Periodic Reporting for period 1 - PrintNano4Fluo (Metal nanoapertures by nanoimprint to enhance single molecule fluorescence detection)
Reporting period: 2023-10-01 to 2025-03-31
Single molecule fluorescence techniques have revolutionized the modern approaches to biophysics. However, nearly all single molecule experiments remain performed on diffraction-limited microscopes, which sets major limits for single molecule measurements. The fluorescence brightness remains low, constraining the temporal resolution to the millisecond range. Moreover, the femtoliter detection volume (1e-15 L) imposes diluted nanomolar concentrations to isolate a single molecule. This is not compatible with the vast majority of biochemical reactions occurring at 1000x higher concentrations in the micromolar range.
To overcome the limits set by diffraction and improve the light-matter interaction at the nanoscale, nanophotonic tools known as optical nanoantennas have been introduced. High fluorescence brightness and zeptoliter volumes (1e-21 L) have been achieved, opening promising avenues for real-time single molecule analysis. However, despite the large interest raised by the optical nanoantennas, their use in single molecule biophysics remains a burgeoning field, with even scarcer industrial applications.
For the single molecule community, the major issue is the accessibility to nanophotonics samples of high reproducibility, moderate cost and ease of use. For the nanophotonics community, the main challenges concern the complexitiy, high operational costs and advanced equipment and technical skills needed for the overall nanofabrication process. Current nanofabrication approaches rely on focused ion beam (FIB) and electronic lithography (E-beam). Both require expensive equipments (> 700 k€) with high maintenance costs. FIB can fabricate 3D samples, but its processing time is slow and serial. E-beam lithography is faster, but the geometries are mainly restricted to 2D and the resist removal is an issue for deep structures, limiting the nanophotonics gain.
After two decades of parallel developments in single molecule fluorescence techniques and nanophotonics, these two fields remain largely disconnected. The aim of the PrintNano4Fluo project is to bridge this gap and bring nanophotonics tools dedicated to improve single molecule fluorescence detection experiments. The goal is to fabricate nanophotonic structures featuring a high performance at a low production cost so as to provide the single molecule community with a broad access to dedicated samples.
To overcome the limits set by diffraction and improve the light-matter interaction at the nanoscale, nanophotonic tools known as optical nanoantennas have been introduced. High fluorescence brightness and zeptoliter volumes (1e-21 L) have been achieved, opening promising avenues for real-time single molecule analysis. However, despite the large interest raised by the optical nanoantennas, their use in single molecule biophysics remains a burgeoning field, with even scarcer industrial applications.
For the single molecule community, the major issue is the accessibility to nanophotonics samples of high reproducibility, moderate cost and ease of use. For the nanophotonics community, the main challenges concern the complexitiy, high operational costs and advanced equipment and technical skills needed for the overall nanofabrication process. Current nanofabrication approaches rely on focused ion beam (FIB) and electronic lithography (E-beam). Both require expensive equipments (> 700 k€) with high maintenance costs. FIB can fabricate 3D samples, but its processing time is slow and serial. E-beam lithography is faster, but the geometries are mainly restricted to 2D and the resist removal is an issue for deep structures, limiting the nanophotonics gain.
After two decades of parallel developments in single molecule fluorescence techniques and nanophotonics, these two fields remain largely disconnected. The aim of the PrintNano4Fluo project is to bridge this gap and bring nanophotonics tools dedicated to improve single molecule fluorescence detection experiments. The goal is to fabricate nanophotonic structures featuring a high performance at a low production cost so as to provide the single molecule community with a broad access to dedicated samples.
We have developed a dedicated novel protocol to fabricate regular arrays of nanoapertures milled into an opaque metallic film, also known as zero-mode waveguide arrays. The proof-of-principle could be established through this ERC-funded Proof-of-Concept project. Single-molecule fluorescence experiments validate a 200-fold volume reduction for the detection of single molecules together with a 8-fold increase in the fluorescence brightness per molecule, making the optical performance of our nanodevices comparable with those obtained with advanced (ebeam, FIB) nanofabrication tools costing 100x more.
To reach these results, we had to overcome several unexpected technical difficulties which occurred while translating well-referenced approaches for fabricating microstructures towards the realization of nanostructures. The change of dimensions from micro- to nano- scales turned out to be technically more challenging than initially planned, yet our multidisciplinary team could successfully overcome those technical issues and validate experimentally the protocol envisioned in the submitted research proposal.
The initial results are promising, showing high efficiency in the parallelization of samples and their large-scale production. We could fabricate 1x1 mm² arrays of zero-mode waveguide nanoapertures of 220 nm diameter with a 3 µm pitch separating the nanoapertures. Achieving a similar result with focused ion beam (FIB) would have taken one full day of instrument operation, while we could achieve our preparation in less than one hour, at only a fraction of the cost required to operate an electron beam lithography (eBeam) instrument.
Please note that a patent application about our technology is being prepared, hence we cannot openly communicate about the technical details of our process.
To reach these results, we had to overcome several unexpected technical difficulties which occurred while translating well-referenced approaches for fabricating microstructures towards the realization of nanostructures. The change of dimensions from micro- to nano- scales turned out to be technically more challenging than initially planned, yet our multidisciplinary team could successfully overcome those technical issues and validate experimentally the protocol envisioned in the submitted research proposal.
The initial results are promising, showing high efficiency in the parallelization of samples and their large-scale production. We could fabricate 1x1 mm² arrays of zero-mode waveguide nanoapertures of 220 nm diameter with a 3 µm pitch separating the nanoapertures. Achieving a similar result with focused ion beam (FIB) would have taken one full day of instrument operation, while we could achieve our preparation in less than one hour, at only a fraction of the cost required to operate an electron beam lithography (eBeam) instrument.
Please note that a patent application about our technology is being prepared, hence we cannot openly communicate about the technical details of our process.
There is currently a strong demand from the single molecule community for nanophotonic samples that exceed the diffraction limit. The PrintNano4Fluo project has the potential to meet this demand by rethinking the nanofabrication process, which is the main limiting factor.
Thanks to our dedicated nanofabrication protocol, we can offer a series of nanophotonic samples for different fluorescence studies on single molecules: measurements in solution based on diffusion, measurements on fixed molecules, multi-colour studies, spatial multiplexing, etc. Each of these configurations can benefit from a nanophotonic sample to replace the glass coverslip currently used. The benefits are (i) a range of concentrations that can be exploited x1000 to probe reactions that are impossible to follow with current microscopes and (ii) a better signal-to-noise ratio to improve sensitivity and temporal resolution. The main target is the scientific community studying fluorescence on the scale of a single molecule. Industrial extensions for DNA sequencing and/or protein sequencing are also possible. Additionally, some of our samples may also serve to characterize and calibrate the performance of optical microscope setups with nanoscale precision.
The work carried out as part of the PrintNano4Fluo ERC Proof-of-Concept project has demonstrated the feasibility of the approach and the reproducibility of the results. Further developments remain needed to (1) improve the control of the homogeneity of the results on our samples, (2) reduce the diameter of the nano-apertures, (3) integrate microlenses to improve the signal and (4) benchmark the optimised samples with end-user collaborators. Filing of an international patent following our invention is an important step tp protect the IP developed during this project, and is currently underway with the CNRS IP agency.
The project currently continues as part of the “prematuration” program funded by CNRS.
Thanks to our dedicated nanofabrication protocol, we can offer a series of nanophotonic samples for different fluorescence studies on single molecules: measurements in solution based on diffusion, measurements on fixed molecules, multi-colour studies, spatial multiplexing, etc. Each of these configurations can benefit from a nanophotonic sample to replace the glass coverslip currently used. The benefits are (i) a range of concentrations that can be exploited x1000 to probe reactions that are impossible to follow with current microscopes and (ii) a better signal-to-noise ratio to improve sensitivity and temporal resolution. The main target is the scientific community studying fluorescence on the scale of a single molecule. Industrial extensions for DNA sequencing and/or protein sequencing are also possible. Additionally, some of our samples may also serve to characterize and calibrate the performance of optical microscope setups with nanoscale precision.
The work carried out as part of the PrintNano4Fluo ERC Proof-of-Concept project has demonstrated the feasibility of the approach and the reproducibility of the results. Further developments remain needed to (1) improve the control of the homogeneity of the results on our samples, (2) reduce the diameter of the nano-apertures, (3) integrate microlenses to improve the signal and (4) benchmark the optimised samples with end-user collaborators. Filing of an international patent following our invention is an important step tp protect the IP developed during this project, and is currently underway with the CNRS IP agency.
The project currently continues as part of the “prematuration” program funded by CNRS.