Periodic Reporting for period 1 - SIMPHONICS (Single-Molecule Acousto-Photonic Nanofluidics)
Período documentado: 2022-06-01 hasta 2024-11-30
Reading biomolecular signatures and understanding their role in health and disease is one of the greatest scientific challenges in genome and proteome biology. Yet, complete protein analysis at the single-molecule level remains an unmet milestone. This pursuit is fundamentally hindered by the huge dynamic range of protein expression in cells and the insufficient spatio-temporal resolution of current analysis methods.
Next-generation single-molecule techniques that can precisely manipulate and sequence proteins in space and time are urgently needed to reach this goal. Among these, nanopore platforms are at the forefront, leading in terms of read length, throughput and sensitivity. However, the major challenges associated with translocation speed control and the precise-readout in solid-state nanopore devices, remain prohibitive.
In SIMPHONICS, I will resolve these issues by developing the first integrated platform that combines nanopore transport measurements, spatially modulated acoustic wavefields and single-molecule fluorescence time traces to confine, scan and optically fingerprint proteins in a non-invasive and massively parallel manner. The feasibility of this method will be established by attaining three main objectives: 1) Confining and controllably manipulating individual molecules using acoustic nanotweezers; 2) On-demand engineering of 2D material optical emitters as ultrabright fluorescent probes for energy transfer based detection, and 3) Identifying proteins/peptides from their optical signatures in multi-color Förster resonance energy transfer (FRET) during acoustophoresis. With this powerful and unique platform, I will harness the vast potential of acousto-photonic interactions in monolithic nanopore devices. Successful achievement of the project objectives will result in a high-throughput and non-destructive protein fingerprinting platform and signify a considerable leap forward in our quest to unravel the human proteome.
Next-generation single-molecule techniques that can precisely manipulate and sequence proteins in space and time are urgently needed to reach this goal. Among these, nanopore platforms are at the forefront, leading in terms of read length, throughput and sensitivity. However, the major challenges associated with translocation speed control and the precise-readout in solid-state nanopore devices, remain prohibitive.
In SIMPHONICS, I will resolve these issues by developing the first integrated platform that combines nanopore transport measurements, spatially modulated acoustic wavefields and single-molecule fluorescence time traces to confine, scan and optically fingerprint proteins in a non-invasive and massively parallel manner. The feasibility of this method will be established by attaining three main objectives: 1) Confining and controllably manipulating individual molecules using acoustic nanotweezers; 2) On-demand engineering of 2D material optical emitters as ultrabright fluorescent probes for energy transfer based detection, and 3) Identifying proteins/peptides from their optical signatures in multi-color Förster resonance energy transfer (FRET) during acoustophoresis. With this powerful and unique platform, I will harness the vast potential of acousto-photonic interactions in monolithic nanopore devices. Successful achievement of the project objectives will result in a high-throughput and non-destructive protein fingerprinting platform and signify a considerable leap forward in our quest to unravel the human proteome.
• Functionalization of DNA origami nanostructures with solid-state optical emitters of hBN via site-specific pi-pi stacking interactions. This is a powerful approach because it maintains the structural fidelity and stability of the nanopore, while enabling the direct integration of optical probes at the nanopore edge, where the sensitivity is highest.
• The binding strength of single-strand DNA on hBN surfaces can be modulated by the buffer conditions, the ssDNA length and sequence and the topography of the hBN (reported in publication 5 and in a work under review). This is a simple tuning knob for enhancing the re-usability of the devices.
• Scanning acoustic force microscopy (SAFM) is an ideal technique to map the acoustic wavefield on supported and suspended 2D material membranes at higher spatial resolution. For the first time, we were able to show that the pitch of the standing wave across suspended graphene drums is ~4 smaller than on bare piezoelectric substrates. The standing wave pattern is strongly affected by the membrane thickness and tension, as well as the shape of the cavity. (manuscript in preparation).
• Developing bulk acoustic wave devices from completely off-the-shelf components (total cost per device €9).
• Single-molecule localization of ssDNA on hBN surfaces using single-particle tracking to study the nanodynamics of single-molecules on atomically-smooth surfaces. The diffusion behaviour crucially depends on the DNA length, topography of the surface and density of atomic point defects.
• Method to extract trapping stiffness of acoustic tweezers from optical imaging directly in acoustic resonator device.
• Method for preventing quenching of fluorescent molecules on graphene by introduction of thickness tunable hBN spacing layers.
• The binding strength of single-strand DNA on hBN surfaces can be modulated by the buffer conditions, the ssDNA length and sequence and the topography of the hBN (reported in publication 5 and in a work under review). This is a simple tuning knob for enhancing the re-usability of the devices.
• Scanning acoustic force microscopy (SAFM) is an ideal technique to map the acoustic wavefield on supported and suspended 2D material membranes at higher spatial resolution. For the first time, we were able to show that the pitch of the standing wave across suspended graphene drums is ~4 smaller than on bare piezoelectric substrates. The standing wave pattern is strongly affected by the membrane thickness and tension, as well as the shape of the cavity. (manuscript in preparation).
• Developing bulk acoustic wave devices from completely off-the-shelf components (total cost per device €9).
• Single-molecule localization of ssDNA on hBN surfaces using single-particle tracking to study the nanodynamics of single-molecules on atomically-smooth surfaces. The diffusion behaviour crucially depends on the DNA length, topography of the surface and density of atomic point defects.
• Method to extract trapping stiffness of acoustic tweezers from optical imaging directly in acoustic resonator device.
• Method for preventing quenching of fluorescent molecules on graphene by introduction of thickness tunable hBN spacing layers.
The discovery that hBN is an atomically smooth, non-quenching surface that enable studies of the dynamics of single-molecule using wide-field fluorescence is highly significant in the single-molecule field. It can become a new tool for characterizing DNA-protein interactions at high resolution and throughput. To date, it was only studies via simulations.