Periodic Reporting for period 2 - SIMONANO2 (Single Molecule Analysis in Nanoscale Reaction Chambers)
Reporting period: 2022-08-01 to 2024-01-31
What is the problem/issue being addressed?
The project aims to develop a new platform for studying individual biological molecules. Today, it is possible to detect single molecules, but they need to be confined in a non-invasive manner to extend the observation time and obtain more information. With the SIMONANO2 platform, the molecules will be confined in a nanoscale chamber (one attoliter) without any forces acting on them and without attaching them to a surface. This is a very non-invasive approach. It will also be possible to exchange the liquid environment around the molecules without washing them away. Last but not least, we aim to precisely control the number of molecules confined in the nanochamber.
Why is it important for society?
If successful, the SIMONANO2 platform will enable new possibilities to study single (or very few) biological molecules. This means that we will learn much more about life on the molecular level because more information is obtained in comparison with an ensemble measurement. For instance, we can understand better how diseases emerge and how medical drugs work. The long-term impact can thus be large in the life sciences and in the pharmaceutical industry.
What are the overall objectives?
The objectives can be summarized as:
- construct the nanochamber for trapping molecules, with two nanopore openings, whereof one is smaller (~10 nm) than the other (~100 nm)
- modify the surfaces chemically to prevent molecules from adsorbing or diffusing away
- use electrical control to push molecules into the chamber and detect when they enter
- use fluorescence microscopy to verify that the molecules remain trapped
- perform some studies on biologically relevant protein systems, i.e. beyond proof-of-concept of trapping
The project aims to develop a new platform for studying individual biological molecules. Today, it is possible to detect single molecules, but they need to be confined in a non-invasive manner to extend the observation time and obtain more information. With the SIMONANO2 platform, the molecules will be confined in a nanoscale chamber (one attoliter) without any forces acting on them and without attaching them to a surface. This is a very non-invasive approach. It will also be possible to exchange the liquid environment around the molecules without washing them away. Last but not least, we aim to precisely control the number of molecules confined in the nanochamber.
Why is it important for society?
If successful, the SIMONANO2 platform will enable new possibilities to study single (or very few) biological molecules. This means that we will learn much more about life on the molecular level because more information is obtained in comparison with an ensemble measurement. For instance, we can understand better how diseases emerge and how medical drugs work. The long-term impact can thus be large in the life sciences and in the pharmaceutical industry.
What are the overall objectives?
The objectives can be summarized as:
- construct the nanochamber for trapping molecules, with two nanopore openings, whereof one is smaller (~10 nm) than the other (~100 nm)
- modify the surfaces chemically to prevent molecules from adsorbing or diffusing away
- use electrical control to push molecules into the chamber and detect when they enter
- use fluorescence microscopy to verify that the molecules remain trapped
- perform some studies on biologically relevant protein systems, i.e. beyond proof-of-concept of trapping
We have developed a reliable method for fabrication of nanochambers where we can control all dimensions quite precisely. Interestingly, this was achieved by another (simpler) method than that in the original proposal. The nanochambers have also been characterized by transmission electron microscopy (not shown here).
Also, we have developed a surface modification protocol for attaching short polymer chains to silica, which will be important for preventing adsorption of the proteins. This method will also be used to block the smaller pore so that proteins cannot spontaneously translocate without an applied voltage.
Furthermore, we have successfully completed a simpler nanochamber design based on a single opening, using thermo-responsive polymers that can act as gates. These chambers can be opened or closed with respect to proteins with temperature control. By adsorbing proteins to the chamber walls in the open state and then closing the gates, it is possible to trap a large number of proteins. Although this system cannot be used for controlling the content with single molecule precision, it still has several advantages compared to existing platforms. Also, it is possible to image multiple nanochambers in parallel. We used these chambers to study enzymatic cascade reactions with all enzymes in solution phase. Reactants and products were continuously transported through the polymer brush barrier.
Finally, we have preliminary results suggesting that there is a threshold behavior in the translocation event frequency dependence on applied voltage (for single nanopores). This is extremely important because it shows that single molecules can be injected to a nanochamber (and detected) controllably - the main goal of the project. These results will be followed up upon and verified using the complete nanochamber structure (dual pores).
Also, we have developed a surface modification protocol for attaching short polymer chains to silica, which will be important for preventing adsorption of the proteins. This method will also be used to block the smaller pore so that proteins cannot spontaneously translocate without an applied voltage.
Furthermore, we have successfully completed a simpler nanochamber design based on a single opening, using thermo-responsive polymers that can act as gates. These chambers can be opened or closed with respect to proteins with temperature control. By adsorbing proteins to the chamber walls in the open state and then closing the gates, it is possible to trap a large number of proteins. Although this system cannot be used for controlling the content with single molecule precision, it still has several advantages compared to existing platforms. Also, it is possible to image multiple nanochambers in parallel. We used these chambers to study enzymatic cascade reactions with all enzymes in solution phase. Reactants and products were continuously transported through the polymer brush barrier.
Finally, we have preliminary results suggesting that there is a threshold behavior in the translocation event frequency dependence on applied voltage (for single nanopores). This is extremely important because it shows that single molecules can be injected to a nanochamber (and detected) controllably - the main goal of the project. These results will be followed up upon and verified using the complete nanochamber structure (dual pores).
As explained above, we have progressed in several ways beyond state of the art. Some of the results are published already, but most are not. The most important objective in the project, which is control of content at the single molecule level, is not yet achieved, but we believe it may be possible based on the promising results so far. (For sure, nothing indicates it would not be possible.) We have also established contact with several molecular biologists to evaluate our platforms for addressing "real" biological problems. For instance, we plan to look at the oligomerization of intrinsically disordered proteins in nanoscale confinement. Here the main feature of our nanochambers compared to state of the art is that all the proteins under observation will remain confined, which extends the measurement time greatly. For comparison, using fluorescence correlation spectroscopy or conventional nanopore sensors, individual protein oligomers can only be observed, but only for about a millisecond before they diffuse away again.