Bright and biologically powered chemiluminescent labels for cell and tissue imaging

Projektinformationen

BioPoweredCL

ID Finanzhilfevereinbarung: 949087

DOI

10.3030/949087

EK-Unterschriftsdatum 15 Oktober 2020

Startdatum 1 Oktober 2021

Enddatum 30 September 2027

Finanziert unter

• EXCELLENT SCIENCE - European Research Council (ERC)

Gesamtkosten

€ 1 449 750,00

EU-Beitrag

€ 1 449 750,00

Koordiniert durch

UNIVERSITA DEGLI STUDI DI PADOVA
Italy

Periodic Reporting for period 2 - BioPoweredCL (Bright and biologically powered chemiluminescent labels for cell and tissue imaging)

Berichtszeitraum: 2023-04-01 bis 2024-09-30

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

The project BioPoweredCL tackles the inherent limitations of existing biomedical imaging techniques. Currently, achieving high sensitivity, spatial/temporal resolution, and significant tissue penetration simultaneously requires either harmful radiations, toxic labels, or extremely costly equipment. While optical imaging techniques strike a balance in terms of cost and performance, their scalability for comprehensive human body imaging is restricted. The primary issues include shallow photon penetration due to tissue absorption and scattering, and autofluorescence leading to lack of selectivity, particularly in the visible light spectrum. Even advancements into the red/near-infrared (NIR) regions have not entirely overcome these challenges, as they still suffer from limited spatial resolution.
Enhancing biomedical imaging is vital for societal health and well-being. Improved imaging techniques can revolutionize medical diagnostics and therapeutic monitoring, leading to earlier detection of diseases, better tracking of treatment efficacy, and potentially improved survival rates. Moreover, reducing the dependence on harmful radiation and expensive machinery makes advanced healthcare more accessible and safer for a broader population. In addition to medical benefits, innovations in imaging can inspire new technologies and applications in other fields, such as environmental monitoring and biological research.
The BioPoweredCL project aims to develop an innovative optical imaging technique using new near-infrared (NIR) luminophores that harvest energy from the cellular respiration chain, thereby emitting light without being consumed. The overarching goals of the project include:
Developing Novel NIR Emitters: The project focuses on creating NIR emitters capable of self-assembling into bright aggregates whose redox potentials match the cellular electrochemical window. This ensures that the luminophores can function effectively within the biological environment.
Overcoming Current Imaging Limitations: By eliminating the need for an external excitation source and enabling the continuous regeneration of the luminophore, the project aims to overcome the limitations associated with fluorescence and bioluminescence imaging. This includes addressing issues related to photon penetration, autofluorescence, and spatial resolution.
High Sensitivity and Spatial Resolution: The new technique aims to provide high sensitivity and spatial resolution up to the molecular scale, facilitating detailed in vitro and whole-body imaging. This capability could transform how diseases are diagnosed and monitored, allowing for more precise and targeted interventions.
Sustained Light Emission for Long-term Investigation: The project aspires to enable persistent light emission for prolonged periods, making it suitable for long-term studies and continuous monitoring of biological processes.
Replacing Harmful Radiations: The novel approach holds the potential to replace existing imaging procedures that rely on harmful ionizing radiations such as X-rays and γ-rays, thereby reducing the associated health risks.
The approach involves leveraging recent advancements in chemiluminescence, electroluminescence, and electrochemiluminescence. The innovative concept of regenerative chemiluminescence is central to this project, where the luminophore undergoes repeated cycles of reduction and oxidation, driven by endogenous cellular metabolites like NADH and reactive oxygen species (ROS). This process not only sustains light emission but also avoids the need for an electrochemical cell or exogenous substrates.
A significant challenge is ensuring the reactivity of synthesized molecules towards biological oxidants and reductants while maintaining their stability and light-emitting properties. The project involves extensive molecular redesign and experimentation to overcome kinetic barriers and enhance the efficiency of these reactions.
In conclusion, BioPoweredCL aims to revolutionize biomedical imaging by introducing a cost-effective, high-resolution, and safe imaging technique. The successful realization of this project could lead to groundbreaking advancements in medical diagnostics and beyond, significantly impacting both healthcare and various scientific fields.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

Our research initially focused on studying the photophysical properties of luminophores, which are materials that emit light. Normally, when excited states are generated through charge recombination reactions—without using light—both singlet (25%) and triplet (75%) excited states are produced. For optimal efficiency, it's crucial that triplet excited states contribute to the light emission, but traditional organic fluorescent compounds can't achieve this. Given the limitations of phosphorescent transition complexes, we turned our attention to a newer generation of organic compounds capable of efficiently capturing triplet excited states through a process called thermally activated delayed fluorescence (TADF).
TADF molecules are special light-emitting compounds made up of donor and acceptor groups. The donor group affects the molecule's ability to oxidize, while the acceptor group affects its ability to reduce. In these molecules, the donor and acceptor groups are arranged in such a way that they create a very small energy gap between their highest and lowest energy states. This small energy gap is close to the amount of thermal energy available at room temperature.
Because of this small energy gap, the molecule's singlet (bright) and triplet (dim) excited states can easily switch between each other. This means that the triplet states, which usually don’t emit light in regular fluorescent compounds, can still contribute to the overall light emission in TADF molecules by transferring their energy to the singlet states.
The color of the emitted light and the amount of energy needed to produce it depend on the energy gap between these states. To explore this further, our first step was to create a variety of TADF compounds by changing the donor and acceptor groups. We then tested these new compounds to see how they performed in terms of their electrochemical and light-emitting properties, as well as their efficiency in electrochemiluminescence (ECL).
Additionally, we aimed to make these TADF compounds water-soluble and capable of being attached to biological molecules, which are important features for bioanalytical applications.
Our efforts led to several publications and new collaborations, focusing on the use of these compounds in light electrochemical cells (LECs). Although we observed a significant reduction in brightness when moving from nonpolar to polar organic solvents, the TADF characteristics remained intact. Since TADF molecules are also extensively researched in photocatalysis, our findings open new possibilities for using TADF in water-based photocatalysis.
These results have been published in scientific journals and presented at major international conferences such as EuCheMS and PhotoIUPAC. However, progressing to the next phase of our project—replacing electrodes with chemical reactions—proved more challenging than we anticipated. The synthesized molecules showed zero reactivity towards chemical oxidants and reductants, not because of thermodynamic issues, but due to kinetic factors. This prompted us to undertake a complete redesign of the molecules.
We explored various classes of fluorescent organic compounds and coordination metal complexes, known for their strong chemical reactivity in reduction and oxidation processes. We developed organic compounds that can be oxidized in air and emit blue light when reduced, as well as compounds that can be reduced with ascorbic acid and emit red light when oxidized. In all cases, the luminophore remains intact, allowing the cycle to be repeated. We believe we have demonstrated the proof of concept, but further work is needed to make these compounds suitable for biological systems and to improve their light generation efficiency.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

Some compounds that usually show weak or no photoluminescence can surprisingly become highly photoluminescent when they self-assemble into larger structures. This intriguing phenomenon is called aggregation-induced emission (AIE). In our research, we found something unexpected: by altering the redox chemistry of these compounds—meaning we first oxidize them and then reduce them back—we can create luminescent supramolecular gels. These gels cannot be made without going through this specific redox reaction.
This finding is significant because it shows that the outcome of the supramolecular polymerization process, where small molecules join together to form larger, complex structures, can be influenced by the redox reactions involved. Interestingly, when the reduction part of the reaction releases a lot of energy (is highly exergonic), the self-assembly process emits light.
Moreover, we discovered that we can not only use reducing agents to form these supramolecular structures on demand, but also achieve the reduction through UV light irradiation, allowing for highly controlled formation in terms of both space and time. Additionally, these supramolecular structures exhibit new functions that their monomer counterparts do not possess, such as the ability to absorb light in the green region, making them more effective antennas. Selective irradiation of the structures in the presence of the oxidized monomer leads to the targeted elongation of the structures, a result that was not predicted beforehand.
Our discovery marks an important step forward in the field of supramolecular chemistry, which studies how molecules come together to form complex structures. It also has implications for creating life-like materials, which mimic natural processes and structures. This new strategy for engineering supramolecular structures could open up exciting possibilities for developing advanced materials with tailored properties, a highly desirable goal in current scientific research.