WO2016001698A1

WO2016001698A1 - Process for polarizing nmr active nuclei, polarizing amplifier and apparatus for polarizing nmr active nuclei

Info

Publication number: WO2016001698A1
Application number: PCT/IB2014/002163
Authority: WO
Inventors: Aaron ROSSINI; Alexandre ZAGDOUN; Dominik KUBICKI; Lyndon Emsley; Anne Lesage
Original assignee: Centre National de la Recherche Scientifique CNRS; Ecole Normale Superieure de Lyon; Universite Claude Bernard Lyon 1
Current assignee: Centre National de la Recherche Scientifique CNRS; Ecole Normale Superieure de Lyon; Universite Claude Bernard Lyon 1
Priority date: 2014-06-30
Filing date: 2014-06-30
Publication date: 2016-01-07
Anticipated expiration: 2016-12-30

Abstract

The invention provides for a process for polarizing NMR active nuclei within an analyte of interest by dynamic nuclear polarization (DNP), said process comprising the steps of: a. providing a polarizing solution; b. contacting the analyte of interest with the polarizing solution; c. submitting the polarizing solution to DNP conditions, characterized in that it comprises the step of providing a DNP amplifying solid material dispersed or immersed in the polarizing solution, in that the DNP amplifying solid material has a real component of relative permittivity higher than that of the polarizing solution, and in that the volumetric fraction of the DNP amplifying solid material in the final mixture of polarizing solution, analyte and DNP amplifying material is in the range of 10 to 80%, preferably between 15 to 75%, most preferably between 40 to 70 %.

Description

Process for polarizing NMR active nuclei, polarizing amplifier and apparatus for polarizing NMR active nuclei

The invention relates to a process for polarizing NMR active nuclei within an analyte of interest by dynamic nuclear polarization (DNP). Such process may be used in the context of a process for analyzing an analyte of interest by nuclear magnetic resonance (NMR). The invention also relates to a polarizing amplifier suitable for a DNP process, and to an apparatus with such polarizing amplifier.

Nuclear magnetic resonance (NMR) spectroscopy is a method of chemical analysis that can reveal information on molecular structure and geometrical arrangement in space. NMR is however an intrinsically insensitive analytical technique because the detected signal is proportional to a very weak population difference between two nuclear energy levels. One way to improve the sensitivity is to increase the difference in energy levels by employing high magnetic fields, the sensitivity increasing with the field to the power 3/2. But even under ultra-high field magnets (21 T), the number of atoms whose spin magnetic moment is oriented in the direction of the magnetic field is only slightly larger than those polarized against (in the opposite direction of) the field. This small excess of ground state nuclei gives the sample only a small overall polarization in the direction of the magnetic field. Other efforts to increase the sensitivity of NMR include adding together the results of many spectra of the same sample so that the signal may be enhanced in comparison with random noise at the cost of longer analysis time. Particularly useful for this purpose is the Fourier Transform method (U.S. 3,475,680).

Recently the technique of dynamic nuclear polarization (DNP) has been used to increase the nuclear magnetic polarization of the sample, and thereby increase the sensitivity of the NMR experiment. DNP can be used to enhance the intensity of NMR signals, since NMR signals arise from transitions between nuclear spin states which have a very low energy difference and which are thus very weakly polarized at room temperature, leading to weak signals.

DNP refers to all the methods where the electron spin polarization is transferred to nuclear spins by the application of a resonant or off resonant microwave excitation of the electronic spin transitions. The technique can also be applied to significantly enhance the sensitivity of magnetic resonance imaging (Golman et al. PMAS July 25, 2006, vol 103 n°30, 11270-11275, incorporated therein for reference). DNP relies on the use of presence of unpaired electrons, which are more highly polarized in a magnetic field than most nuclei owing to the much larger gyromagnetic ratio of the electron compared with nuclei. Unpaired electrons are thus roughly 658 times more polarized than proton spins under the same conditions. DNP is the method whereby electron polarization is transferred to nuclei. This polarization can be transferred to the nuclei of the sample when the microwave irradiation is sufficient to induce transitions between the electron magnetic energy levels of the polarizing agent.

Currently, two main approaches can be applied to obtain highly polarized nuclear spins in solution by DNP.

Overhauser induced DNP (" ODNP ") is an established technique with the first experiments dating back decades {Overhauser, 1953). Solutions that contain a polarizing agent with unpaired electrons (a radical) can be directly hyperpolarized by this approach. This effect is however limited to low magnetic fields, typically 0.35 T (above 1 T, the modulation of the electron- nuclei dipolar couplings is not efficient for magnetization transfer by the Overhauser effect). It has been shown by Griffin and coworkers (G. 1 Gerfen et al., J. Chem. Phys., 1995, 102, 9494, and D. A. Hall et al, Science 1997, 276, 930, incorporated therein for reference) that, at higher magnetic field and in particular cases where a scalar coupling is established between the electron and a nucleus, the modulation of this through-bond interaction can give rise to a Overhauser effect and to significant NMR signal amplification. The ODNP approach is typically applied to probe the dynamics of water molecules in close proximity to an electron spin label (5 to 10 A) (B. D. Armstrong, S. Han, J. Am. Chem. Soc, 2009, 131, 4641, incorporated therein for reference). More recently, ODNP has been performed at high magnetic fields using either i) shuttle DNP spectrometers, which enables to excite the electron spins at low magnetic and then shuttles the sample to high magnetic field for NMR detection (M. Reese et al, 1 Am. Chem. 2009 Soc. 131, 15086-15087, incorporated therein for reference) or ii) a high-field DNP spectrometer, which performs simultaneously the microwave excitation and NMR detection and where the sample is contained in small capillaries of a few nanoliters (Denysenkov, et al, AppL Magn. Reson. 2008, 34 289-299; C. Griesinger et al, Progress in Nuclear Magnetic Resonance Spectroscopy 2012, 64, 4-28, incorporated therein for reference).

J, H. Ardenkjasr-Larsen et al developed a more recent approach (J. H. Ardenkjaer-Larsen et al, PNAS, 2003, 100, 10158-10163, incorporated therein for reference), in which the polarization of the nuclear spins is performed at low temperature in the solid- state. The sample is then (i) either studied directly using solid-state NMR techniques or (ii) is dissolved rapidly to obtain a solution in which the nuclear spins of the molecules of interest are strongly polarized and then the polarized sample is shuttled over to a different magnetic field for solution NMR or MRI experiments. The sensitivity increase was achieved by the use of a polarizing agent containing unpaired electron(s). The polarizing agent was dissolved in a glass forming solution containing the substance to be analyzed, and the solution cooled to 1.5 Kelvin in a polarizing magnetic field of 3.35 Tesla. The cold solution was irradiated with radio frequency radiation at the Larmor frequency of the electron (94 GHz). Then the sample was warmed by the addition of room temperature solvent, and this new solution is then transferred to a second magnetic field within a few seconds where the spectra (or magnetic resonance images) are obtained. The irradiation step transferred the high polarization of the unpaired electron of the free radical to the nuclei of the sample and the hyper polarization was retained for several seconds while the sample was warmed and dissolved in the diluting solvent. The dissolution experiment is usually performed in an ex situ DNP polarizer, consisting in a magnet, a cryostat to cool down the sample to a temperature often close to 1.5 K (sometimes around 100 K when liquid nitrogen based cooling systems are available) and a microwave source. Solid-state NMR experiments make use of polarization in-situ inside the NMR spectrometer. After polarization by microwave irradiation, and rapid dissolution, the hyperpolarized liquid sample is transferred to a high-resolution NMR spectrometer, where the NMR signal is detected. The NMR signals can be amplified by factors larger than 10 000. The radicals used to obtain the polarization can either be neutralized chemically or filtered out of solution. This method has been mostly be used for low-nuclei (¹³C and ¹⁵N) and the detection of hyper-polarized protons using dissolution DNP remains challenging due to the shorter nuclear relaxation times. Single scan methods have been applied to obtain multidimensional correlation spectra.

Others authors describe the use of radicals on solid supports to (hyper)polarize solutions and flowing fluids by DNP. First, Dorn et al, in J. Am. Chem. Soc, 110, 2294 (1988); Anal. Chem. 70, 2623-2628 (1998), incorporated therein for reference, have studied the polarization of several flowing organic solvents by ODNP using TEMPO immobilized on polymer beads and silica gel. This approach is dubbed SLIT DNP for flow Solid-Liquid Intermolecular Transfer DNP. The advantage of this approach is that a radical-free hyperpolarized solution is obtained. In favorable cases, ¹³C (scalar-dominated) enhancements of 1-2 orders of magnitude could be obtained (for example in mixtures of benzene and several chlorocarbons which were continuously "recycled" through the DNP spectrometer). This approach is however not general (i.e. is limited to cases where a transient coupling with the radical occurs) and a complex apparatus is needed.

An agarose gel containing covalently bound radicals (TEMPO) has also been developed by S. Han et al. (J MR, 2008, 190, 307-315, incorporated therein for reference) to polarize aqueous solutions (stagnant or in continuous flow) by ODNP, at room temperature and low magnetic field (0.35 T). The mobility of the radicals in the gel is sufficient for efficient polarization transfer via the Overhauser effect, without the radicals being released into the solution. However, as the radicals are immobilized and the mobility of the solution is reduced within the gel, the proton enhancements observed for the water signal are lower than those obtained when TEMPO is directly dissolved in the sample. This method has so far been limited to the enhancement of the water NMR signal.

In the both techniques developed by Dorn et al. and Hans et al., the solution is polarized at low field before being subsequently transported into a high-resolution magnet. Therefore, this approach is an ex-situ polarization technique. The method has not been demonstrated yet as a general approach to detect small concentrations of substrates in solutions.

More recently Lafon et al. described in Applied Magn. Reson. 2012, 43, 237, incorporated therein for reference ,the use of in situ solid-state DNP NMR to polarize a commercially available TEMPO containing solid support, SiliaCAT(R) TEMPO which is a non- structured material containing 0,7 mmol radicals by grams of material. The sample did not contain any other component, and the notion of polarizing a substrate or an analyte of interest with such a material is not considered.

Other non-structured materials comprising TEMPO groups are also used as catalytic materials for selective oxidation of alcohols (US 6,797,773).

A DNP process can provide a theoretical maximum NMR signal enhancement of γ_ε/γ_η, where y_e and γ_η are the gyromagnetic ratios of the electron and nucleus in question (γ_ε/γΐΗ = 658, γ_ε/γΐ3ο = 2618, y_e/yi5N = 6494). In both dissolution and magic angle spinning (MAS) DNP experiments, intrinsically diamagnetic samples may be doped with exogenous radical polarizing agents such as stable trityl or nitroxide radicals. In dissolution DNP experiments carbon-13 may be directly polarized, and polarizations above 10 % are routinely obtained with low sample temperatures (< 5 K). Jannin et al. recently reported polarization of 71% achieved by cross-polarization from protons to carbon-13 at 1.2 K in a magnetic field of 6.7 T. Similarly, with state of the art biradical polarizing agents, proton DNP enhancements (ε_Η, defined, as conventional in the art, as the ratio of the intensity of the proton NMR signals obtained with and without microwave irradiation, i.e. the gain in NMR signal intensity from performing DNP) of up to 200 and 235 have been reported for organic or aqueous biradical solutions, respectively, with magnetic fields of 5-9.4 T and sample temperatures of ca. 85-105 K. Further detail on such experiments can be found in the following publications which are incorporated therein for reference:

- Zagdoun, A., et al., Large Molecular Weight Nitroxide Biradicals Providing Efficient Dynamic Nuclear Polarization at Temperatures up to 200 K. Journal of the American Chemical Society, 2013. 135 (34): p. 12790- 12797 ;

- Zagdoun, A., et al., A Slowly Relaxing Rigid Biradical for Efficient Dynamic Nuclear Polarization Surface-Enhanced NMR Spectroscopy: Expeditious Characterization of Functional Group Manipulation in Hybrid Materials. Journal of the American Chemical Society, 2012. 134: p. 2284- 2291 ;

- Sauvee, C, et al., Highly Efficient, Water-Soluble Polarizing Agents for Dynamic Nuclear Polarization at High Frequency. Angewandte Chemie- International Edition, 2013. 52: p. DOI:10.1002/anie.201304657 ;

- Kiesewetter, M.K., et al., Dynamic Nuclear Polarization with a Water- Soluble Rigid Biradical. Journal of the American Chemical Society, 2012. 134(10): p. 4537-4540.

A proton DNP enhancement of 235 corresponds to 36% of the maximum theoretical DNP enhancement of 658. These large MAS DNP solid- state NMR signal enhancements have enabled the characterization of a diverse range of chemical systems such as functionalized porous materials, polymers, nanoparticles, pharmaceuticals, and several biomolecular systems, that would have otherwise been inaccessible. Similarly, dissolution DNP has enabled many novel magnetic resonance experiments that hold great promise for medical diagnostics, such as the improved detection of cancers and better understanding of metabolic pathways. The key to all of these applications is obtaining large DNP enhancements that translate into greatly improved sensitivity for magnetic resonance experiments. As of today, there is still room for improvement in how much NMR signal enhancement may be achieved through DNP. An object of the invention is to provide for a new DNP process achieving, all things being equal, better enhancement than the known DNP processes.

In view of this object, the invention therefore provides a process for polarizing NMR active nuclei within an analyte of interest by dynamic nuclear polarization (DNP), said process comprising the steps of:

a. providing a polarizing solution containing a polarizing agent in a solvent,;

b. contacting the analyte of interest with the polarizing solution; c. submitting the polarizing solution to DNP conditions including application of an electromagnetic field in the microwave range of 9 to 789 GHz, preferably 80 to 600 GHz, before, during or after contacting the analyte of interest with the polarizing solution,

characterized in that it comprises the step of providing a DNP amplifying solid material dispersed or immersed in the polarizing solution, in that the DNP amplifying solid material has a real component of relative permittivity higher than that of the polarizing solution, and in that the volumetric fraction of the DNP amplifying solid material in the final mixture of polarizing solution, analyte and DNP amplifying material is in the range of 10 to 80%, preferably between 15 to 75%, most preferably between 40 to 70 %.

According to other optional features of such a process, which can be provided alone or in combination:

- The DNP amplifying solid material may have a real component of the relative permittivity at least two units larger than that of the polarizing solution, preferably at least three units higher than that of the polarizing solution; such difference ensures a significant improvement in the amplification of the enhancement.

- The DNP amplifying solid material is preferably distinct from the analyte.

- The analyte is preferably distinct from the solvent, from the polarizing agent and from the DNP amplifying solid material. - The polarizing agent is preferably not bonded to the DNP amplifying solid material.

- The DNP amplifying solid material may have a real component of the relative permittivity higher than 3, preferably higher than 4.5; examples of DNP amplifying materials having such characteristic have shown to perform well in terms of enhancement amplification.

- The DNP amplifying solid material may have a dielectric loss tangent is lower than that of the polarizing solution; this allows reducing the increase in temperature of the preparation when submitted to DNP microwave irradiation, the lower temperature being favorable to enhancement amplification.

- The DNP amplifying solid material has a dielectric loss tangent less than 0.01, preferably less than 0.002; examples of DNP amplifying materials having such characteristic have shown to perform well in terms of enhancement amplification.

- The DNP amplifying solid material may be present in the preparation under the form of discrete particles.

- At least 80% of the mass of the DNP amplifying solid material particles may have a size such as to be contained within a circumscribing sphere, the diameter of which is between 0.001 mm and 2 mm, more preferably between 0.1 mm and 0.5 mm.

- The DNP amplifying solid material may be dispersed in the polarizing solution under the form of discrete particles, and at least 80% of the mass of these particles may have a size such as to be contained within a circumscribing sphere, the diameter of which is less than 2 times the wavelength of the electromagnetic field to be used for the DNP, preferably less than 0.5 times the wavelength of the electromagnetic field to be used for the DNP.

- The DNP amplifying solid material may be under the form of at least one unitary solid macroscopic structure;

- The unitary solid macroscopic structure may have a size such as to be contained within a circumscribing sphere, the diameter of which is more than 0.7 mm, more preferably more than 2.4 mm, said unitary solid macroscopic structure being at least partly immersed in the polarizing solution.

- The unitary solid structure may be in the form of a macroscopic lattice structure enclosing voids;

- At least some voids of the unitary solid structure may have a size such as to be contained within a circumscribing sphere, the diameter of which is more than 0.01 mm, more preferably more than 0.1 mm.

- The unitary solid macroscopic structure is formed so as to exhibit a ratio between the volume of the DNP amplifying solid material compared to the volume circumscribing the macroscopic structure which is comprised between 10% and 70%, more preferably between 10 % and 50%.

- The DNP amplifying solid material may comprise one or more of the following substances :

- crystalline potassium bromide (KBr);

sapphire (a-AI203);

sodium chloride (NaCI);

- calcium fluoride (CaF2);

quartz (Si02);

zinc selenide (ZnSe).

- The polarizing agent may comprise at least one free radical having unpaired electrons.

- The polarizing agent may comprise at least two free radicals having unpaired electrons.

- Prior to the step of submitting the polarizing solution to DNP conditions, the process may comprise a step of generating unpaired electrons in the polarizing agent by submitting the polarizing solution to an energy field, for example to UV radiation, other ionizing radiation, heat, electrical discharges, electrolysis, and/or chemical reactions.

- The polarizing agent may comprise one or more of the following substances :

- TEMPO (2,2,6,6-Tetramethylpiperidin-l-oxyl); - TOTAPOL (l-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol);

- AMUPol;

- TEKPol (bis-phenylcyclohexyl-TEMPO-bisketal);

- bCTbK (bis-cyclohexyl-TEMPO-bisketal);

- bTBK (bis-TEMPO-bisketal);

- Functionalized derivatives of triphenylmethyl radicals.

- The polarizing agent is preferably not bonded to the DNP amplifying solid material.

- The solvent may comprise an organic solvent;

- The solvent may comprise one or more of the following substances :

- TCE (1,1,2,2-tetrachloroethane), (with ca. 5% methanol or chloroform optionally added to improve glass formation)

- Dimethyl sulfoxide (DMSO) with deuterium oxide (D20) and water.

- 1,2-dichlorobenzene

- 1,1,2,2-tetrabromoethane

- 1,4-dibromobutane

- pyruvic acid.

According to another aspect of the invention, it is also provided a process for analyzing an analyte of interest by nuclear magnetic resonance (NMR), the process comprising the step of polarizing NMR active nuclei of the analyte by dynamic nuclear polarization (DNP) through a process having one or several of the above mentioned features.

According to another aspect of the invention, it is also provided a polarizing amplifier suitable for a dynamic nuclear polarization (DNP) process, characterized in that:

- it comprises at least one unitary solid macroscopic structure comprising of a DNP amplifying solid material, the unitary solid macroscopic structure having a size such as to be contained within a circumscribing sphere, the diameter of which is more than 0.7 mm, more preferably more than 2.4 mm. - the DNP amplifying solid material has a real dielectric constant higher than 3, preferably higher than 4.5.

According to other optional features of such a polarizing amplifier, which can be provided alone or in combination:

- The DNP amplifying solid material may have a dielectric loss tangent less than 100, preferably less than 20.

- The unitary solid structure may be in the form of a macroscopic lattice structure enclosing voids having a size such as to be contained within a circumscribing sphere, the diameter of which is more than 0.7 mm, more preferably more than 2.6 mm.

- The unitary solid macroscopic structure may be formed so as to exhibit a ratio between the volume of the DNP amplifying solid material and the volume circumscribing the macroscopic lattice structure comprised between 10% and 70%, preferably between 10% and 50%, more preferably comprised between 10% and 30%.

According to another aspect of the invention, it is also provided an apparatus for performing a dynamic nuclear polarization (DNP) process comprising:

- a microwave source;

- an insulated cryostat or magic angle spinning probe;

- a sample holder for receiving a polarizing solution comprising a solvent and a polarizing agent to be polarized;

- a waveguide/transmission line system to transport the microwaves to the sample holder,

characterized in that a polarizing amplifier having one or several of the features above is provided in the sample holder so as to be contacted by a polarizing solution.

Other features of the invention will be described in the following description of some embodiments of the invention, with reference to the annexed drawings where :

- Figure 1 illustrates several steps for a method for determining particle size; - Figure 2 is a graph representing some results of DNP enhancements which may be obtained thanks to the invention;

- Figure 3a and Figure 3b are graphs showing variations of enhancement factor and of temperature according to variation in applied microwave power;

- Figure 4 is a schematic view representing the basic principles of one embodiment of a polarizing amplifier according to the invention.

According to an aspect of the invention, a polarizing solution is prepared which is suitable for enhancing the polarization of nuclear magnetic resonance (NMR) active nuclei by dynamic nuclear polarization (DNP).

NMR active nuclei include isotopes that contain an odd number of protons and/or of neutrons, which have an intrinsic magnetic moment and angular momentum, in other words a non-zero spin. NMR active nuclei thus include protons and deuterons (1H and 2H, respectively), ¹³C, ¹⁵N, ¹⁹F, ³¹P etc... . An NMR process will analyze the NMR of such nuclei which are contained in an analyte of interest. The DNP process will include transfer of the polarization of a polarizing agent to the NMR active nuclei.

The polarizing solution is prepared by dissolving a polarizing agent in a solvent or mixture of solvents that is/are usually chosen so that, when frozen, the resulting solution forms a glass, i.e. a largely amorphous structure.

The polarizing agent comprises one or more chemical species with unpaired electrons or in which it is possible, at some stage of the process, to generate such unpaired electrons. Indeed, it will be seen that, prior to the step of submitting the polarizing solution to DNP conditions, the process may comprise a step of generating unpaired electrons in the polarizing agent by submitting the polarizing solution to an energy field, for example to UV radiation, other ionizing radiation, heat, electrical discharges, electrolysis, and/or chemical reactions.

The polarizing agent will typically contain at least one free radical, also referred to more simply as radical, i.e. an atom, molecule, or ion that has unpaired valence electrons or an open electron shell. Examples of such radicals having unpaired electrons include the hydroxyl radical (ΗΟ·), the superoxide anion (·02), i.e. an oxygen molecule (02) with one ext2ra electron, and most commonly nitroxide radicals (R₂- ΝΟ·). Free radicals may be formed in various manners, including synthesis with very dilute or rarefied reagents, reactions at very low temperatures, or breakup of larger molecules, which can be achieved through any process that delivers enough energy to the parent molecule, such as UV and ionizing radiation, heat, electrical discharges, electrolysis, and chemical reactions.

A polarizing agent may contain species having more than one free radical. Such species having two free radicals are often called bi-radicals.

In the scientific literature, many polarizing agents have been already proposed which are suitable for use in a DNP experiments, and which would be thus suitable for implementing the invention. Such suitable polarizing agents include, as a non-limiting list:

- TEMPO (2,2,6,6-Tetramethylpiperidin-l-oxyl)

TOTAPOL (l-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol)

AMUPol

TEKPol (bis-phenylcyclohexyl-TEMPO-bisketal)

bCTbK (bis-cyclohexyl-TEMPO-bisketal)

bTBK (bis-TEMPO-bisketal)

Functionalized derivatives of tri phenyl methyl radicals.

The polarizing solution to be used in the invention may comprise several different polarizing agents.

Also, many solvents have been suggested in the scientific literature which would be suitable for preparing a polarizing solution according to the invention. Many of the most common solvents used in polarizing solutions are based upon mixtures of water and alcohols (e.g., glycerol, ethanol, etc.). Other such suitable solvents/solutions include, as a non-limiting list:

TCE (1,1,2,2-tetrachloroethane), (with ca. 5% methanol or chloroform optionally added to improve glass formation)

Dimethyl sulfoxide (DMSO) with deuterium oxide (D2O) and water.

1,2-dichlorobenzene 1,1,2,2-tetrabromoethane

1,4-dibromobutane

pyruvic acid

The polarizing solution to be used in the invention may comprise several different solvents.

The polarizing solution may typically be prepared by mixing the necessary solvents and dissolving the required amount of the polarizing agent.

The concentration of polarizing agent in the polarizing solution may be in the range of 2 to 80 mmol/L, more preferably in the range of 4 to 20 mmol/L.

At some point in the process, the analyte of interest is to be contacted with the polarizing solution. This may be performed in various ways, including but not limited to, by dissolving the analyte in the polarization solution, by dispersing the analyte in the polarization solution, by immersing the analyte in the polarization solution, by impregnating the analyte with the polarizing solution, a combination hereof, etc..

The polarizing solution is to be submitted to DNP conditions including application of an electromagnetic field in the microwave range, for the purpose of effectively polarizing the polarizing solution and possibly any analyte in contact with the solution. Before such a step, if the polarizing agent has been provided under a form where it does not exhibit the required free radicals, the polarizing agent or the polarizing solution may be subjected to a radical generating step, which may comprise submitting the polarizing agent or solution to an energy field, for example to UV radiation, other ionizing radiation, heat, electrical discharges, electrolysis, and/or chemical reactions.

It is known to the skilled man in the art that, in a DNP process, the polarizing solution may be polarized in presence of the analyte (one step process), or may be polarized before being set in presence of the analyte (two step process). In the one step process, polarization of the polarization agent and transfer of this polarization to the analyte is performed simultaneously. In such a case, the step of submitting the polarizing solution to DNP conditions may be performed after the step of contacting the analyte of interest with the polarizing solution.

In the two step process, polarization of the polarizing agent is obtained in a first step, in the absence of the analyte. Transfer of polarization to the analyte is then performed after the step of contacting the analyte of interest with the polarizing solution.

In a variant, the step of submitting the polarizing solution to DNP conditions is performed simultaneously with the step of contacting the analyte of interest with the polarizing solution.

The step of submitting the polarizing solution to DNP conditions is performed according to conventional techniques known from the prior art. It includes application of an electromagnetic field in the microwave range of 9 to 789 GHz (determined by the magnetic field strength of the applied field), preferably in the range of 80 to 600 GHz. Experiments have shown good results at 94 GHz, 140 GHz, 263 GHz and 527 GHz.

The DNP conditions may comprise submitting the polarizing solution, with or without the analyte, to a DNP temperature where the polarizing solution is frozen. The polarizing solution may exhibit at this temperature a largely amorphous (glassy) structure. The DNP temperature may be in the order of 1 to 30 K if a liquid helium based freezing system is used, or in the order of 78 to 120 K if a liquid nitrogen based freezing system is used.

According to an aspect of the invention, a DNP amplifying solid material is dispersed or immersed in the polarizing solution. Importantly, in the present invention, the DNP amplifying solid material may comprise a several different materials having the required properties. Preferably the DNP amplifying solid material remains non-dissolved or largely non-dissolved during the DNP process. The combination of the polarizing solution with the DNP amplifying solid material immersed or dispersed in the polarizing solution may be called the polarizing preparation. The DNP amplifying solid material is present in the preparation so as to interfere in the DNP process when the microwave field is applied to cause the polarization.

According to one aspect, the DNP amplifying solid material has a real component of relative permittivity higher than that of the polarizing solution. Preferably, the DNP amplifying solid material has a real component of relative permittivity at least two units larger than that of the polarizing solution, more preferably at least three units higher than that of the polarizing solution.

Defined differently, in absolute terms, the DNP amplifying solid material may preferably have a real component of the relative permittivity higher than 3, more preferably higher than 4.5.

Preferably, the real component of relative permittivity of the DNP amplifying material and/or that of the polarizing solution are measured or estimated at the DNP conditions. However, the estimation of the real component of relative permittivity may be based on measurements made at other conditions, especially for example at room temperatures, and/or frequencies in the microwave range, but not necessarily equal to those used in the DNP experiments. It is known that the real component of relative permittivity usually shows a small variation with frequency in the microwave region.

There are several standard methods known and used to measure the values of the relative permittivity and loss tangents for millimeter and sub- millimeter frequencies. They include dispersive Fourier transform spectroscopy, Fabry-Perot open-resonators, Mach-Zender IMPATT/Gunn diode spectrometer, etc....

As will be shown hereunder, it has been identified by the inventors that the addition of such a DNP amplifying material to the polarizing solution has amplified the DNP enhancement factor (defined as the ratio of NMR signal intensities obtained with and without the application of DNP) obtained with such polarizing solution by at least two, and in some cases nearly three. For example proton DNP enhancement factors obtained for the NMR resonances in experiments were compared where one sample was a bulk solution of a given bi-radical as polarizing agent, another sample comprised the same polarizing solution filled into a rotor containing solid particles of crystalline potassium bromide (KBr), and still another sample contained solid particles of sapphire (α-ΑΓ203). For a sapphire rotor containing only a sample of 16 mM (i.e. 16 mmol/l) TEKPol 1,1,2,2-tetrachloroethane (TCE) solution, a proton DNP enhancement of 200 was obtained. In all cases the observed enhancement is significantly higher when the solid particles are present in the sample, reaching a value as high as 530 (or more than 2.5 times the reference value for the bulk solution) when a TEKPol TCE polarizing solution is mixed with KBr particles as a DNP amplifying solid material. Furthermore, the effect is not limited to non-polar organic solvents such as (TCE) but is also observed in water-based solvent systems, as exemplified by the AMUPol biradical as polarizing agent dissolved in a 60:30:10 solvent mixture glycerol- d₈/D₂0/H₂0 mixed with sapphire solid particles as a DNP amplifying solid material. Also, it is clear that the effect does not change significantly from one polarizing agent to another, as it is roughly the same with TEKPol, AMUPol, bCTbK or bTbK. Similar results are obtained with other DNP amplifying solid materials such as NaCI or CaF₂.

By selecting a DNP amplifying material have such dielectric properties, it is believed that, when exposed to the microwave irradiation during the DNP process, the DNP amplifying solid material modifies locally the electromagnetic field in the preparation, which promotes the amplification of the DNP polarization enhancement.

It is determined that the volumetric fraction of the DNP amplifying solid material in the final mixture of polarizing solution, analyte and DNP amplifying material should be in the range of 10 to 80%, preferably between 15 to 75%, most preferably between 40 to 70 %. The volumetric fraction will be chosen based on a compromise between the amplifying effect obtained thanks to the DNP amplifying solid material, and the volume of analyte which may still be analyzed. Indeed, the signal strength obtained in a NMR analysis will be the dependent on the product of the quantity of potential NMR active nuclei in the analyte, which depends on volume of the analyte in the NMR analyzed sample, by the proportion of these potentially active nuclei which are effectively active, i.e. polarized.

Therefore, it is foreseen that a positive effect of the invention can be achieved already at a volumetric fraction of 10 %, especially if highly dielectric DNP amplifying material is used, although 15% percent would achieve a more noticeable amplifying effect. With DNP amplifying material dispersed in the polarizing solution, considerable amplification effect has been shown at a volumetric fraction of 65%, but it is anticipated that with other forms of incorporation of the DNP amplifying material, especially under the form of a unitary solid macroscopic structure as will be described below, a volumetric fraction of less than 50%, preferably less than 30 % may achieve a significant amplification of the enhancement factor. In other words, it is expected that the unitary solid macroscopic structure will be more efficient than randomly dispersed particles, at least in part due to a more controlled distribution of the DNP amplification material in the polarizing solution. Also, the lower volume factions would seem most useful when using highly dielectric DNP amplifying material, for having higher real component of relative permittivity than those of the materials used in the experiments which will be used thereafter.

It should be mentioned that the DNP amplifying solid material is preferably distinct from the analyte of interest.

Also, the analyte of interest is preferably distinct from the solvent, from the polarizing agent and from the DNP enhancing solid material.

Also, in the invention, the polarizing agent is preferably not bonded to the DNP amplifying solid material, especially not covalently bonded.

Preferably, the DNP amplifying solid material has a dielectric loss tangent, which may be defined as the ratio of the imaginary component of the permittivity to the real component of the permittivity, which is lower than that of the polarizing solution. In absolute terms, the DNP amplifying solid material may have a dielectric loss tangent lower than 100, preferably less than 20. Preferably, the dielectric loss tangent of the DNP amplifying material and/or that of the polarizing solution are measured or estimated at the DNP conditions. However, the estimation of the dielectric loss tangent may be based on measurements made at other conditions, for example conditions near the DNP conditions where it is known that the real component of relative permittivity is analogous to that at the DNP conditions.

By providing a DNP amplifying solid material having such a dielectric loss tangent, which can be considered as low, it is provided that, when exposed to the microwave irradiation during the DNP process, the DNP amplifying solid material generates little heat, preferably less heat that the polarizing solution. This low heat generation by the DNP amplifying material is believed to be favorable for the polarization to occur.

The DNP amplifying solid material may be present under various solid forms, ranging from finely ground powder to larger particles, i.e. under the form of numerous solid particles to be dispersed in the polarizing solution, or to the form of at least one generally bigger unitary macroscopic solid at least partly immersed in the polarizing solution.

In some embodiments, the DNP amplifying solid material is present in the preparation under the form of discrete particles. These particles may be of various size, between two experiments, or even within a given experiment. For example, in some experiments, at least 60%, but preferably at least 80% of the mass of these particles have a size such as to be contained within a circumscribing sphere, the diameter of which is between 0.001 mm and 2 mm, more preferably between 0.1 mm and 0.5 mm. An optimal size of the particles is believed to be connected to the modification of the electromagnetic field when the microwave radiation is applied for the DNP process. The circumscribing sphere of an object is the sphere having the smallest diameter which can fully contain the said object.

A practical method for measuring the size of the particles, representative of their circumscribing sphere is now described in relation to Figure 1, parts (A) to (D). Figure 1 shows several steps of a method to determine particle size of KBr particles which were used in the experiments.

In part (A) is shown that a first step of the measurement method is to acquire a light microscope image of a representative amount of particles laid on a flat horizontal surface. Part (B) shows that, from the image, one acquires the fitted outlines of the particles thus observed. From these outlines, a fitted area S of the particles is derived. A distribution of the fitted area S of the particles can be determined as exemplified in part (C) of Figure

1. Also, from the fitted area S, an extrapolated particle edge length ^{a =z} Js can be determined. Part (D) of Figure 1 illustrates an example of the distribution of particle sizes where extrapolated particle edge length a is taken as an image of the particle size.

It is believed that when the DNP amplifying solid material contains discrete particles, they may have an optimal size when a significant proportion of such particles have a size connected to the wavelength of the electromagnetic field to be used for the DNP process. It is presently believed that an optimal size of the particles would be such as to be contained within a circumscribing sphere, the diameter of which is less than 2 times the wavelength of the electromagnetic field to be used for the DNP, preferably less than 0.5 times the wavelength of the electromagnetic field to be used for the DNP process. In this context, a significant proportion of the particles is believed to be at least 60%, preferably at least 80% of the mass of these particles.

In other embodiments of the invention, the DNP amplifying solid material is under the form of at least one unitary solid macroscopic structure. Such a unitary solid structure presents an advantage in being easier to handle during experiments, especially easier to separate from the polarizing solution and from the analyte after the DNP process.

Such a structure is to be at least partly immersed in the polarizing solution, so as to positively interfere in the DNP process when the microwave field is applied to cause the polarization. The one or several unitary solid macroscopic structure(s) thus form a polarizing amplifier.

Preferably, said unitary solid macroscopic structure has a size such as to be contained within a circumscribing sphere, the diameter of which is more than 0.7 mm, preferably more than 2.4 mm. These dimensions are believed to provide an optimum amplification effect on the DNP enhancement.

In some embodiments, the unitary solid structure is in the form of a macroscopic lattice structure enclosing voids. Preferably at least some of the voids have a size such as to be contained within a circumscribing sphere, the diameter of which is more than 0.01 mm, more preferably more than 0.1 mm, so as to be more easily filled with the polarizing solution and/or analyte. Preferably, the voids create an open-cell three dimensional structure so as to be porous at the macroscopic level and so that the polarizing solution and/or analyte may fill the voids in the structure.

Preferably, the unitary solid macroscopic structure is formed so as to exhibit a ratio between the volume of the DNP amplifying solid material and the volume circumscribing the macroscopic structure between 10% and 70%, more preferably between 10 % and 50%. This ratio is an image of the "porosity" of the macroscopic structure. It is also an image of the volumetric fraction of the DNP amplifying solid material in the final mixture of polarizing solution. A unitary solid macroscopic structure macroscopic lattice structure enclosing voids has the advantage of perfectly controlling the spatial dispersion of the DNP amplifying material in the solution. Especially with the ratio above, is believed to provide optimum DNP enhancement on a bigger volume of analyte, for a given capacity of a sample holder, thereby increasing the NMR signal that can be obtained from the analyte.

Preferably, the size of the unitary solid macroscopic structure, or the combined size of the several macroscopic structures used side by side in a given DNP process, is such that it spreads over a substantial portion of, preferably most of, and most preferably the totality of the experimental zone occupied by the polarizing solution in the DNP apparatus during the DNP process.

Figure 4 illustrates the basic principle for construction a unitary solid macroscopic structure 10 which be used as a polarizing amplifier in the context of the invention. The polarizing amplifier may be made a block of DNP amplifying solid material have the requisite dimension, i.e. so as to be received in the sample holder of DNP apparatus, and preferably for occupying a substantial volume in that sample holder. Preferably, this unitary solid macroscopic structure comprises voids, which may be obtained by perforations made in the block of DNP material. In the shown example, the perforations comprise cylindrical drillings 20 which cross the block of material from side to side, so as to have open extremities 30 in two opposite sides of the block of DNP material, so as to form open drillings. Preferably, each drilling has a diameter "a" of at least 0.01 mm, preferably at least 0.1 mm. The drillings may have a diameter in the order of 1mm. The drillings may be all parallel one to the other, but they are preferably perforated along at least several distinct directions, for 2 or 3 orthogonal directions. Drillings of different directions may intersect.

The drillings thus confer to the polarizing amplifier a macroscopic lattice structure (i.e. visible with the eye) having voids which, when contacted with the polarizing solution, will be filled with the polarizing solution. Preferably, the size of the voids also allows the analyte to enter in the voids.

Of course, a polarizer having such a macroscopic lattice structure could be made differently and could for example have a form resembling more that of a mesh, preferably a three-dimensional mesh.

The DNP amplifying material may comprise one or more of the following substances:

crystalline potassium bromide (KBr)

sapphire (a-AI2O3)

sodium chloride (NaCI)

calcium fluoride (CaF2)

quartz (SiO₂)

zinc selenide (ZnSe)

The DNP amplifying material may be crystalline, semi-crystalline or amorphous.

The DNP amplifying material is distinct from the polarization agent, and the polarizing agent is not bonded to the DNP amplifying solid material. The process for polarizing NMR active nuclei within an analyte of interest by dynamic nuclear polarization (DNP), involving the use of DNP amplifying material as described, can be used in view of a NMR analysis. Indeed, an analyte which will have been treated according to the process will deliver much better response to NMR analysis.

The process and the polarizing amplifier as described above may be used in an apparatus for performing a dynamic nuclear polarization (DNP) process. Such apparatus usually comprises a high frequency microwave source (usually a gyrotron or diode), an insulated cryostat or magic angle spinning probe, and a waveguide/transmission line system to transport the microwaves to the sample holder. Normally, the polarizing solution and analyte are contained within a sample holder, which is usually a sapphire rotor for MAS DNP experiments or a poly(l,l,2,2-tetrafluoroethylene) cylinder for liquid helium temperature dissolution DNP experiments. The sample holder contains the polarizing solution, which comprises a solvent and a polarizing agent, and usually the analyte to be polarized. For implementation of the process according to the invention, the DNP amplifying solid material is held in the sample holder so as to be contacted with the polarizing solution and analyte. If the DNP amplifying material is under the form of a polarizing amplifier exhibiting a unitary macroscopic structure, then this polarizing amplifier is also to be held in the sample holder.

Thanks to the inventive process an improvement in DNP enhancements can be achieved in DNP experiments, especially in MAS DNP experiments of frozen solutions, by incorporating dielectric solids into the sample holder, especially dielectric solid particles, in to the samples. In this way enhancements up to εΗ = 530 have been obtained, or 80% of the theoretical maximum. This provides a gain in DNP enhancement between 1.5 to 2.5 compared to standard experiments performed on pure bulk solvent solutions.

EXPERIMENTS All DNP experiments were carried out on a commercial Bruker Avance III 400 MHz NMR spectrometer equipped with a 263 GHz gyrotron microwave source using a 3.2 mm triple resonance MAS probe at sample temperatures around 100 K. Such an apparatus is described in "Solid-state dynamic nuclear polarization at 263 GHz: spectrometer design and experimental results", M Rosay et al. ,Phys. Chem. Chem. Phys., 2010, 12, 5850-5860, incorporated therein for reference. In general the microwave power was optimized to obtain the largest DNP enhancements, although the variation in enhancement with microwave power is discussed below for some samples. The magnet sweep coil was used to set the magnetic field so that microwave irradiation occurred at the maximum positive enhancement for a sample of TOTAPOL. Proton DNP enhancements were measured on spectra acquired with a spin echo pulse sequence with a single rotor cycle echo delay to remove probe background signals. The ¹³C cross-polarization DNP enhancement (e_c,cp) and the Proton DNP enhancement (εΗ) were measured with a standard ramped CP pulse sequence. The DNP enhancement factors e_c,cp and SH are defined as the ratio of the intensity of the NMR signals with and without microwave irradiation in the ¹³C CPMAS spectra and 1H spin echo spectra, respectively. Since in a ¹³C CPMAS experiment all of the NMR signal is derived from H nuclei, S_C,CP also provides a measure of the DNP enhancement of the proton nuclei and should be equal to ε_Η.

The polarizing preparation samples were prepared by placing a weighed amount of dry DNP amplifying material into the sapphire rotor of the spectrometer. The materials herein used as DNP amplifying solid materials were chosen based on their dielectric constant and loss tangent to cover various possible mutual relations of these two parameters. The polarizing solution was then added onto the loosely packed crystalline material with a micro-pipette and the liquid was distributed throughout the rotor by gently stirring with a syringe or copper wire. All samples were weighed to determine the precise composition of the sample. Samples were topped with a Teflon insert to minimize solution leakage from the rotors. All samples were weighed before and after performing the experiments to confirm that no loss of solution occurred. Exact compositions of the samples are given in Table 1 below.

Table 1 In Table 1: the various DNP amplifying materials were as follows:

KBr(l) : crystal diameter 0.3-0.5 mm on average (purchased from Sigma Aldrich, Inc., ref 221864);

KBr (2) : crystal diameter 0.2-0.3 mm on average (purchased from Acros Organics Inc., ref 20639);

KBr (3) : finely ground KBr (purchased from Acros Organics, Inc.)

NaCI : crystals diameter 0.3-0.5 mm on average (purchased from Sigma

Aldrich, Inc., ref S9888);

Sapphire : ground sapphire recovered from a failed 3.2 mm Bruker DNP rotor. In the experiments, the DNP amplifying solid material was in crystalline form.

Specifically, it was previously observed that repeated insertion and ejection of the rotor containing bulk TEKPol/TCE solutions led to improved MAS DNP enhancements (see Zagdoun et al., Journal of the American Chemical Society 2013, 135, 12790). One result of these cycles is to obtain better glass formation (amorphous structure) in pure TCE when frozen, since the quality of the glass formed by pure TCE is highly variable. It was observed here that glass formation in bulk TCE can be improved by addition of ~5% d₄-methanol by volume, and these conditions are used throughout. It has been observed the repeatedly inserting the sample into the cold MAS stator and ejecting the sample to the base of the probe (i.e. freeze-thaw cycling) of the sample leads to the progressive increase in the proton relaxation time 7i from 2.7 s to 3.5 s. This increase is accompanied by gradual growth of the enhancement factor from around 260 to 530 shown for TEKPOL in TCE with KBr. This result is attributed to progressive removal of dissolved oxygen from the solution since there is a pure nitrogen atmosphere inside the probe.

Samples were thus partially de-gassed inside the low temperature DNP probe by leaving them under a constant nitrogen flow from the sample eject gas for approximately 5 minutes prior to first insertion. Insert-eject cycling for each sample was performed until a constant relaxation time Ti value was measured for the *H nuclei of the solvent, in order to assess the amount of dissolved oxygen present in the polarizing solution. Of course, other degassing procedures may be employed with a view to remove the dissolved oxygen. Sample temperatures were determined by measuring ⁷⁹Br longitudinal relaxation times of crystalline KBr. The dependence of the sample temperature on the microwave power delivered to the sample was also determined by measuring the ⁷⁹Br ΤΊ values.

Results of the experiments are presented in the Table 2. In those results, the following can be noted:

- Reference samples REF1 to REF6 are samples containing only the polarizing solution, without DNP amplifying solid material. Polarizing solutions had ca. 16 mM polarizing agent concentrations;

- The DNP amplifying solid volume fraction (%) was calculated by weighing the amount of solid particles and the amount of polarizing solution added the rotor, then using densities to convert to volumes;

-The particle length was measured by optical microscopy as an image of the size of the particles, according to the methodology described above;

- The ¹³C cross-polarization DNP enhancement (e_c,cp) and the Proton DNP enhancement (ε_Η) as measured are reported in that order in the same column. If one of the two enhancements was not available, it is indicated with a dash. Values are to be considered with plus or minus 10% error.

- e_r is the real component of the relative permittivity for the DNP amplifying solid at microwave frequencies. Sample DNP Amp. Crystal Maximum Max sample ere

N° Solid Volume Length 8C,CP/sH temperature

Fraction (%) (mm) (K)

REF1 0 - 205/- 114.5 -

SP1 64 0.2-0.3 -/414 - 4,9

SP2 64 0.3-0.5 530/540 - 4.9

SP3 64 Finely 385/353 113 4.9

ground

SP4 63 0.3-0.5 491/445 - 5.9

SP5 64 0.3-0.5 391/486 - 9.6

REF2 0 - 238/- 139 -

SP6 42 0.3-0.5 -/377 112 9.6

SP7 55 341/371 111.5 6.8

REF3 0 - 58/- 108 -

SP8 46 0.3-0.5 98/87 105 9.6

REF 4 0 - 146/- - -

SP9 63 0.3-0.5 300/379 - 4.9

SP11 47 0.3-0.5 297/273 111.5 5.9

REF5 0 - 53/- 107 -

SP11 60 0.3-0.5 152/148 107 4.9

Table 2

Figure 2 illustrates some results extracted from the results of Table 2.

For some of the sample of Table 2 samples, Figure 2 shows a comparison of the proton MAS DNP enhancement factors (ε_Η) obtained for the solvent NMR resonances in experiments at 9.4 T with sample temperatures of 100 K observed on the solvent resonances for various bi-radical polarizing agents dissolved in bulk solvents, and when the solution is filled into rotors containing solid particles of either potassium bromide (KBr), sapphire (a- AI2O3), calcium fluoride (CaF₂), and sodium chloride (NaCI).

In all cases the observed enhancement is significantly higher when the solid particles are present in the sample, reaching a value as high as 540 (or nearly three times the bulk reference value) for the biradical polarizing agent TEKPol dissolved in 1,1,2,2-tetrachloroethane (TCE) mixed with potassium bromide (KBr). Furthermore, it can be seen that the effect is not limited to non-polar organic solvents such as TCE but is also observed in water-based systems, as exemplified by the AMUPol biradical¹⁶ dissolved in 60:30:10 glycerol-£¾/D₂0/H₂0 mixed with ground sapphire. Also, it is clear that the effect does not change significantly from one polarizing agent to another, as the amplification of the DNP enhancement is roughly the same when comparing bulk solutions of TEKPol, AMUPol, bCTbK or bTbK and solutions dispersed on the DNP amplifying solid materials. Similar results are obtained with other DNP amplifying solid materials such as NaCI or CaF₂.

In this light, there are several mechanisms that might explain the effects observed here. The first would be that the solids used here are more transparent to microwaves than the solutions, and therefore that better overall microwave penetration into the samples is obtained when they are mixed with the solid particles. The second is that the structure caused by the solid particles concentrates the microwaves in the regions containing the frozen solutions. A third explanation could be that bulk sample heating is reduced in the samples containing the particles due to less microwave absorption.

Figure 3a shows the variation, as a function of the applied microwave power, in proton enhancement factor for the solvent resonance for 16 mM/l TEKPol in TCE:methanol-<¾ (94:6 v/v) either in bulk or in presence of KBr crystals. Enhancement factors of the sample containing KBr are systematically higher than in bulk solution for all the microwave powers used.

Figure 3b shows the variation, as a function of the applied microwave power, in sample temperature resonance for 16 mM TEKPol in TCE:methanol- <¾ (94:6 v/v) either in bulk or. in presence of KBr crystals. Sample temperature was measured by spin-lattice relaxation rate of ⁷⁹Br (in case of bulk solutions a small amount of KBr was placed at the bottom of the rotor). The lines are guides for the eye. Figure 3b shows that the sample temperatures are quite similar.

In both cases this confirms that the difference in enhancement observed between the bulk and the heterogeneous samples does not arise from differences in overall sample temperature that would lead to higher CE DNP performance at lower temperatures. Figure 3b also shows that, in contrast to the solutions, a sample of pure ground KBr does not change temperature significantly over this range of powers. This suggests that the samples including KBr are indeed more transparent to microwaves, suggesting that both of the first two mechanisms discussed above may play a role.

The invention is not limited to the described examples in view of the various modifications which can be easily derived therefrom without departing from the ambit of the invention.

Claims

1 - Process for polarizing NMR active nuclei within an analyte of interest by dynamic nuclear polarization (DNP), said process comprising the steps of:

a. providing a polarizing solution containing a polarizing agent in a solvent,;

b. contacting the analyte of interest with the polarizing solution;

c. submitting the polarizing solution to DNP conditions including application of an electromagnetic field in the microwave range of 9 to 789 GHz, preferably 80 to 600 GHz, before, during or after contacting the analyte of interest with the polarizing solution,

2 - Process for polarizing NMR active nuclei according to claim 1, characterized in that the DNP amplifying solid material has a real component of the relative permittivity at least two units larger than that of the polarizing solution, preferably at least three units higher than that of the polarizing solution.

3 - Process for polarizing NMR active nuclei according to claim 2, characterized in that the DNP amplifying solid material is distinct from the analyte.

4 - Process for polarizing NMR active nuclei according to claim 3, characterized in that the analyte is distinct from the solvent, from the polarizing agent and from the DNP amplifying solid material. 5 - Process for polarizing NMR active nuclei according to any preceding claim, characterized in that the polarizing agent is not bonded to the DNP amplifying solid material.

6 - Process for polarizing NMR active nuclei according to any preceding claim, characterized in that the DNP amplifying solid material has a real component of the relative permittivity higher than 3, preferably higher than 4.5.

7 - Process for polarizing NMR active nuclei according to any preceding claim, characterized in that the DNP amplifying solid material has a dielectric loss tangent is lower than that of the polarizing solution.

8 - Process for polarizing NMR active nuclei according to claim 7, characterized in that the DNP amplifying solid material has a dielectric loss tangent less than 0.01, preferably less than 0.002.

9 - Process for polarizing NMR active nuclei according to any preceding claim, characterized in that the DNP amplifying solid material is present in the preparation under the form of discrete particles.

10 - Process for polarizing NMR active nuclei according to claim 9, characterized in that at least 80% of the mass of the DNP amplifying solid material particles have a size such as to be contained within a circumscribing sphere, the diameter of which is between 0.001 mm and 2 mm, more preferably between 0.1 mm and 0.5 mm.

11 - Process for polarizing NMR active nuclei according to any preceding claim, characterized in that the DNP amplifying solid material is dispersed in the polarizing solution under the form of discrete particles, and in that at least 80% of the mass of these particles have a size such as to be contained within a circumscribing sphere, the diameter of which is less than 2 times the wavelength of the electromagnetic field to be used for the DNP, preferably less than 0.5 times the wavelength of the electromagnetic field to be used for the DNP.

12 - Process for polarizing NMR active nuclei according to any preceding claim, characterized in that the DNP amplifying solid material is under the form of at least one unitary solid macroscopic structure. 13 - Process for polarizing NMR active nuclei according to claim 12, characterized in that the unitary solid macroscopic structure has a size such as to be contained within a circumscribing sphere, the diameter of which is more than 0.7 mm, more preferably more than 2.4 mm, said unitary solid macroscopic structure being at least partly immersed in the polarizing solution.

14 - Process for polarizing NMR active nuclei according to any of claim 12 or 13, characterized in that the unitary solid structure is in the form of a macroscopic lattice structure enclosing voids.

15 - Process for polarizing NMR active nuclei according to claim 14, characterized in that at least come voids of the unitary solid structure have a size such as to be contained within a circumscribing sphere, the diameter of which is more than 0.01 mm, more preferably more than 0.1 mm.

16 - Process for polarizing NMR active nuclei according to any of claims 14 or 15, characterized in that the unitary solid macroscopic structure is formed so as to exhibit a ratio between the volume of the DNP amplifying solid material compared to the volume circumscribing the macroscopic structure which is comprised between 10% and 70%, more preferably between 10 % and 50%.

17 - Process for polarizing NMR active nuclei according to any preceding claim, characterized in that the DNP amplifying solid material comprises one or more of the following substances :

crystalline potassium bromide (KBr);

sapphire (a-AI203);

sodium chloride (NaCI);

calcium fluoride (CaF2);

quartz (Si0₂);

zinc selenide (ZnSe).

18 - Process for polarizing NMR active nuclei according to any preceding claim, characterized in that the polarizing agent comprises at least one free radical having unpaired electrons. 19 - Process for polarizing NMR active nuclei according to claim 18, characterized in that the polarizing agent comprises at least two free radicals having unpaired electrons.

20 - Process for polarizing NMR active nuclei according to any preceding claim, characterized in that, prior to the step of submitting the polarizing solution to DNP conditions, the process comprises a step of generating unpaired electrons in the polarizing agent by submitting the polarizing solution to an energy field, for example to UV radiation, other ionizing radiation, heat, electrical discharges, electrolysis, and/or chemical reactions.

21 - Process for polarizing NMR active nuclei, characterized in that the polarizing agent comprises one or more of the following substances :

TEMPO (2,2,6,6-Tetramethylpiperidin-l-oxyl)

TOTAPOL (l-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol)

AMUPol

TEKPol (bis-phenylcyclohexyl-TEMPO-bisketal)

bCTbK (bis-cyclohexyl-TEMPO-bisketal)

bTBK (bis-TEMPO-bisketal)

Functionalized derivatives of triphenylmethyl radicals

22 - Process for polarizing NMR active nuclei according to any preceding claim, characterized in that the polarizing agent is not bonded to the DNP amplifying solid material.

23 - Process for polarizing NMR active nuclei according to any preceding claim, characterized in that the solvent comprises an organic solvent.

24 - Process for polarizing NMR active nuclei according to any preceding claim, characterized in that the solvent comprises one or more of the following substances :

Dimethyl sulfoxide (DMSO) with deuterium oxide (D2O) and water. 1,2-dichlorobenzene 1,1,2,2-tetrabromoethane

1,4-dibromobutane

pyruvic acid

25 - Process for analyzing an analyte of interest by nuclear magnetic resonance (NMR), the process comprising the step of polarizing NMR active nuclei of the analyte by dynamic nuclear polarization (DNP) according to claims 1 to 24.

26 - Polarizing amplifier suitable for a dynamic nuclear polarization (DNP) process, characterized in that:

it comprises at least one unitary solid macroscopic structure comprising of a DNP amplifying solid material, the unitary solid macroscopic structure having a size such as to be contained within a circumscribing sphere, the diameter of which is more than 0.7 mm, more preferably more than 2.4 mm.

the DNP amplifying solid material has a real dielectric constant higher than 3, preferably higher than 4.5.

27 - Polarizing amplifier according to claim 26, characterized in that the DNP amplifying solid material has a dielectric loss tangent less than 100, preferably less than 20.

28 - Polarizing amplifier according to claim 26 or 27, characterized in that the unitary solid structure is in the form of a macroscopic lattice structure enclosing voids having a size such as to be contained within a circumscribing sphere, the diameter of which is more than 0.7 mm, more preferably more than 2.6 mm.

29 - Polarizing amplifier according to claim 28, characterized in that the unitary solid macroscopic structure is formed so as to exhibit a ratio between the volume of the DNP amplifying solid material and the volume circumscribing the macroscopic lattice structure comprised between 10% and 70%, preferably between 10% and 50%, more preferably comprised between 10% and 30%.

30 - Apparatus for performing a dynamic nuclear polarization (DNP) process comprising: a microwave source;

an insulated cryostat or magic angle spinning probe;

a sample holder for receiving a polarizing solution comprising a solvent and a polarizing agent to be polarized;

a waveguide/transmission line system to transport the microwaves to the sample holder,

characterized in that a polarizing amplifier according to claims 26 to 29 is provided in the sample holder so as to be contacted by a polarizing solution.