Plasmon-Enhanced Nanochannels for Protein Isoforms Tracking and Identification

Project Information

Plas-ID

Grant agreement ID: 101152070

DOI

10.3030/101152070

Project closed

EC signature date 3 April 2024

Start date 1 May 2024

End date 30 April 2026

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 184 698,24

Coordinated by

TECHNION RESEARCH AND DEVELOPMENT FOUNDATION LTD
Israel

Periodic Reporting for period 1 - Plas-ID (Plasmon-Enhanced Nanochannels for Protein Isoforms Tracking and Identification)

Reporting period: 2024-05-01 to 2026-04-30

Summary of the context and overall objectives of the project

The project aims to realize a powerful sensing platform based on nanochannels capable of identify proteins and biomarkers at the single-molecule level. Nanochannel technologies have already enabled DNA manipulation, such as linearization, stretching and sorting, and are at the core of diagnostic devices used for optical genome mapping. Recently, nanochannel-based devices are emerging as powerful sensing tools not only for genomics but also for the identification of proteins and other biomarkers with unprecedented sensitivities at ultra-low concentrations and ultra-small volumes. As the field of liquid biopsy keeps expanding, nanochannel-based technologies are expected to play a central role as key sensing elements while enabling easy interfacing of other nanofluidic elements for upstream samples preparation.
In this project, we have designed and realized nanofluidic devices based on nanochannels. These devices have been assembled and successfully used for the identification of protein mixtures by performing single-molecule fluorescence imaging. The confinement properties of these devices have been exploited to squeeze the optical sensing volume achieving single-molecule resolution and to study how the physical properties of molecules, including their diffusion, are affected under the confinement regime. To improve the capabilities of the device for proteins separation an acrylamide-based gel was also introduced to perform miniaturized gel electrophoresis along the channel. These devices were successfully tested for the discrimination and quantification of VEGF isoforms, which is not currently possible with conventional techniques, such as ELISA, and to detect other proteins, such as CCl2, RBP4, Clusterin and Serpin A4 which are biomarkers for Age-related Macular Degeneration. This technique is now able to separate, track and identify thousands of proteins at the single molecule Level in complex biological samples, requiring ultra-small volumes (only few picoliters) and ultra-low concentrations (in the picomolar range). Currently, these devices are ready to be challenged with more sophisticated applications including liquid biopsy and diseases monitoring. Besides their potential for advanced diagnostics, nanochannel-devices can be used as powerful tools for fundamental research due to the single-molecule sensitivity and thus, they might help elucidating the different functions of proteins isoforms in cell physiology and their over/downregulation during disease progression.
Societal impacts. The development of a single-molecule diagnostic tool capable of detecting multiple proteins in a single run and discriminating proteins isoforms has high potential for early-diagnosis base on liquid biopsy with high benefit to society healthcare. Currently, there is gaining attention on the development of new sensing devices able to identify biomarkers and other molecules from small volumes of samples, including blood, urine, sweat and tears to allow frequent and comprehensive monitoring of diseases and to drastically reduce the invasiveness of screening, the duration of the analysis and the required infrastructure compared to conventional diagnostic techniques.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

For WP1 Nanochannels devices were fabricated in house on a wafer scale by UV lithography starting from double sided wafers with a layer composition of Si(300 μm)/SiO2(500 nm)/SiNx(50 nm). After the fabrication of the wafer, a glass slide is sealed to the frontside of each chip through anodic bonding at 1000 V and at 400 °C. The backside of the chip is bonded to a PDMS elastomer by plasma activation to form fluidic compartments. Importantly, the height of the nanochannels devices is only few hundreds of nanometers and is precisely controlled by performing BOE etching of the SiO2 layer. These devices are used to perform single-molecule fluorescence imaging, taking advantage of the reduced optical volume formed by the sub-wavelength height. The nanochannels can be functionalized with an acrylamide-based gel to reduce the speed of molecules driven in the channel by electrophoresis.
For WP2 recombinant proteins, including VEGF121 and VEGF 165, as well as other proteins relevant in the progression of Age-related Macular Degeneration disease, which are CCL2, RBP4, Clusterin and SerpinA4, were labelled on specific amino acid residues after their denaturation. Therefore, the primary structure exposing the amino acid sequence of each protein was available to label specific amino acid residues, specifically cysteines and lysines with chemoselective labelling techniques. In particular, cysteines were labelled with Atto542 through maleimide chemistry (the sulphydryl side chain of a cysteine acts as a nucleophile to attach the maleimide double bond of the dye) and lysins were through NHS ester chemistry (the unprotonated amine group of the lysine attacks the carbonyl carbon of the NHS ester of the dye).
For WP3 nanochannels devices were mounted on a custom-made single-molecule fluorescence imaging setup equipped with two lasers with wavelength 532 nm and 645 nm. The sample was loaded in the nanochannel and the migration of the proteins in the gel was driven by electrophoresis, namely by applying an external voltage. In this regard, the device allows us to perform miniaturized gel electrophoresis. The chip was illuminated with the two lasers working in alternated conditions (frame rate 25 ms) and the fluorescence emitted by the proteins during their migration was recorded on an EM-CCD camera. Because the height of the nanochannel is subwavelength the optical volume is squeeze at the point that we can resolve single molecules. We recorded movies of proteins moving in the nanochannels and from the analysis of the acquired movies we could form tracks related to the motion of each individual protein. From analysis of the trajectories (performed in Track-Mate and MATLAB), we were able to extract information on the arrival time of the proteins in a certain location of the chip, the average velocity and the average green, red and FRET signals. Due to our labelling strategy, the intensity of the emitted fluorescence gives information on the primary structure of the protein, specifically the green fluorescence is related to the cysteines number, the red signal to the lysins number and the FRET signal to their reciprocal arrangement, respectively. Therefore, these technique enables a single-molecule multiparametric characterization of proteins, which can be used for proteins identification from complex biological samples and other mixtures.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Due to its capabilities for single-molecule protein sensing in small volumes, the technology proposed in this project could be used for early-diagnosis and disease monitoring decreasing the healthcare costs related to diagnostics compared to conventional imaging techniques and other methodologies, including ELISA and Mass Spectroscopy. Furthermore, the global liquid biopsy market size was estimated at USD 13.60 billion in 2025 and is projected to reach USD 32.57 billion by 2033 (https://www.grandviewresearch.com/industry-analysis/liquid-biopsy-market).