a biosensor platform will be developed, that is not limited to a special target but that can be easily adapted to a wide range of applications. The platform should not only impress in its flexibility in application, but also with its ease of use, high selectivity and sensitivity as well as the speed of detection. All above-mentioned benefits are possible based on the capabilities of DNA-Origami. The so-called DNA origami, a folded DNA single strand, enables the possibility to combine the advantages of biomolecular sensors and the high-precision optical measurement method of surface-enhanced Raman spectroscopy (SERS). Individual, exchangeable bio-recognition elements (e.g. DNA aptamer, antibodies; bio-RE) with integrated atomic clusters can be easily integrated into specially aligned nanoparticle (NP) arrangements like a pin on a (nano-)breadboard, 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 (paper, polymer or textiles).
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
In our project a biosensor platform will be developed, that is not limited to a special target but that can be easily adapted to a wide range of applications. The platform should not only impress in its flexibility in application, but also with its ease of use, high selectivity and sensitivity as well as the speed of detection. All above-mentioned benefits are possible based on the capabilities of DNA-Origami. The so-called DNA origami, a folded DNA single strand, enables the possibility to combine the advantages of biomolecular sensors and the high-precision optical measurement method of surface-enhanced Raman spectroscopy (SERS). Individual, exchangeable bio-recognition elements (e.g. DNA aptamer, antibodies; bio-RE) with integrated atomic clusters can be easily integrated into specially aligned nanoparticle (NP) arrangements like a pin on a (nano-)breadboard, 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 (paper, polymer or textiles).
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
In the first year of the DeDNAed project, work was carried out on the development of (I) the bio-RE ,(II) metallic NP synthesis, (III) DNA origami and (IV) immobilisation system design. This involved the synthesis of a suitable oligonucleotide (oligo) sequence for the bio-RE consisting of a complementary attachment sequence to the DNA origami, the DNA aptamer for AF-B1 detection, and the sequence, which allows the synthesis of atomic clusters (AC). In addition to research on a method for AC synthesis within an antibody for IL-6 detection as a second potential bio-RE, both single-crystal gold NP and polycrystalline gold NP were also synthesized and functionalized with thiloated complementary oligos for binding to the DNA origami.
The DNA origami structure design was adapted for optimal position of bio-RE immobilization to ensure adequate gold NP anchor points for all contemplated gap sizes. Based on this four DNA origami designs with different NP spacing were synthesized. In addition, initial experiments of NP attachment were performed considering the desired spacing.
For the subsequent integration of the biosensor, three different material systems with varying binding-resistant (BR) layers were developed by nanotechnology. Their selectivity in binding affinity towards the binding-attractive (BA) layer was investigated by immobilization tests with un-functionalized DNA templates. Two of the three systems were found to be sufficiently selective. Further process adaptations to reduce the binding affinity of the BR layer are currently in progress, as are tests to increase the binding affinity of the BA layer.
From the scientific work, three deliverables (D) regarding the design of the bio-RE (D2.1) the DNA Origami (D3.1) and the immobilization system (D5.1) have been prepared so far.
In the context of project management as well as dissemination and exploitation, next to the Consortium Agreement three other administrative documents to ensure efficient project development have been developed and published in an open-access repository (Zenodo).
(1) Project Quality Plan
(2) Data Management Plan
(3) Exploitation and dissemination plan of Results
Furthermore, a project website was created, as well as an online presence on Twitter and LinkedIn.
The DNA origami structure design was adapted for optimal position of bio-RE immobilization to ensure adequate gold NP anchor points for all contemplated gap sizes. Based on this four DNA origami designs with different NP spacing were synthesized. In addition, initial experiments of NP attachment were performed considering the desired spacing.
For the subsequent integration of the biosensor, three different material systems with varying binding-resistant (BR) layers were developed by nanotechnology. Their selectivity in binding affinity towards the binding-attractive (BA) layer was investigated by immobilization tests with un-functionalized DNA templates. Two of the three systems were found to be sufficiently selective. Further process adaptations to reduce the binding affinity of the BR layer are currently in progress, as are tests to increase the binding affinity of the BA layer.
From the scientific work, three deliverables (D) regarding the design of the bio-RE (D2.1) the DNA Origami (D3.1) and the immobilization system (D5.1) have been prepared so far.
In the context of project management as well as dissemination and exploitation, next to the Consortium Agreement three other administrative documents to ensure efficient project development have been developed and published in an open-access repository (Zenodo).
(1) Project Quality Plan
(2) Data Management Plan
(3) Exploitation and dissemination plan of Results
Furthermore, a project website was created, as well as an online presence on Twitter and LinkedIn.
In this project, we want to combine the advantages of SERS and DNA origami to develop a new generation of biosensors that extend existing concepts for nano arrangements on DNA origami for SERS. We would like to create highly ordered 2D arrays from at least three NPs in order to create hot spots of defined quantities and local distribution in the light-exposed areas. In this way we create the indispensable prerequisites for a detection (qualitatively and quantitatively) directly from a supernatant solution and enable a direct correlation between analyte concentration and the SERS signal. The heterogeneous functionalization consisting of a highly ordered SERS array and an individually adaptable bioRE as a biosensor has not yet been described in the literature and offers great innovation potential due to its selectivity and sensitivity as well as its speed and individuality. The variability of the DNA origami as a binding matrix for various bioRE and NP offers the possibility to construct individual structures and arrays and to adapt them to the requirements of the measurement method.
Further, to the best of our knowledge, a method for a surface immobilization of a (functionalized) DNA origami on paper, cotton or similar substrates has never been described in the literature. However, opens up the possibility of use as wipe tests or for wearables.
The direct detection of the analyte from the solution and the transfer to application-related surfaces represent two essential steps for the further development of current research on SERS and DNA origami-supported diagnostics, which will bring a market launch into the existing biosensor technology significantly closer. The detection of various model analytes with high research relevance such as interleukin-6 (IL-6), aflatoxin, cancer DNA and recombinant surface proteins of influenza is intended to verify the high application potential of our sensor platform in the point-of-care diagnosis. However, these new research results not only limit their application to sensors and medical technology, but can also be used for optics or telecommunications.
Further, to the best of our knowledge, a method for a surface immobilization of a (functionalized) DNA origami on paper, cotton or similar substrates has never been described in the literature. However, opens up the possibility of use as wipe tests or for wearables.
The direct detection of the analyte from the solution and the transfer to application-related surfaces represent two essential steps for the further development of current research on SERS and DNA origami-supported diagnostics, which will bring a market launch into the existing biosensor technology significantly closer. The detection of various model analytes with high research relevance such as interleukin-6 (IL-6), aflatoxin, cancer DNA and recombinant surface proteins of influenza is intended to verify the high application potential of our sensor platform in the point-of-care diagnosis. However, these new research results not only limit their application to sensors and medical technology, but can also be used for optics or telecommunications.