Thermal modulation and fluorescence/Raman reading-out for kinetically analyzing networks of chemical/biological reactions – T-KiNet

We want to introduce a versatile method to characterize mechanisms, and measure the associated rate constants, from analyzing the response of a reactive system to thermal excitations. Our strategy will be applied to networks of reactions, in which the species reacting together define the different nodes (up to three of them will be considered). In particular, this approach will open a way for fast and non-invasive measurements of fluxes in complex sets of reactions, yielding to what could constitute an unprecedented chemical amperometer. It will also make possible to check whether a reactive network is in equilibrium or in a nonequilibrium steady state, a situation frequently encountered in biological media. Although our non-invasive analytical approach is fully compatible with in vivo constraints, this project will focus on in vitro analysis. However we will work with biologically-relevant reactants and the next step should be to address network analysis in living systems.
This project integrates multiple individually ambitious developments. Beyond their relevance for the whole, those developments will be relevant for themselves, being of possible use on a short term for a vast community of scientists. Theoretical, instrumental, and reactive system issues will be examined:
• The theoretical developments will address in a general perspective the characterization of biologically-relevant reactive networks from analyzing the first- and second-order DC and AC responses of the reactant concentrations to temperature oscillation. The possibility to design discriminating criteria for classes of networks will be evaluated, with the goal of being able to assign a given chemical mechanism to an unknown network. It will be examined which plan of experiments should be implemented to qualitatively and quantitatively characterize the dynamics of a reactive network.
• We will fabricate various miniaturized devices to modulate, in a wide frequency range (0.1-104 Hz), the temperature inside an observation chamber. In a first design, a resistive layer will be flown by an alternating current in order to heat the observation cell in AC mode. Alternately, we will take benefit from using aqueous solutions as solvents to directly inject a tailored alternating current into the buffer and thus modulate its temperature by Joule effect. Such heated microchambers should increasingly find interest to access the thermodynamic and kinetic properties of various processes of biological relevance, making them desirable for biological assays.
• We will use fluorescence emission and Raman spectroscopy to report on the concentration of reactants. Experiments run in parallel with both observables will cross-validate our analytical protocols. In particular, to access a rich amount of information at high frequency and low concentration, we have retained Coherent Anti-Stokes Raman Scattering (CARS) and Stimulated Raman Scattering (SRS) as Raman methodologies. Combined with synchronous and phase-sensitive detection schemes, made possible by the temperature modulation, they should appear as selective and powerful analytical tools.
• The validation of our network analysis protocols will require unprecedented reactive systems that will (i) exhibit triangular mechanisms with rate constants spanning different ranges of relaxation times; (ii) possess favorable properties facilitating their observation by fluorescence emission and Raman spectroscopy. More precisely, we will devise synthetic models based on oligonucleotide association in order to test our methodology. Then we will implement our protocols to characterize the reactivity network of two systems which are relevant to systems pharmacology and biomolecular folding. Beyond bringing the proof of principle of our analytical approach, we anticipate our results to lead new mechanistic insights in the investigated processes.