WO2021156063A1 - Procédé de préparation d'un échantillon comprenant des spins nucléaires hautement polarisés et utilisations et dispositifs destinés audit procédé - Google Patents

Procédé de préparation d'un échantillon comprenant des spins nucléaires hautement polarisés et utilisations et dispositifs destinés audit procédé Download PDF

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WO2021156063A1
WO2021156063A1 PCT/EP2021/051265 EP2021051265W WO2021156063A1 WO 2021156063 A1 WO2021156063 A1 WO 2021156063A1 EP 2021051265 W EP2021051265 W EP 2021051265W WO 2021156063 A1 WO2021156063 A1 WO 2021156063A1
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epr
sample
polarization
radical
polarization system
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Jonas MILANI
Mika TAMSKI
Jean-Philippe Ansermet
Christophe Roussel
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Ecole Polytechnique Federale De Lausanne (Epfl)
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/28—Details of apparatus provided for in groups G01R33/44 - G01R33/64
    • G01R33/282—Means specially adapted for hyperpolarisation or for hyperpolarised contrast agents, e.g. for the generation of hyperpolarised gases using optical pumping cells, for storing hyperpolarised contrast agents or for the determination of the polarisation of a hyperpolarised contrast agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
    • G01N24/12—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using double resonance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/62—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using double resonance

Definitions

  • the present invention relates to a method for the preparation of a sample comprising highly polarized nuclear spins, as well as uses of such a method and devices for carrying out such a method.
  • Dynamic Nuclear Polarization designates an array of techniques that seek to enhance Nuclear Magnetic Resonance (NMR) signals by transferring electron’s spin polarization to nuclei thanks to the irradiation of electromagnetic waves around the Electron Paramagnetic Resonance (EPR).
  • EPR Electron Paramagnetic Resonance
  • Overhauser identified a possible mechanism that he considered applicable for electron and nuclear spins in metals. His prediction was verified and extended to liquids with radicals in solution.
  • the Overhauser Effect can be obtained in liquids when working with two dipolar-coupled nuclei that have very different gyromagnetic ratios for example between electron’s spin and other nuclear spins.
  • OE designates a mechanism of hyperpolarization when electromagnetic waves are applied at the frequency of EPR.
  • Solid Effect SE
  • Cross Effect CE
  • Thermal Mixing TM
  • Hyperpolarization by dissolution dynamic nuclear polarization is a method to enhance the nuclear magnetic resonance signals by several orders of magnitude, typically >10'000 times.
  • the presence of a radical is required during the hyperpolarization process.
  • radicals become unwanted after the process to preserve the hyperpolarized sample, as they induce high relaxation and so a significant loss of the polarization over time.
  • Ji, X., et al. (Nat Commun 8, 13975, 2017) report, that nuclear spin hyperpolarization of relabelled metabolites by dissolution dynamic nuclear polarization can enhance the NMR signals of metabolites by several orders of magnitude, which has enabled in vivo metabolic imaging by MRI.
  • the polarization process must be carried out close to the point of use. They introduce a concept that markedly extends hyperpolarization lifetimes and enables the transportation of hyperpolarized metabolites.
  • the hyperpolarized sample can thus be removed from the polarizer and stored or transported for use at remote MRI or NMR sites. They show that hyperpoiarization in alanine and glycine survives 16 h storage and transport, maintaining overall polarization enhancements of up to three orders of magnitude. This method is used and limited for nano-crystalline suspensions in toluene.
  • Gajan et al. (PNAS October 14, 2014 111 (41) 14693-14697) report that hyperpoiarization of substrates for magnetic resonance spectroscopy (MRS) and imaging (MRI) by dissolution dynamic nuclear polarization (d-DNP) usually involves saturating the ESR transitions of polarizing agents (PAs; e.g., persistent radicals embedded in frozen glassy matrices).
  • PAs polarizing agents
  • This approach has shown enormous potential to achieve greatly enhanced nuclear spin polarization, but the presence of PAs and/or glassing agents in the sample after dissolution can raise concerns for in vivo MRI applications, such as perturbing molecular interactions, and may induce the erosion of hyperpoiarization in spectroscopy and MRI.
  • d-DNP can be performed efficiently with hybrid polarizing solids (HYPSOs) with 2, 2, 6, 6- tetramethyl-piperidine-1-oxyl radicals incorporated in a mesostructured silica material and homogeneously distributed along its pore channels.
  • the powder is wetted with a solution containing molecules of interest (for example, metabolites for MRS or MRI) to fill the pore channels (incipient wetness impregnation), and DNP is performed at low temperatures in a very efficient manner.
  • molecules of interest for example, metabolites for MRS or MRI
  • HYPSO is physically retained by simple filtration in the cryostat of the DNP polarizer, and a pure hyperpolarized solution is collected within a few seconds.
  • the resulting solution contains the pure substrate, is free from any paramagnetic or other pollutants, and is ready for in vivo infusion.
  • the material presented in this publication is a porous silicate linked with permanent radicals. With it, it is possible to get pure hyperpolarized sample in liquid (and only in liquid). However, the critical point is during the fast dissolution, especially during the few milliseconds when the sample is heated by water but still in contact with electrons. This process leads to a significant loss of hyperpolarized signal. It is also not possible to use this material to store the hyperpolarized sample. The method presented here allows the removal of the radical, and thus storage of the sample.
  • US2016033590 discloses a method for the preparation of a sample comprising highly polarized nuclear spins, comprising at least the following steps: a) provision of molecules with 1 ,2-dione structural units and/or molecules with 2,5-diene- 1 ,4-dione structural units in the solid state; b) generation of radicals from these molecules by photo induced electron transfer by a first electromagnetic irradiation in the visible or ultraviolet frequency range in the solid state; c) dynamic nuclear polarization in the presence of a magnetic field in the solid state by applying a second electromagnetic irradiation with a frequency adapted to transfer spin polarization from the electrons to the nuclear spins leading to a highly polarized state thereof.
  • US2004049108 relates to devices and method for melting solid polarized sample while retaining a high level of polarization.
  • a sample is polarized in a sample-retaining cup in a strong magnetic field in a polarizing means in a cryostat and then melted inside the cryostat by melting means such as a laser connected by an optical fibre to the interior of the cryostat.
  • Electrochemical reactions change the oxidation state of a redox mediator at an electrode either by reduction or oxidation reactions.
  • This change in oxidation state can be used to generate or remove a paramagnetic species with at least one unpaired electron, which can be studied by Electron Paramagnetic Resonance (EPR) spectroscopy at low fields, or more recently, at high fields.
  • EPR Electron Paramagnetic Resonance
  • the creation/removal of radicals by means of electrochemistry gives an opportunity to create and to annihilate them as required.
  • the proposed invention comprises methods to create radicals by electrochemistry to perform dynamic nuclear polarization and/or to remove them in situ. This includes the creation/annihilation of radicals at electrodes with different surface areas and morphologies.
  • the chemical species responsible for generating or providing the radicals may or may not be bonded to the electrode surface with covalent bonds, or be present as a part of a polymer layer deposited to the electrode surface. With radicals bonded to the electrode surface and polymers, it is possible to use this method in solid, liquid or gas/vapor phases in stop flow or continuous flow experiments.
  • the process proposed by our invention comprises typically the following three steps:
  • Hyperpolarization process this step can include DNP;
  • the process proposed by our invention can comprise the following three steps:
  • Hyperpolarization process this step can include DNP;
  • the process proposed by our invention can also comprise the following three steps
  • this step can include DNP;
  • the process is combined involving generation, DNP and subsequent removal of the radical by electrochemistry in situ.
  • the sample can directly be used for analysis in the same probe or with a continuous flow setup in another spatial location.
  • the sample can then be transferred in an external low temperature storage device such as a dewar with a low magnetic field.
  • an external low temperature storage device such as a dewar with a low magnetic field.
  • Dynamic nuclear polarization will give a significant advantage on nuclear magnetic resonance (NMR) analysis or Magnetic Resonance Imaging (MRI) scanning. Basically, it increases the signal by a factor > 10 ⁇ 00 times for the technique of dissolution-DNP (d- DNP) which is used to rapidly dissolve the frozen sample with hot water or deuterated water and inject the solution in a standard NMR spectrometer. This technique can give enhancement up to 50’000-fold in the condition of final spectrometer in liquid state.
  • the proposed invention gives the opportunity to create and store long lived hyperpolarized samples for NMR or MRI detection. It is a way to provide cheap hyperpolarization for final users (labs or hospitals). This becomes feasible because one is able to remove the radical from the sample after the polarization transfer, significantly extending the lifetime of the sample.
  • DNP helps NMR analysis and MRI scanning by different ways.
  • NMR signal to noise ratio
  • S/N The signal to noise ratio
  • DNP offers an opportunity to reduce this measurement time to hours or even minutes. For example, in a situation where averaging of 10 ⁇ 00 scans would be needed for an acceptable signal, an increase of 100 in the S/N by other means, such as DNP, would allow the measurement to be performed with a single scan. This is a significant improvement for the entire pharmaceutical industry and will lead to significantly reduced R&D costs.
  • Gadolinium is a commonly used contrast agent. Although usually well tolerated by the subjects, in cases such as spleen disease the side effects can vary from mild to severe. Also, a study in 2019 showed significant risks of fetal exposure during pregnancy, and the safety of this contrasting agent remains to be established. Gd is also known to accumulate in the brain and cause long-term damage. DNP hyperpolarized solutions can also act like a contrast agents, introducing a non-invasive option for Gd.
  • the big advantage is that it is possible to choose the target molecule to be polarized, for example water for angiography, as shown by Denysenkov et. al. (Sci Rep 7, 44010, 2017). The method proposed here will remove the radical before the injection in a continuous flow setup and so prevent the toxicity related to radical species.
  • PET scan Alternative to PET scan: The detection of metastasis is essential to significantly enhance the prospects of successful cancer treatment.
  • a common method today is to use PET scan, a powerful and precise method to detect metastasis.
  • the process consists of injection of a radioactive isotope, which according to CHUV documents exposes the patient to equivalent of 7.8 years of natural irradiation. For this reason, PET scan is never applied to a healthy subject, and is highly inadvisable during pregnancy.
  • DNP has provided very promising results for metastasis detection with the additional benefit of being non-invasive. Thus, DNP can contribute to the detection of very early stage cancer in healthy subjects, preventing and reducing the cost of expensive treatments.
  • DNP equipment are rather expensive for a lab or a hospital.
  • the proposed invention can significantly decrease the cost for the use of DNP, thus democratizing the technique.
  • Previously electrochemistry has been combined with NMR and Electron Paramagnetic Resonance (EPR) spectroscopies to study for example reaction intermediates and products, or kinetics related to electrode processes.
  • EPR Electron Paramagnetic Resonance
  • electrochemical methods have never been used in the context of DNP as an in-situ source of radical species, although many electrochemical reactions intrinsically involve the generation of radical intermediates and/or products.
  • a setup has been assembled comprising an EPR resonator and an NMR coil to combine microwave irradiation to NMR experiment. Also, we have developed and experimented with electrochemical cells that can be assembled to a standard NMR tubes and inserted to our EPR/NMR/DNP setup.
  • the present invention relates to the following subject matter:
  • a method for the preparation of a sample comprising highly polarized nuclear spins using at least one polarization system which using an electrochemical reaction can be converted from a diamagnetic form without an EPR-active radical into a paramagnetic EPR-active radical form thereof, and/or which is paramagnetic and comprises an EPR-active radical and using an electrochemical reaction can be converted into a diamagnetic form without an EPR-active radical.
  • the polarization system can be a system containing for example a stable spin label which, without being influenced by an external potential, is in a paramagnetic state and is therefore EPR-active and can be used for DNP, but which can be converted by applying an external potential electrochemically into a system which is diamagnetic and will therefore not disturb the system for example in a subsequent NMR experiment or lead to excessive relaxation.
  • the polarization system can also be one which is diamagnetic without being influenced by an external potential, but which can be converted into a paramagnetic state by applying an electrochemical potential. According to a preferred variant, such a polarization system can be reconverted into the diamagnetic form by applying a correspondingly different electrochemical potential.
  • the method as proposed is involving at least one step of in situ electrochemically forming an EPR-active radical form of a diamagnetic polarization system and/or of in situ electrochemically converting a paramagnetic polarization system comprising an EPR-active radical into a diamagnetic form without an EPR-active radical.
  • the basic idea is therefore to use an electrochemical reaction for the in-situ generation and/or for the removal of the paramagnetic species used for the DNP process.
  • a radical is created at the electrode.
  • the radical then diffuses to the bulk solution in liquid state, or stays at the electrode surface if a covalently linked or a polymer- based polarization system is used.
  • a high surface area electrode with covalent linked radical on its surface, or a polymer based radical is preferably used for a frozen sample. Even if the radical is fixed, the hyperpolarization diffuses by spin diffusion into the bulk, as well. By applying an opposite electric potential, one is able to oxidize or reduce the radical to quench it.
  • the method for the preparation of a sample comprising highly polarized nuclear spins is comprising at least the following steps: a) bringing a polarization system, which using an electrochemical reaction can be converted into an EPR-active radical form thereof, in contact or close proximity with said sample; b) in situ electrochemically forming an EPR-active radical form of said polarization system; c) applying DNP techniques in the presence of a magnetic field by applying electromagnetic irradiation with a frequency adapted to transfer spin polarization from the electrons of the polarization system to the nuclear spins of the sample leading to a highly polarized state thereof.
  • the method comprises at least the following steps: x) bringing a polarization system, which is paramagnetic and comprises an EPR-active radical, in contact or close proximity with said sample; c) applying dynamic nuclear polarization techniques in the presence of a magnetic field by applying electromagnetic irradiation with a frequency adapted to transfer spin polarization from the electrons of the polarization system to the nuclear spins of the sample leading to a highly polarized state thereof d) in situ electrochemically converting said polarization system, which is paramagnetic and comprises an EPR-active radical into a diamagnetic form without an EPR-active radical.
  • step x) can be provided by the above-mentioned sequence a) and b), while c) is the same as the above-mentioned step c), so the paramagnetic property is basically switched on for the DNP process and then switched off for subsequent handling of the sample comprising highly polarized nuclear spins, in particular for subsequent nuclear magnetic resonance measurements or imaging measurements.
  • steps a), b), x), c) and d) are carried out in frozen/solid state, liquid state or gaseous/vapour state of the sample. If the state is liquid state or gaseous/vapour state, the system can be used in a continuous flow mode.
  • step c) is followed by d) in situ electrochemically re-converting the EPR-active radical form of the polarization system into a non-radical form of the polarization system.
  • Step c) or step d) as given above can be followed by e) spatially separating the polarization system and the highly polarized sample, preferably in that either the polarization system is immobilized on a carrier and the sample is in a mobile (liquid or gaseous) state and separated from the carrier, or in that the sample is immobilized on a carrier and the polarization system is in a liquid state and separated from the carrier.
  • the polarization system including the highly polarized sample in frozen/solid state is transferred into an external low temperature storage device such as a dewar with a magnetic field. The sample is stored for a desired time.
  • step c) can be preferably applied with a frequency essentially equal to the difference or the sum of the Larmor frequencies of the electron spins and the nuclear spins, respectively, or at the Larmor frequencies of the electron spins in the presence of the applied magnetic field, and/or wherein the magnetic field is a static, preferably essentially homogeneous magnetic field with a strength of at least 0.1 T or of 0.2 T or of at least 0.3 T. Possible are also higher fields such as 1 - 10T or 5 - 9.5T.
  • the (preferably covalently linked) polarization system is preferably one selected from the following group: polycyclic heteroaromatic systems, preferably molecules based on substituted or unsubstituted imidazole, pyrazole, triazole, tetrazole, pyridine, diazine, triazine, tetrazine, pentazine, azepine, diazepine, preferably dimeric or trimeric forms thereof, preferably substituted and unsubstituted bipyridine, most preferably N,N'-dimethyl-4,4'-bipyridinium, or a member of the quinone family, especially for non-aqueous work.
  • polycyclic heteroaromatic systems preferably molecules based on substituted or unsubstituted imidazole, pyrazole, triazole, tetrazole, pyridine, diazine, triazine, tetrazine, pentazine, azepine, di
  • the radical can be selected for example from TEMPOL (4- hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl) and its derivatives: PROXYL,
  • the electrodes containing a polymer layer can be based on conducting backbone such as but not limited to poly(aniline), poly(pyrrole), poly(thiophene), poly(p-phenylene), poly(vinylcarbazole), Poly(phenylthiophene), Poly(phenylene vinylene), Poly(o- aminophenol) and poly(1,8-diaminonaphthalene), or non-conducting backbone such as Poly(methyl acrylate), Poly(vinyl ether), Polyethylene glycol), Poly(vinylidene fluoride) or polystyrene.
  • conducting backbone such as but not limited to poly(aniline), poly(pyrrole), poly(thiophene), poly(p-phenylene), poly(vinylcarbazole), Poly(phenylthiophene), Poly(phenylene vinylene), Poly(o- aminophenol) and poly(1,8-diaminonaphthalene), or non-conducting backbone such as Poly(methyl
  • the polymers above, conducting or non-conducting can be carrying a redox probe as part of the polymer back bone or as a pendant molecule (redox polymer) such as but not limited to N,N’-diphenyl-2,3,5,6-tetraketopiperazine (PHP), N,N’-diailyl-2,3,5,6-tetraketopiperazine (AP), N,N’-di-n-propyl-2,3,5,6-tetraketopiperazine (PRP), or a polyimide-based erylene- 3,4,9, 10-tetracarboxylic dianhydride(PTCDA).
  • PDP N,N’-diphenyl-2,3,5,6-tetraketopiperazine
  • AP N,N’-diailyl-2,3,5,6-tetraketopiperazine
  • PRP N,N’-di-n-propyl-2,3,5,6-tetraketopiperazine
  • the polymers above, conducting or non-conducting can be in EPR active form carrying a radical spin label as part of their backbone or as a pendant molecule (radical polymer) such as but not limited to TEMPOL (4-hydroxy-2, 2,6,6- tetramethylpiperidin-1-oxyl) and its derivatives: PROXYL, Spirobisnitroxide, Arylnitroxide, nitronylnitroxyl, Galvinoxyl, Arylnitroxide or other radicals such as but not limited to poly(2,2,6,6-tetramethylpiperidinyloxy methacrylate) and nitronyl nitroxide radicals.
  • TEMPOL 2-hydroxy-2, 2,6,6- tetramethylpiperidin-1-oxyl
  • PROXYL Spirobisnitroxide
  • Arylnitroxide nitronylnitroxyl
  • Galvinoxyl Galvinoxyl
  • Arylnitroxide or other radicals such as but not limited to poly(2,2,6,6-
  • the polymer membranes can be grown to the electrode surface by electropolymerization, condensation polymerization, step-growth polymerization, or any other suitable method.
  • step c), and/or subsequent to step d) as given above and/or subsequent to step e) as given above the highly polarized nuclear spins are preferably measured in a nuclear magnetic resonance (NMR) or in a magnetic resonance imaging (MRI) experiment, wherein preferably protons, 13C and/or 15N nuclei or a combination thereof is measured.
  • NMR nuclear magnetic resonance
  • MRI magnetic resonance imaging
  • steps a)-c), and if used at least one of steps d) and e), as well as the nuclear magnetic resonance (NMR) or magnetic resonance imaging (MRI) experiment can but are not necessarily carried out in one same probe head, but the analysis can be done in a commercial spectrometer or MRI scanner.
  • NMR nuclear magnetic resonance
  • MRI magnetic resonance imaging
  • the molecule carrying the electrons responsible for providing the hyperpolarization can be grafted to a Working Electrode (WE) surface.
  • WE Working Electrode
  • This can be achieved for example by 1): grafting an electrochemical mediator or a stable radical to the WE surface via covalent bonding, or 2) depositing a polymer layer with conducting electrons, redox mediators, or stable radicals to the WE surface.
  • the unpaired electrons are intrinsically present in the polymer membrane, and the membrane is part of the WE itself.
  • the electrons can be removed from the electrode surface by applying a positive enough potential to the WE.
  • the WE is analogous to a positive plate of a parallel plate capacitor, which is depleted from electrons relative to the negative plate.
  • the redox mediator can be used to produce a radical mediator according to step b) above, followed by the annihilation of the radical after the polarization transfer according to step d) above.
  • a radical polymer is used as described in step x) above, after the polarization transfer the radical is annihilated according to step d) above.
  • the potentials are applied with respect to a Reference/Counter electrode, depending on the type of the setup. The principle is the same whether we work with liquid or frozen samples.
  • the electrode material to which the redox mediators or radicals are linked can be for example a noble metal such as Au, Ag or Pt, but also ferro- or ferrimagnetic materials can be incorporated.
  • said polarization system which using an electrochemical reaction can be converted form a diamagnetic form without an EPR-active radical into a paramagnetic EPR-active radical form thereof and/or which is paramagnetic and comprises an EPR-active radical and using an electrochemical reaction can be converted into a diamagnetic form without an EPR-active radical, is chemically and/or physically attached to a working electrode or incorporated into a coating on the working electrode used for the electrochemical formation, and/or as part of a polymer, coated or forming part of the working electrode surface.
  • the present invention relates to a Nuclear magnetic resonance (NMR), magnetic resonance spectroscopy (MRS) or magnetic resonance imaging (MRI) experiment, preferably in vitro or in vivo, wherein in a first step a hyperpolarized sample is prepared using a method as detailed above, and the hyperpolarized sample is used in the experiment, preferably for enhancing the signal-to-noise ratio.
  • NMR Nuclear magnetic resonance
  • MRS magnetic resonance spectroscopy
  • MRI magnetic resonance imaging
  • the present invention relates to the use of a method according as detailed above for diagnostic purposes (e.g. MRI), in vivo, in vitro, for electrochemical systems, for surface analysis, for the monitoring of electrochemical processes and their mechanisms and kinetics and/or identification of species generated during electrochemical processes, for the analysis of devices for storing energy, for the analysis of catalytic processes, in particular taking place on the surface
  • the present invention relates to a combined EPR/NMR device or EPR/MRI device, suitable and adapted to apply, in the measurement area, not only a static magnetic field as well as the electromagnetic irradiation required for EPR and NMR, but also an electrochemical potential for the in situ generation of a radical from a polarization system, which using an electrochemical reaction can be converted into an EPR-active radical form thereof, in particular a device suitable and adapted to carry out a method as detailed above. Further embodiments of the invention are laid down in the dependent claims.
  • Fig. 1 shows a schematic presentation of the experimental setup
  • Fig. 2 shows in a) cyclic voltammogram of a 1 mM solution of MV 2+ in acetonitrile with
  • Electrochemical Overhauser Effect DNP (EC-OE-DNP):
  • the coupling factor p is a function of the rates wo, wi and wz, which are the 1 H dipolar relaxation rate factors for the two 1 ⁇ 2-spin system and on wo which is the 1 H relaxation rate factor in the absence of radical.
  • the leakage factor f depends of Tio and T which are respectively the relaxation rate constant of solvent without and with free radical presence
  • k is the relaxivity constant
  • C is the concentration of free radical.
  • the saturation factor s is a function of the power P, the number of hyperfine lines n and a, a factor related to the electron Ti.
  • the mass transport is expressed by the Nernst-Planck equation, which describes the total mass density flux / as the sum of the diffusion, electrostatic migration and convection, where v is the fluid velocity, z the ionic species valence, e the elementary charge, k B the Boltzmann constant, T the temperature. Because of this mass flux, the distribution of radicals across the sample volume depends on the time and distance from the radical- producing electrode. The electrostatic potential f is negligible because of the screening effect of the electrolyte.
  • the diffusion constant D in the system was determined to be 1.3x10 5 cnr 2 /s at a 10 pm diameter Pt ultra-micro-electrode. If we consider that the concentration decreases from 10 15 cm 3 to zero in 0.1 cm the diffusion contribution to / is of about 10 9 cm 2 /s. Therefore, the slightest convection leads to a far greater J in our electrochemical cell than diffusion does. Indeed, after a couple of seconds of electrolysis, the flux of the bright blue methyl viologen radical (MV +' ) became visibly turbulent. This means that, during the DNP experiment, the radical was not homogeneously distributed throughout the sample volume, and the observed DNP enhancement is a spatial average over different local concentrations.
  • MV 2+ methylviologen dichloride
  • TBAP tetrabutylammonium perchlorate
  • TSAHP tetrabutylammonium hexafluorophosphate
  • ACN anhydrous acetonitrile
  • FIG. 1 displays a schematic representation of the experimental setup.
  • the electrochemical cell was assembled into a 4 mm inner-diameter NMR tube.
  • the working electrode (WE a in Fig. 1) was a 250 pm diameter coiled silver wire with ca. 0.5 cm 2 of surface area.
  • a Pt wire tip was used as a pseudo reference (RE, not shown) and a Pt coil as counter electrode (CE b in Fig. 1).
  • a cotton wool plug (c in Fig. 1) was placed between the WE and the CE to prevent the convection of methyl viologen radicals (MV +‘ ), thus confining the electrochemically generated radical ions to the sensitive part of the NMR measurement, i.e. in the volume occupied by the WE.
  • a uniform magnetic field B of 0.3322 T is applied perpendicular to the tube.
  • the setup is made with a working electrode (WE) and counter electrode (CE).
  • a reference electrode (RE) can be added to the setup for a better potential control.
  • the polarizable sample is in contact with the WE.
  • the sample can be solid, liquid or vapor/gas.
  • the working electrode can be fabricated out of metal, alloy or semiconductor. It can be flat, porous or composed of nanowires.
  • the working electrode can be functionalized with stable spin labels (radicals), redox mediators capable of generating a stable radical, membranes, or a polymer layer can be deposited to the electrode surface.
  • Microwave source (MW) irradiates the sample and can be completed with or without wave-guide, horn, resonator or cavity.
  • the entire setup is in a magnetic field, created by resistive coils, superconducting coil or permanent magnet.
  • the NMR detection can be performed with radiofrequency coils, but is not necessary for the DNP process.
  • the electrochemical Overhauser (EC-OE)-DNP experiment was conducted within a Varian electromagnet in a magnetic field B of 0.33 T.
  • a coupling loop (d in Fig. 1) provided continuous microwave irradiation, at the EPR resonance frequency of the MV +‘ (9.3 GHz), to a sapphire resonator (e in Fig. 1).
  • An NMR coil was placed in the vicinity of the WE for a simultaneous 14 MHz excitation/detection using a spin-echo technique.
  • MV +' was produced in-situ in controlled quantities, and was used to transfer the polarization from the unpaired electrons to the nuclei of ACN protons.
  • the EPR spectrum of MV +' was measured with a Magnettech MiniScope MS 400 benchtop EPR spectrometer inside a ca. 1 mm inner diameter borosilicate tube.
  • Figure 2a shows a cyclic voltammogram of a 1 mM solution of MV 2+ at a gold electrode in acetonitrile with 200 mM of TBAP as supporting electrolyte against a Pt pseudo RE.
  • the two visible redox processes are the reduction of MV 2+ into MV +' at around -0.158 V, and the consecutive reduction of MV +' into MV° at around -0.559 V. Both processes are electrochemically reversible.
  • the corresponding processes are depicted on Fig. 2a and the structure of the MV 2+ is shown in the inset.
  • MV +' can also be generated by stepping the electrode potential beyond -0.559 V to produce MV° which then comproportionates in the vicinity of the electrode surface according to: MV°+ MV 2+ 2 MV +' with an equilibrium constant of 1 c 10 7 in ACN.
  • Figure 2b shows an X-band EPR spectrum of MV +' recorded at a radical concentration of about 100 mM in acetonitrile with 200 mM of TBAP as supporting electrolyte.
  • the spectrum shows a hyperfine splitting of ca. 0.137 mT, typical for the ring protons of MV +' although in acetonitrile, the larger line width masks most of the hyperfine couplings visible in aqueous samples.
  • Figure 3 presents the result before and after (average of 20 scans) electro-generating the radicals. After the radical production, the much shorter Ti of the protons led to a slightly higher MW-off signal. The OE-DNP signal of the ACN protons is 23-fold enhanced.
  • the concentration of generated radicals was estimated by determining the Ti of the sample protons, which in liquid phase is sensitive to the concentration of radical species, before and after applying the EC potential.
  • the calculated concentration was 3.7 mM.
  • the Ti were measured by saturation recovery.

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  • Magnetic Resonance Imaging Apparatus (AREA)

Abstract

L'invention concerne un procédé de préparation d'un échantillon comprenant des spins nucléaires hautement polarisés, consistant au moins : a) à mettre un système de polarisation, qui à l'aide d'une réaction électrochimique peut être converti en une forme de radical actif en RPE de ce dernier, en contact ou à proximité immédiate dudit échantillon ; b) à former de manière électrochimique in situ une forme de radical actif en RPE dudit système de polarisation ; c) à appliquer des techniques de polarisation nucléaire dynamique en présence d'un champ magnétique par l'application d'un rayonnement électromagnétique à une fréquence adaptée afin de transférer une polarisation de spin depuis les électrons du système de polarisation vers les spins nucléaires de l'échantillon, ce qui conduit à un état hautement polarisé de ce dernier ; d) à convertir de manière électrochimique in situ ledit système de polarisation, paramagnétique et comprenant un radical actif en RPE, en une forme diamagnétique sans radical actif en RPE.
PCT/EP2021/051265 2020-02-07 2021-01-21 Procédé de préparation d'un échantillon comprenant des spins nucléaires hautement polarisés et utilisations et dispositifs destinés audit procédé WO2021156063A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2024002754A1 (fr) * 2022-06-29 2024-01-04 Evonik Operations Gmbh Procédé pour vérifier l'authenticité d'un produit par spectrométrie par résonance paramagnétique électronique

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WO2021156063A1 (fr) Procédé de préparation d'un échantillon comprenant des spins nucléaires hautement polarisés et utilisations et dispositifs destinés audit procédé

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