# LogicLab Symposium 'Molecular Biosensing: from Theory to Practice'

Europe/Berlin
Online
Registration
Registration
• Wednesday, 17 November
• 14:00 17:45
Day 1: 1st day
• 14:00
Symposium Opening 10m
• 14:10
Fluorescent Sensors and Logic Systems 25m
Speaker: Prof. Amilra Prasanna de Silva (Professor of Organic Chemistry at Queens University, Belfast, United Kingdom)
• 14:35
Speaker: Prof. Amilra Prasanna de Silva (Professor of Organic Chemistry at Queens University, Belfast, United Kingdom)
• 14:55
A NIR excitable fluorescent probe for a versatile intracellular nitric oxide detection 10m

Understanding intracellular dynamics and concentrations of diverse intracellular analytes is key to develope novel diagnostic tools. Fluorescence-based molecular probes have proben to be key for intracellular imaging and sensing analytes of biological interest. Most of the currently available fluorescent probes are excited using ultraviolet or visible light, which results in high photodamage to living cells. Alternatively, near-infrared (NIR) light provides high photostability, low autofluorescence, high biological tissue penetration and minimal photodamage. In the development of diagnostic tools for cancer, one of the most relevant reactive nitrogen species is nitric oxide (NO) since its effect is strongly related to its concentration. Fluorescent NIR excitable molecular probes seem to be ideal candidates for the intracellular detection of NO and could ultimately yield to valuable insights on the biological role played by NO.
The aim of this work is to develop a NIR excitable molecular probe for the intracellular detection of NO via a photoinduced electron transfer (PET) mechanism. The probe showed good sensitivity (LOD = 78 nM) and selectivity towards NO. Additionally, the fluorescence intensity of the probe was stable in a range of pHs from 4 to 9; and the detection of NO in acidic environments was succesfully evidenced. The NO probe was able to detect NO in a variety of macrophages including RAW264.7 cells, by means of confocal microscopy and multiphoton microscopy (NIR excitation). The NO probe is currently being used to detect intracellular NO in other cellular environments including endothelial cells.

References:
[1] S. K. Choudhari et al., World J. Surg. Oncol.,2013 11, 118,https://doi.org/10.1186/1477-7819-11-118.
[2] H. Yu et al., J. Am. Chem. Soc.,2012, 134, 42, https://doi.org/10.1021/ja308967u.

Speaker: Carla Arnau del Valle (University of East Anglia)
• 15:05
Synthesis and cytotoxicity evaluation of a ‘V-shaped’ fluorescent 4-Amino-1,8- naphthalimide Tröger’s base derived Ru(II)-curcumin organometallic conjugate 10m

The design, synthesis and application of luminescent metal complexes as efficient theragnostic
agents have great significance in the field of medicinal chemistry [1]. Luminescent d‐metal ion
complexes are well-known for their therapeutic activities because of their unique
photochemical and photophysical characteristics [2]. 4-amino‐1,8‐naphthalimide derived
Tröger’s base (TBNap) derivatives are novel organic scaffolds that are famous for their robust
DNA binding affinity, quick cellular uptake, and can also act as apoptosis inducers in cancer
cells [3-5]. We have developed a novel luminescent N-4-pyridyl-4-amino-1,8-naphthalimide
Tröger’s base (TB Nap) with unique chiral cleft shape geometry using N-4-pyridyl-4-amino1,8-naphthalimide as the precursor through facile synthetic strategy. Knowing the significance
of ruthenium metal complexes and the naturally available therapeutic agent "curcumin"
and TBNap in the field of cancer therapy, we designed a novel TBNap-containing Ru(II)
curcumin organometallic conjugate (TB-Ru-Cur) by the self-assembly of TBNap with
previously reported arene-Ru(II)-curcuminato complex, Ru-Cur. TB-Ru-Cur displayed a fast
cellular uptake, highly luminescent characteristics, and cytotoxicity against various cancer cell
lines such as HeLa cells, HCT-116, and HepG2 cancer cells with an efficiency much higher
than clinically used cisplatin. In summary, the work herein demonstrates that the TB‐Ru‐
Cur
can act as a potent anticancer theragnostic agent thereby bridging the gap between
therapeutic and diagnosis properties.

Speaker: Binduja Mohan (Department of Chemistry, Indian Institute of Technology Palakkad (IITPKD), Palakkad)
• 15:15
Break 20m
• 15:35
Chemical Tools for Biosensing with Subcellular Resolution 25m
Speaker: Dr Pablo Rivera Fuentes (Assistant Professor of Chemical Biology, EPFL, Switzerland)
• 16:00
Speaker: Dr Pablo Rivera Fuentes (Assistant Professor of Chemical Biology, EPFL, Switzerland)
• 16:10
pH-driven lateral organization of biomimetic cell membranes 10m

Many studies have been devoted to investigation of phase separation and formation of lipid domains, which play crucial role in many biological processes. In the following study we measured the size and distribution of liquid-ordered (Lo)¬ phase domains in the wide range of buffer pH 1.7-9.0 and showed that there is a strong correlation between the size of the formed Lo phase domains and pH of the buffer hydrating the lipid bilayer. Although the increase of the buffer pH led to the formation of bigger and more round domains, the size of the vesicles that are building blocks for the assembly of the lipid membrane did not change, and the formation of different size of domains was confirmed to occur at the stage of membrane rearrangement on the solid support. We present that the use of different pH did not have an influence on the dynamics of lipids composing both ordered and disordered phase, presenting a constant diffusion coefficient over the whole tested pH range. These findings demonstrate that by using more acidic or basic pH during the formation of lipid membranes we can influence the size and shape of the formed domains without any external modification of the solid support or altering the membrane composition. Finally, we show that once formed the architecture of the lipid membrane is stable even upon replacement of the aqueous medium to the buffer of neutral pH, which makes this method of domains formation applicable in the studies involving binding of proteins or incorporation of other pH-sensitive molecules.

Speaker: Ms Emilia Krok (Poznan University of Technology, Institute of Physics)
• 16:20
Lateral diffusion of lipids senses local hydration in biomembranes 10m

From various electrical and mechanical studies, supported lipid bilayers (SLBs) have been realized to possess brilliant implementation potential in terms of technological application as biosensors and biocoatings1. Structure and dynamics of SLBs are modulated not only by lipid-lipid, lipid-protein interactions, but the interplay between lipids and the water hydrating the membrane plays a crucial role altering the lateral diffusion of lipids in biomembrane2. Using fluorescence microscopy imaging and Fluorescence Recovery After Photobleaching (FRAP) techniques, we have studied phase separated SLBs at varying local hydration states by changing relative humidity of the environment. Our study shows that the lateral mobility of liquid disordered phase lipids varies accordingly with change in local hydration state during dehydration as well as rehydration3. A six-fold decrease in diffusion coefficient values have been observed from fully hydrated membrane to 0% relative humidity condition. Moreover, the change of diffusion coefficient of lipids with hydration state is absolutely reversible and repeatable, i.e. the mobility change occurs in multiple dehydration and rehydration cycles. This significant dependence and high sensitivity down to a few water molecules per lipid offers a huge potential for SLBs to be utilized as hydration sensor on a molecular level and can be used to investigate local hydration heterogeneity in biomimetic systems4.
References:
(1) Holden, M. A.; Jung, S. Y.; Yang, T.; Castellana, E. T.; Cremer, P. S. Creating Fluid and Air-Stable Solid Supported Lipid Bilayers. J. Am. Chem. Soc. 2004, 126, 6512–6513, doi:10.1021/ja048504a
(2) Pasenkiewicz-Gierula, M.; Baczynski, K.; Markiewicz, M.; Murzyn, K. Biochimica et Biophysica Acta - Biomembranes. Elsevier B.V. October 1, 2016, pp 2305–2321, doi: 10.1016/j.bbamem.2016.01.024
(3) Chattopadhyay, M.; Krok, E.; Orlikowska, H.; Schwille, P.; Franquelim, H. G.; Piatkowski, L. J. Am. Chem. Soc. 2021, 143, 14551–14562,
doi: 10.1021/jacs.1c04314
(4) Chattopadhyay, M.; Orlikowska, H.; Krok, E.; Piatkowski, L. Biosens. 2021, Vol. 11, 11, 241,
doi: 10.3390/bios11070241

• 16:30
Long-term quantification of membrane biophysical properties using an exchangeable polarity-sensitive sensor 10m

Polarity-sensitive fluorescent sensors constitute useful tools to quantify key biophysical properties of biomembranes, such as lipid order. However, prolonged acquisitions using these dyes are universally limited by dye photobleaching, i.e. the irreversible destruction of the sensor by the excitation light. This problem is aggravated in the case of super-resolution microscopy, due to the high laser powers used. Exchangeable membrane dyes have been recently proposed as a remedy to photobleaching, avoiding it by only temporarily binding to their target. Here, we show NR4A, a new exchangeable membrane dye, reports on the molecular order and dynamics of lipid membranes with no photobleaching-induced signal loss. We use super-resolution fluorescence correlation spectroscopy (STED-FCS) to simultaneously quantify membrane dynamics and lipid packing, which correlate in model and live cell membranes. Last, to showcase potential applications of polarity-sensitive exchangeable dyes, we use live 3D-STED imaging to monitor lipid exchange during membrane fusion (Figure 1).

Speaker: Pablo Carravilla (Leibniz Institute of Photonic Technology)
• 16:40
Break 20m
• 17:00
Nanoscale biophysical properties of plasma membrane and how to measure them 25m
Speaker: Dr Erdinc Sezgin (Assistant Professor in Biophysyics at Karolinska Institutet, Sweden)
• 17:25
Speaker: Dr Erdinc Sezgin (Assistant Professor in Biophysyics at Karolinska Institutet, Sweden)
• 17:35
Closing remarks 10m
• Thursday, 18 November
• 14:00 17:45
Day 2: 2nd day
• 14:00
Introduction 10m
• 14:10
In Silico Photochemical Experiments with Non-Born-Oppenheimer Molecular Dynamics 25m
Speaker: Dr Basile F. E. Curchod (Assistant Professor in Theoretical Chemistry at Durham University, United Kingdom)
• 14:35
Speaker: Dr Basile F. E. Curchod (Assistant Professor in Theoretical Chemistry at Durham University, United Kingdom)
• 14:45
Computational investigation of Methacrylic acid as chemical agent for radiation detection by polymerization 10m

Ion-beam cancer therapy is used for the treatment of cancer. The initial ions in the beam are entering the biological material with energies of a few million eV and are generating secondary electrons by ionization processes. The secondary electrons can cause more ionization or initiate excitation processes, which can cause break some chemical bonds and cause the death of the affected cells. For the adjustment of the dose in ion-beam cancer therapy, it is crucial to have knowledge about these processes. In real biological system this is difficult to achieve, because of the diffusion processes involved.
A methodology, which avoids these problems is the technique of radiation detection by polymerization. In this technique a gel is used, that contains chemical agents, which can polymerize after electronic excitation or ionization. After the irradiation of a probe with the ion-beam, an MRI scanner can be used to locate the distribution of the polymers, which gives an image about the dose distribution. For this purpose the chemical agents in the gel should designed in such a way, that they polymerize at similar energies, at which chemical bonds in biological material (e. g. DNA and amino acids) are broken.
One of the most popular gels for radiation detection by polymerization is MAGIC (methacrylic and ascorbic acid in gelatin initiated by copper) [1]. In our project we want to study the effects energetic electrons on methacrylic acid by means of Monte Carlo simulations [2]. As input parameters the Monte Carlo simulations require the knowledge of cross section data, which describes the various interactions [2]. We will present preliminary results for the cross section for vibrational excitation and ionization, which we have computed with the help of standard quantum chemistry program packages, as described in e. g. [3 - 5].

Bibliography:
[1] M.P. Fong, C. K. Derek, D.D. Mark, J.C. Gore, Polymer gels for magnetic resonance imaging of radiation dose distributions at normal room atmosphere. Phys. Med. Biol. (2001), 46, 3105. DOI: 10.1088/0031-9155/46/12/303
[2] S. Taioli, P. E. Trevisanutto, P. de Vera, S. Simonucci, I. Abril, R. Garcia-Molina, M. Dapor, Relative Role of Physical Mechanisms on Complex Biodamage Induced by Carbon Irradiation. J. Phys. Chem. Lett. (2021), 12, 487−493. DOI: 10.1021/acs.jpclett.0c03250
[3] J. Franz, F. A. Gianturco, I. Baccarelli, Low-energy positron scattering from gas-phase uracil, Eur. Phys. J. D (2014) 68: 183, DOI: 10.1140/epjd/e2014-40796-0
[4] N. F. Lane, The theory of electron-molecule collisions. Rev. Mod. Phys. (1980), 52, 29. DOI: 10.1103/RevModPhys.52.29
[5] W. Hwang, Y.-K. Kim, M. E. Rudd, New model for electron-impact ionization cross sections of molecules, J. Chem. Phys. (1996), 104, 2956 – 2966. DOI: 10.1063/1.471116

Speaker: Mrs Katarzyna Wiciak-Pawłowska
• 14:55
Radiative lifetime of a BODIPY dye as calculated by TDDFT and EOM-CCSD methods: Solvent and vibronic effects 10m

Speaker: Rengel Cane Sia (Gdańsk University of Technology)
• 15:05
Red to Blue Upconversion Nitric Oxide Sensing and the Localization of Sensors in Liposomes 10m

Over the past decades, endothelial dysfunction and atherosclerosis have become a significant issue across the world. Nitric oxide (NO), along with calcium, is a primary physiological mediator for detecting endothelial dysfunction as NO plays a crucial role in the proper functioning of endothelial cells. On the other hand, triplet-triplet annihilation upconversion (TTA-UC) has become a powerful tool with promising applications like photocatalysis, solar energy conversion, drug delivery, bio-imaging, and photoactivated chemotherapy. Because of its low energy activation, for example red light, it causes less cell damage and deeper light penetration. Furthermore, in a cellular setting, the anti-stokes wavelength shift during upconversion allows it to be distinguished from the autofluorescence from cells.
Here we will be discussing how we have developed a TTA-UC nitric oxide sensor and the challenges involved in making future TTA-UC sensors. Simulation of the membrane using molecular dynamics calculations to describe the structure of the system is very expensive. Using the novel COSMOplex software, many simulation applications of self-organizing systems are possible at a significantly lower computational cost. This is made possible by using ensembles of thermodynamic states, compared to explicitly modeling the whole system using molecular dynamics. We used the COSMO suite of software to exploit this significantly lower computation costs.
In this communication, we will be discussing the development of our new upconversion nitric oxide sensor capable of sensing nitric oxide in liposome formulation and modeling a simplistic lipid membrane to calculate the relative positions of the different modifications of the sensor in the membrane.

Speaker: Aswin Reena Chandrababu
• 15:15
Break 20m
• 15:35
Biotechnologically reconstructed human tissue models addressing requirements of regulators in toxicity testing of chemicals, cosmetics, ingredients, and formulations. 15m
Speaker: Dr Silvia Letasiova (Senior scientist and manager director at MatTek Slovakia)
• 15:50
Speaker: Dr Silvia Letasiova (Senior scientist and manager director at MatTek Slovakia)
• 16:00
Investigating the effect of novel NO-releasing ruthenium-terpyridine compound's cytotoxicity and redox profile in human fibroblasts 10m

Nitric oxide (NO) is a crucial molecule for the function of the vascular system. NO releasing compounds are widely used for researching the effects of NO as well as therapies of cardiovascular and skin disorders. The amount of NO released is the conclusive element for wanted effects and because of this, Ruthenium-terpyridine is a very important compound as it is possible to change concentration of NO release with L2 ligand site. The derivative HE-10, a ruthenium (II) nitrosyl-complex incorporating 4′-phenyl-terpyridine and o-benzoquinone diimine (Ru(ptp)(o-bqdi)NO3), is a newly synthesized photoinducible NO releaser. We aimed to investigate the cytotoxic and prooxidant effects likely related to NO release induced from HE-10 with white LED light stimulation in the VH10 fibroblasts.

Our findings indicate that cytotoxicity of HE-10 was increased by exposure to white LED light, as confirmed by lower IC50 value obtained from the HE-treated light-exposed cells compared to the unexposed ones. Cytotoxicity followed by increased intracellular ROS/RNS production, G2/M cell cycle arrest and protein nitration, depending on both, light exposure and concentration. Moreover, light-exposure- enhanced toxic effect of HE-10 on VH10 cells was most likely to be NO-dependent as confirmed by the induced NO2- release in the medium, intracellular oxidation of H2DCF-DA probe sensitive to ROS and RNS in fibroblasts, and increased protein nitration levels. Cell cycle analysis showed that HE-10 decreased cell proliferation and antiproliferative effect was enhanced with light exposure. Our findings indicate that HE-10 is capable of selective cytotoxicity induced by light exposure through releasing NO.

Speaker: Hande Özbaşak
• 16:10
Photoaffinity Labeling of proteins by Photoreactive Probes in Live cells and Tissues 10m

Histone deacetylases (HDACs) constitute an epigenetic enzyme family that is implicated in cancer and a variety of other immunological, neurological, cardiac, and endocrine diseases. Development of novel HDAC isoform-selective inhibitors is necessary to improve their in vivo potency while reducing toxicity. The isoform selectivity of HDAC inhibitors is routinely measured in biochemical recombinant enzyme inhibition assays using synthetic substrates, which does not account for regulation of HDAC activity though post-translational modifications and multiprotien complex formation.
Photoaffinity labeling is a valuable tool to study ligand-target interactions within complex proteomes. A set of chemically diverse photoreactive probes (PRPs) were synthesized. The PRPs were tested for in vitro HDAC inhibitory activity in comparison to their parent compounds. Their ability to label HDACs within a complex proteome was studied in cell-based and tissue-based models.
The results showed that a subset of the PRPs retained HDAC isoform selectivity with respect to their parent compounds as demonstrated by their inhibitory profile against recombinant enzymes and labeling of HDAC isoforms in live cells. Other PRPs, however, while maintaining HDAC inhibition of recombinant enzymes, differed in selectivity with respect to their photolabeling of HDAC isoforms in live cells. This highlights the need for a cell-based approach during the early development stages of novel HDAC isoform-selective inhibitors. In addition, the physicochemical properties of four of the PRPs indicated they have drug-like character and could be used for in vivo studies of HDAC target engagement.

Speaker: Shaimaa Aboukhatwa
• 16:20
Profiling Nitric Oxide metabolites in endothelial dysfunction using 3D blood vessel model 10m

Endothelial dysfunction is the prime cause of many pathological conditions such as atherosclerosis, thrombosis, platelet aggregation, inflammation, and defective oxygen mechanism due to hypoxic conditions. The natural mechanism in maintaining vascular endothelium involves various signaling compounds and mechanistic factors such as fluid flow and shear stress. Among all signaling compounds, Nitric Oxide (NO) is considered an essential one in maintaining vascular homeostasis. Hence, analysis of the NO level is necessary for understanding pathological conditions. Evaluating the level of NO to predict the risk of endothelial rupture is crucial for patient management, yet current two-dimensional endothelial cell culture models and methods suffer from several limitations due to NO’s short half-life and lack of fluid flow in the model. This results in less NO expression and displays a non-physiologic phenotype.
This study focuses to compare two-dimensional (2D) and three-dimensional (3D) platforms in terms of NO-specific metabolites level. To measure NO metabolites using UPHPLC/MS in a three-dimensional model, we developed a tracer-based metabolomics strategy in the three-dimensional micro vessels-on-a-chip model with a microfluidic pump, that maintains a unidirectional fluid flow. And we investigated the specific marker isotope metabolites, tracking through the NO substrate L-Arginine in the NO mechanism. We detected significant changes in L-citrulline and L-ornithine levels in stimulation and inhibition of the eNOS (endothelial nitric oxide synthase) enzyme. Compare to the 2D culture, the augmented effects of NO specific metabolite, L-citrulline was determined in 3D blood vessels. We also studied the impact of oxygen in endothelial dysfunction condition over NO metabolism with an in-line oxygen measurement system. This model signifies a more similar physiological environment that displays the difference between 2D and 3D cultures.

Speaker: Kanchana Pandian (ESR_11, PhD)
• 16:30
miRNA biomarker detection in the fight against Cardiovascular Disease 10m

miRNAs are small, 22 nucleotide long RNA sequences. They are found in every cell type and perform the role of regulating gene expression. Research in the field has also identified free miRNA in a variety of bodily fluid. Their availability and their role in gene regulation make them attractive targets as biomarkers for the advanced detection of many diseases such as CVD, one of the leading causes of death globally. Changes in gene expression is the earliest marker for cells entering a disease state and as such miRNA can offer earlier markers than traditional protein biomarkers. This work describes the detection of miRNA related to cardiovascular disease using a sandwich assay design. The use of gold nanoparticle functionalized carbon ink screen-printed electrodes allows the electrode surface to be readily functionalised with capture strands of RNA, and simultaneously provides a low background substrate for the assay. The sandwich assay design uses platinum nanoparticles to catalyse the degradation of hydrogen peroxide. The current generated in this reaction is then quantified using chronoamperometric measurement which correlates with the amount of target miRNA concentration. Micromolar levels of miRNA can be detected using the assay design. The platform also offers single use functionality and rapid testing times (2 minutes total) with the aim of creating an ultrasensitive miRNA quantitative assay. These are both significant advances to gold standard qPCR assays for nucleic acid detection of disease biomarkers.

Speaker: Fionn O Maolmhuaidh (DCU)
• 16:40
Break 20m
• 17:00
Probing liquid and biomimetic interfaces with surface second-harmonic generation 25m
Speaker: Prof. Eric Vauthey (Professor of Physical Chemistry at University of Geneva)
• 17:25
Speaker: Prof. Eric Vauthey (Professor of Physical Chemistry at University of Geneva)
• 17:35
Closing remarks 10m
• Friday, 19 November
• 14:00 17:45
Day 3: 3rd day
• 14:00
Introduction 10m
• 14:10
Visualizing dynamic processes with rapidFLIMHiRes, the ultra fast FLIM imaging method with outstanding 10ps time resolution 20m
Speaker: Dr Fabian Jolmes (Team leader, Application Specialist Microscopy at PicoQuant)
• 14:30
Monitoring excited-state relaxation in a molecular marker in live cells–a case study on astaxanthin 10m

Small molecules are frequently used as dyes, labels and markers to visualize and probe biophysical processes withing cells. Though the optical properties and excited-state relaxation pathways of these molecules have been studied intensively, [1, 2] very little is generally known about the light-driven excited-state reactivity of such systems when placed in live cells. In this work, we introduce an experimental approach to study ps time-resolved excited state dynamics of a benchmark molecular marker, AXT (astaxanthin), in live human cancer cells. AXT stains lipids in cells rendering lipid-containing structures easily detectable by resonance Raman microscopy. Based on shifts in the C=C stretching band of the Raman spectrum [3] it was suggested that AXT undergoes confirmational changes in the cellular organelles as compared to isolated AXT in solution. In contrast, the photoinduced excited-state dynamics in AXT recorded in cellulo
appears to be unchanged compared to the dynamics detected in solution. That indicates that the absence of AXT aggregation in the cells, and the structural distortion of the AXT molecules do not alter the excited-state properties. [4]
In a methodological context we show that our approach to in cellulo transient absorption spectroscopy offers a valuable path to study the impact of the local environment on the photoinduced dynamics in stains, markers and beyond this in light-activated drugs, e.g. for photodynamic therapy, [5] and interactions between phototherapy agents and live cells.

[1] Fuciman, M., et al., Chemical Physics Letters, 2013, 568, 21-25. https://doi.org/10.1016/j.cplett.2013.03.009
[2] Kaczor, A. and Baranska, M. Anal Chem, 2011, 20,7763-7770. https://doi.org/10.1021/ac201302f
[3] Czamara, K., et al., Cell Mol Life Sci, 2021, 7, 3477 - 3484. https://doi.org/10.1007/s00018-020-03718-1
[4] Yang, T., et al., Chem Commun, 2021, 57, 6392-6395. https://doi.org/10.1039/D1CC01907D
[5] Monro, S., et al., Chemical Reviews, 2018, 119, 797-828. https://doi.org/10.1021/acs.chemrev.8b00211

Speaker: Tingxiang Yang
• 14:40
Synthesis of Ruthenium-based nitric oxide releasers towards visible light activation by triplet-triplet annihilation upconversion 10m

Nitric oxide (NO) plays an important role in many biological signalling processes including blood pressure regulation, neurotransmission, inflammatory response, and programmed cell death (apoptosis).[1,2] Although the NO donors have been useful research tools, many of them release NO via spontaneous decomposition, so that it is difficult to control NO release.[3] To overcome this problem, NO donors that are triggered by light (photo-NORMs) have prompted significant synthetic research efforts towards their use as photopharmaceuticals owing to their exceptional control over the location, timing, and dosage.[4] One issue for these photo-NORMs is their activation in the UV part of the spectrum which is harmful and has poor penetration to living tissues. In order to activate this category of molecules using a safer light source, we have developed ruthenium nitrosyl complexes that can be directly activated by 505 nm LED. Then, by rational design of the ligand sphere, the ruthenium nitrosyl complexes are developed towards their integration in a biological environment. Molecular as well as environmental parameters are studied towards NO release based on triplet-triplet annihilation upconversion (TTA-UC). Using TTA-UC the activation of photo-NO release with red-NIR light becomes available, with greater penetration depths and without detrimental effects to the tissue.

References
[1] Fry, N. L., Heilman, B. J. & Mascharak, P. K. Inorganic Chemistry, 2011, 50, 317–324.
[2] Yang, Liu, Shuqi Wu, Bijuan Lin, Tianxun Huang, Xiaoping Chen, Xiaomei Yan, and Shoufa Han, Journal of Materials Chemistry B, 2013, 1, 6115-6122
[3] Hishikawa, Kazuhiro, Hidehiko Nakagawa, Toshiaki Furuta, Kiyoshi Fukuhara, Hiroki Tsumoto, Takayoshi Suzuki, and Naoki Miyata, Journal of the American Chemical Society, 2009, 131, 7488-7489.
[4] Becker, Tobias, Stephan Kupfer, Martin Wolfram, Helmar Görls, Ulrich S. Schubert, Eric V. Anslyn, Benjamin Dietzek, Stefanie Gräfe, and Alexander Schiller, Chemistry - A European Journal, 2015, 21, 15554-15563.

Acknowledgements. This work has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 813920.

Speaker: Mr Hani M. Elbeheiry
• 14:50
DNA-nanoantenna ECL assay development for bacteria detection 10m

Early detection of bacteria forming biofilms can have a significant impact in a variety of fields such as healthcare, food industry and environment protection. Development of highly sensitive, specific, and cost-effective biosensors for bacteria detection is necessary for the prevention of infections, especially due to increase in the number of new multi-drug resistant bacteria. Electrochemiluminescence (ECL) based biosensors (i.e. electrochemically generated light) has attracted much attention due to their high selectivity, controllability, and sensitivity. In this case, novel transition metal complexes based on ruthenium are of high interest since they provide advantages such as strong luminescence, good solubility in several aqueous and non-aqueous solvents, and good ECL efficiency, that makes them excellent luminophores. Furthermore, ECL signal generated by novel ruthenium dyes can be effectively enhanced when is coupled with surface plasmon resonance (SPR). The use of plasmonic nanoparticles as gold NPs can be used to amplify the ECL signal mediated by ruthenium complexes. Nevertheless, several factors (e.g. particle size, shape, distance and etc.,) play a key role in the SPR-ECL process and they can be optimized. Therefore, the objective is to find out the optimal distance between luminophore and particle using DNA as spacer. Characterization of the DNA-nanoantenna ECL system through spectroscopic (UV-VIS and fluorimetry) and electrochemical techniques such as cyclic voltammetry will provide the information needed to build reliable, highly sensitive, selective, and specific optical DNA-based biosensor for and early detection of bacterial infections.

Speaker: Miren Ruiz de Eguilaz (Dublin City University)
• 15:00
Watching life at molecular level through advanced chemical microscopy 25m
Speaker: Prof. Ji-Xin Cheng (Moustakas Chair Professor in Photonics and Optoelectronics, Professor of Biomedical Engineering, Electrical & Computer Engineering, Chemistry, and Physics at Boston University, USA)
• 15:25
Speaker: Prof. Ji-Xin Cheng (Moustakas Chair Professor in Photonics and Optoelectronics, Professor of Biomedical Engineering, Electrical & Computer Engineering, Chemistry, and Physics at Boston University, USA)
• 15:35
Break 25m
• 16:00
Creating upconverting liposomes sensitive to $Ca^{2+}$ 10m

Endothelial dysfunction (ED) is a strong predictor of atherosclerosis. To prevent disease development new method of ED detection should be created. Diagnostics of the dysfunction in one sample requires two parameters sensitive system (Logic Gate). For ED nitric oxide (NO) and calcium cations were chosen as interdependent parameters for ED detection. In this study, we are focusing on $Ca^{2+}$ sensing for in vivo applications.
In vivo sensing is characterized by tough challenges such as the presence of competitive analytes, low signal-to-noise ratio, low light penetration depth, toxicity, among others. Some of these issues may be overcome by combining triplet-triplet annihilation upconversion (TTA-UC) and photo-induced electron transfer (PET) quenching.
Here we present the synthesis and proof-of-concept tests of upconverting liposomes sensitive to $Ca^{2+}$. Liposomes containing PdTPTBPP as photosensitizer and an annihilator with calcium ions-chelating properties showed an increase of upconverted emission in presence of $Ca^{2+}$.

Speaker: Valeriia Andreeva
• 16:10
EdU-labelling: Raman-based click-free detection of endothelial cell proliferation 10m

Endothelial dysfunction (ED) has been linked to the development of many lifestyle diseases such as atherosclerosis, diabetes or hypertension 1 and the phenotype of endothelial dysfunction is often linked to altered capacity of endothelial regeneration unable to repair vascular dysfunction. Therefore, new methods to study endothelial cell proliferation are desirable.
Raman imaging of endothelial cells provides valuable information on the chemical changes associated with disease development 2. Although Raman spectroscopy allows label-free detection of key biological compounds i.e. proteins, lipids and nucleic acids, the use of Raman reporters has been shown to improve sensitivity and selectivity of subcellular organelles imaging and tracking specific molecules 3. One example of this is the detection of alkyne-tagged 5-ethynyl-2′-deoxyuridine (EdU).
EdU is a thymidine analogue that incorporates into newly synthesized DNA during replication. EdU subsequent detection, after its cupper-catalysed cycloaddition reaction “Click Chemistry” with a fluorescent azide, has been used in fluorescence imaging to follow DNA synthesis of proliferating cells 4,5. Due to the alkyne tag, EdU could be easily detected in the Raman spectra of cells as it gives a Raman band at ca. 2122 cm-1 in the “silent region” where there are no interferences in the signal from other biological compounds 6.
This study aims to assess the Raman imaging of endothelial cells (ECs) DNA by following the characteristic alkyne band at ca. 2122 cm-1 in EdU-labelled ECs and to investigate the changes in ECs proliferation using EdU as an indicator of DNA replication in in vitro and ex vivo conditions. We studied the effects of cycloheximide (CHX), a protein synthesis inhibitor that inhibits DNA replication, 7 on EdU-tagged ECs. EdU-labelling has been shown to improve Raman imaging of the nuclei of ECs from different origins. Furthermore, CHX pre-treated cells showed a significantly lower EdU signal from their nuclei. It was clear that cell proliferation was inhibited after CHX treatment and this effect could be detected using Raman EdU-labelling approach, which was not detectable using label-free Raman imaging. When fluorescent detection of Alexa Fluor® 488-tagged EdU was used as a reference method, the intensity of the signal from Alexa Fluor as well as the percentage of EdU positive cells were decreased in CHX pre-treated cells compared to the control. The Raman imaging results are in agreement with the fluorescence method, moreover, the Raman-based approach omits cell permeabilization step and allows EdU detection in live cells and without the need of additional dyes.
In conclusion, the results of this study show the feasibility of click-free detection of EdU-labelled DNA in endothelial cells using Raman spectroscopy, and this method is being further used and optimized to detect endothelial proliferation in the isolated vessel ex vivo.

Acknowledgements: The authors are grateful to MSc Renata Budzynska (JCET) for cell culturing.
This study was supported through LogicLab ITN, funded by the European Union’s Horizon 2020 Research and Innovation Programme under Marie Sklodowska-Curie Grant Agreement 813920.

References
[1] P. Rajendran, T. Rengarajan, J. Thangavel, Y. Nishigaki, D. Sakthisekaran, G. Sethi, I. Nishigaki, The vascular endothelium and human diseases, Int. J. Biol. Sci. 9 (2013) 1057–1069. https://doi.org/10.7150/ijbs.7502.
[2] M. Baranska, A. Kaczor, K. Malek, A. Jaworska, K. Majzner, E. Staniszewska-Slezak, M.Z. Pacia, G. Zajac, J. Dybas, E. Wiercigroch, Raman microscopy as a novel tool to detect endothelial dysfunction, Pharmacol. Reports. 67 (2015) 736–743. https://doi.org/http://dx.doi.org/10.1016/j.pharep.2015.03.015.
[3] A. Adamczyk, E. Matuszyk, B. Radwan, S. Rocchetti, S. Chlopicki, M. Baranska, Toward Raman Subcellular Imaging of Endothelial Dysfunction, J. Med. Chem. 64 (2021) 4396–4409. https://doi.org/10.1021/acs.jmedchem.1c00051.
[4] T. Ishizuka, H.S. Liu, K. Ito, Y. Xu, Fluorescence imaging of chromosomal DNA using click chemistry, Sci. Rep. 6 (2016) 33217. https://doi.org/10.1038/srep33217.
[5] D.P. Basile, J.L. Friedrich, J. Spahic, N. Knipe, H. Mang, E.C. Leonard, S. Changizi-Ashtiyani, R.L. Bacallao, B.A. Molitoris, T.A. Sutton, Impaired endothelial proliferation and mesenchymal transition contribute to vascular rarefaction following acute kidney injury, Am. J. Physiol. Physiol. 300 (2011) F721–F733. https://doi.org/10.1152/ajprenal.00546.2010.
[6] H. Yamakoshi, K. Dodo, M. Okada, J. Ando, A. Palonpon, K. Fujita, S. Kawata, M. Sodeoka, Imaging of EdU, an Alkyne-Tagged Cell Proliferation Probe, by Raman Microscopy, J. Am. Chem. Soc. 133 (2011) 6102–6105. https://doi.org/10.1021/ja108404p.
[7] S. Henriksson, P. Groth, N. Gustafsson, T. Helleday, Distinct mechanistic responses to replication fork stalling induced by either nucleotide or protein deprivation, Cell Cycle. 17 (2018) 568–579. https://doi.org/10.1080/15384101.2017.1387696.

Speaker: Mr Basseem Radwan (Jagiellonian Centre for Experimental Therapeutics (JCET) ul. Bobrzyńskiego 14, 30-348 Kraków, Poland)
• 16:20
Bodipy Derivatives Sensitized Triplet Triplet Annihilation Upconversion in Solution and Liposomes 10m

Bodipy Derivatives Sensitized Triplet Triplet Annihilation Upconversion in Solution and Liposomes
Amrutha Prabhakaran, Ruben Arturo Arellano Reyes, and Tia E Keyes*
aSchool of Chemical Sciences and National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland
Abstract
Triplet-triplet annihilation upconversion (TTA-UC) is a photophysical phenomenon in which two low energy photons from triplet excited states are converted to a high energy photon. This process results in the emission of light at shorter wavelength than the associated excitation wavelength used to excite the process. Unlike other UC schemes, TTA-UC does not require high energy or coherent excitation sources so offers exciting opportunities in healthcare applications including photodynamic therapy (PDT) and biological imaging. However, it does make require careful tuning of sensitizer to optimise process efficiency and designing efficient photosensitizer for TTA-UC is challenging with many constraints due to the thermodynamics and multiplicity requirements of the system. BODIPY derivatives, due to their attractive tuneable photophysical characteristics and versatile, synthetic chemistry are proving useful as TTA sensitizers .1
Herein, we have designed and compared two BODIPY-perylene based sensitizers for TTA-UC. The first sensitizer is functionalised with a heavy Iodine atom, to promote inter-system crossing through spin-orbit coupling, and the second molecule is a heavy-atom-free sensitizer. We demonstrate that both conjugates are suitable for TTA-UC and also for fluorescence imaging to different degrees. In addition, their long-lived triplet state lifetimes and lipophilic character makes them suitable for liposomal encapsulation. We observe intense oxygen sensitive blue emission with the annihilator under green excitation in solution as well as in cell membrane models.2 The biodelivery of the TTA-UC system was further investigated at varied membrane compositions ranging from different alkyl chain length to different melting transition temperature. We observed the most efficient upconversion in the case of highly fluidic zwitterionic membrane.3
Scheme 1: TTA-UC in liposome containing sensitizer and annihilator under 532 nm excitation.
Acknowledgements
We thank Horizon2020 Marie-Skłodowska-Curie Action “LogicLab” (Grant Agreement 813920) for Financial support.
References
1. S. Dartar, M. Ucuncu, E. Karakus, Y. Hou, J. Zhao, and M. Emrullahoglu. Chem. Commun., 2021, 57,6039.
2. S. H. C. Askes, A. Bahreman, S. Bonnet, Angew. Chem.Int. Ed., 2014, 53, 1029.
3. G. B. Berselli, N. K. Sarangi, P. V. Murphy, T. E. Keyes, ACS Appl. Bio. Mater., 2019, 2, 3404.

Speaker: Amrutha Prabhakaran (DCU)
• 16:30
Synthesis and functionalization of ferrite magnetic beads for bioseparation and biosensing applications 10m

The biosensing platforms have potential applications in the diagnosis of disease as sensitive, durable, portable and inexpensive systems. The use of Ferrite Magnetic Beads (FMBs) in biosensors for high-performance clinical diagnosis is gaining popularity due to their low toxicity and ability to be manipulated by an external magnetic field. FMB-based biosensors necessarily involve well-tuned magnetic beads, which consist of a ferrite core, as biolabeling, bioseparation, and biodetection probes in order to generate significant and precise biological signals and also separate analytes for further detection of diseases. In this study, FMBs will be synthesized through chemical routes and functionalized as a bioseparation material. Functionalization of MNPs not only ensures the integrity of FMBs in solution but also prevents interparticle reactions and agglomeration. For producing functionalized MNPs, silica will be used, which allows the binding of biological or other ligands to the surface of NPs. We'll make particles with a variety of sizes, surface charges and coatings, then evaluate their biomolecule binding affinity. The aim of the project is to isolate blood-borne pathogens from human plasma via using aptamer-modified and silica-coated FMBs integrated into a biosensor system that integrates processes such as separating, mixing, detecting biomolecules in a single piece of platform. The proposed project would build on previous studies in this area by evaluating the properties of FMBs that would maximize iron oxide nanoparticle Finally, we also aim to eliminate the stability problems by effectively functionalizing iron oxide nanoparticles and developing effective and orderly magnetic micro/nano-assembly structures.

Speaker: Mr Yasar Ozer Yilmaz
• 16:40
Break 20m
• 17:00
Carotenoid-mediated photophysics in plants 25m
Speaker: Dr Gabriela S. Schlau Cohen (Associate Professor of Chemistry at Massachusetts Institute of Technology (MIT), United States of America)
• 17:25