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WO2020208103A1 - Système d'évaluation de molécules hyperpolarisées dans un échantillon biologique - Google Patents

Système d'évaluation de molécules hyperpolarisées dans un échantillon biologique Download PDF

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Publication number
WO2020208103A1
WO2020208103A1 PCT/EP2020/060086 EP2020060086W WO2020208103A1 WO 2020208103 A1 WO2020208103 A1 WO 2020208103A1 EP 2020060086 W EP2020060086 W EP 2020060086W WO 2020208103 A1 WO2020208103 A1 WO 2020208103A1
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Prior art keywords
sample
sensor
detection
spin moments
substrate
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PCT/EP2020/060086
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English (en)
Inventor
Philipp Neumann
Ilai SCHWARTZ
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Nvision Imaging Technologies Gmbh
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Publication of WO2020208103A1 publication Critical patent/WO2020208103A1/fr

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    • 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
    • G01N24/10—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using electron paramagnetic resonance
    • 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
    • G01N24/08—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
    • G01N24/088—Assessment or manipulation of a chemical or biochemical reaction, e.g. verification whether a chemical reaction occurred or whether a ligand binds to a receptor in drug screening or assessing reaction kinetics
    • 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
    • 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
    • G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
    • G01R33/445—MR involving a non-standard magnetic field B0, e.g. of low magnitude as in the earth's magnetic field or in nanoTesla spectroscopy, comprising a polarizing magnetic field for pre-polarisation, B0 with a temporal variation of its magnitude or direction such as field cycling of B0 or rotation of the direction of B0, or spatially inhomogeneous B0 like in fringe-field MR or in stray-field imaging
    • 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
    • G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
    • G01R33/46—NMR spectroscopy
    • G01R33/465—NMR spectroscopy applied to biological material, e.g. in vitro testing
    • 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
    • G01R33/30—Sample handling arrangements, e.g. sample cells, spinning mechanisms
    • G01R33/302—Miniaturized sample handling arrangements for sampling small quantities, e.g. flow-through microfluidic NMR chips
    • 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
    • G01R33/32—Excitation or detection systems, e.g. using radio frequency signals
    • G01R33/323—Detection of MR without the use of RF or microwaves, e.g. force-detected MR, thermally detected MR, MR detection via electrical conductivity, optically detected MR

Definitions

  • the invention relates to a method for analysing a sample by Nuclear Magnetic Resonance (NMR) spectroscopy, the method comprising the steps of: Providing a magnet, at least one antenna element and a sensor, wherein the sensor comprises a substrate with a multiplicity of detection spin moments in a detection area; Submitting at least part of the sample to polarising of its nuclear spins; Placing at least a part of the sample that has been submitted to polarisation of its nuclear spins in the proximity of the sensor; Generating, by means of a magnet, a magnetic field permeating the sample; Radiating, by means of the at least one antenna element, radio frequency pulses for influencing the nuclear spin moments in the sample and/or influencing the detection spin moments in the substrate; and Acquiring, by means of the sensor, an NMR signal from the sample.
  • NMR Nuclear Magnetic Resonance
  • the invention moreover relates to a device for analysing a sample by NMR spectroscopy, the device comprising a magnet, at least one antenna element and a sensor, wherein the sensor comprises a substrate with a multiplicity of detection spin moments in a detection area.
  • Magnetic detection and imaging devices are important tools in biological and material sciences applications.
  • methods for detecting nuclear magnetic resonance (NMR) have high spectral resolution of the nuclear magnetic resonance spectra generated.
  • NMR nuclear magnetic resonance
  • the nuclear spin moments of a (material) sample are exposed to a strong static magnetic field, as a result of which the nuclear spin moments are excited to effect a precessing movement, so-called Larmor precession.
  • the precession or Larmor frequency is specific to the respective chemical nuclear spin species, and is influenced by the chemical and spatial environment of a respective nuclear spin moment.
  • Inductive methods are often used for detecting nuclear magnetic resonance signals.
  • an induction coil extending around the sample is used, for example, wherein the precessing nuclear spin moments generate alternating magnetic fields that induce an electrical voltage as nuclear magnetic resonance signal in the turns of the induction coil.
  • Such inductive methods for generating nuclear magnetic resonance spectra are suitable in particular for detecting a large magnetic moment (magnetization), such as is generated in particular by a large number of nuclear spin moments aligned (polarized) in the same way.
  • the induced voltage that is to say the nuclear magnetic resonance signal, is proportional to the change over time in a magnetic flux permeating the induction coil.
  • the magnetic flux is substantially proportional to the number of magnetic flux lines which pass through the interior of the induction coil. If the diameter of the induction coil is reduced, the number of flux lines passing through and hence the amplitude of the induced voltage are reduced. In this case, the minimum diameter of the induction coil is typically restricted to the millimetre scale in order to avoid self-induction effects.
  • a detection region in this case should be understood to mean, in particular, a spatial interaction region which permeates the sample and in which nuclear spin moments present bring about a sufficient influencing of the detection spin moment by means of dipolar interactions, said influencing being detectable or measurable. Since the interaction strength of the dipolar interactions scales with the inverse cubic distance (r 3 ), small detection regions of a few cubic nanometres (nm 3 ) and hence high spatial resolutions can be realized.
  • NV centre nitrogen- vacancy centre
  • the detected nuclear magnetic resonance signal is brought about by a statistical polarization of the nuclear spin moments of the sample, that is to say a random net alignment or magnetization of the nuclear spin moments in the detection region.
  • the nuclear magnetic resonance spectra generated have comparatively low spectral (frequency) resolutions with line widths in the kilohertz range (kHz). In this case, the spectral resolution is substantially limited by the comparatively low relaxation time of the detection spin moments.
  • a sequence of radio-frequency pulses is radiated onto the detection spin moment. Afterwards, a signal of the magnetization that occurs in the detection region is detected and stored as detection result in a list.
  • the detection is continuously repeated during a long measurement time (> 1000 s) such that a long list of detection results is generated.
  • the list is subsequently evaluated for example by means of autocorrelation of the detection results and subsequently Fourier-transformed in order to generate the nuclear magnetic resonance spectrum. In this case, the occurring
  • the sequence impresses on the detection spin moment a phase that is dependent on the initial state of the (statistical) magnetization.
  • the sequence substantially acts only as a frequency filter with which the sensitivity of the detection spin moment is set to the frequency range of the Larmor frequencies of the nuclear spin moments.
  • the phase is detected as a detection result, for example as a number of photons emitted by the detection spin moment by means of a photon detector, is stored in the list and synchronized with the external local oscillator before the sequence is performed anew.
  • the spectral resolution of the Qdyne method is substantially only limited by the stability of the local oscillator, as a result of which nuclear magnetic resonance spectra with millihertz (mHz) or microhertz (mHz) frequency resolution or line width in conjunction with spatial resolution in the nanometres range are made possible.
  • the spectral resolution is limited on account of the finite dimensions of the detection region.
  • the molecular diffusion in particular of the translational and rotational diffusion, of the nuclear spin moments in liquid samples, the dipolar interactions of the nuclear spin moments among one another, or nuclear spin moments of different molecules within the sample, are effectively reduced, as a result of which narrower nuclear magnetic resonance spectra with higher resolution are made possible.
  • the problem simultaneously occurs that, in particular on account of the translational diffusion, nuclear spin moments or molecules diffuse out of the detection region during the measurement time and new nuclear spin moments diffuse into the detection region.
  • the phase and/or amplitude of the statistical magnetization are/is altered, and the detection results are thus influenced, as a result of which the line widths of the nuclear magnetic resonance spectrum generated are widened, such that the spectral resolution is disadvantageously reduced.
  • the Qdyne method can also be used to detect a thermal, or preferably, a thermal or a hyperpolarized signal from the sensing volume, similar to microscale NMR inductive coils.
  • a thermal signal might take several hours of integration time for achieving a high signal-to-noise ratio, if the molecules are hyperpolarized before the NMR detection, a significant time and SNR enhancement can be achieved.
  • the concentration sensitivity achieved is considerably too low.
  • the method for spin polarization is unspecific in that it enhances the polarization of all nuclear spin species (1 H and 19F in the case presented above) on all molecules and not only selected ones.
  • Neoplasia 21.1 (2019): 1- 16 many hyperpolarized metabolites can serve as probes for the study and diagnostics of cancerous tumours, including [1- 13 C]pyruvate, have been successfully polarized and used to study multiple biological processes , including [2- 13 C] pyruvate, [1- 13 C] acetate, [1- 13 C]alanine, 13 C urea/[ 13 C, 15 N2]urea, perdeuterated [U- 13 Ce]glucose, [ 13 C]bicarbonate, [1 ,4- 13 C2]fumarate, and several others.
  • the most studied hyperpolarized metabolite probe so far is [1- 13 C]pyruvate, where upon the injection of hyperpolarized pyruvate to the tissue, the detected pyruvate to lactate conversion ratio has a strong correlation to the aggressiveness of the tumour and can therefore be used for tumour characterization and diagnostics as well as treatment assessment.
  • a main limitation with the state of the art technology is the large volume of tissue needed for achieving a high signal-to-noise ratio (SNR) of the SNR.
  • the required number of cells for an SNR larger than 10 for the lactate signal is > 100,000 cells.
  • the invention aims at providing an improved method and apparatus for analysing a biological sample such as a sample comprising one or more biological cell(s). In some aspects, the invention aims at reducing the size of the sample needed for analysis, for example the number of biological cells needed. In particular, the invention in some aspects aims at providing a method and a device that can be used in applications where traditional NMR inductive coils are used, but requires a considerably smaller sample volume. Moreover, in some aspects, the invention aims at improving the concentration sensitivity of the method and the apparatus for analysing a sample by NMR spectroscopy.
  • any reference to one (including the articles“a” and“the”), two or another number of objects is, provided nothing else is expressly mentioned, meant to be understood as not excluding the presence of further such objects in the invention.
  • the reference numerals in the patent claims are not meant to be limiting but merely serve to improve readability of the claims.
  • the problem is solved by a method for analysing a sample by NMR spectroscopy according to claim 1.
  • the method comprises the steps of: Providing a magnet, at least one antenna element and a sensor, wherein the sensor comprises a substrate with a multiplicity of detection spin moments in a detection area; Submitting at least part of the of the sample to polarising of its nuclear spins by means of parahydrogen induced polarisation; Placing at least a part of the sample that has been submitted to polarisation of its nuclear spins in the proximity of the sensor; Generating, by means of a magnet, a magnetic field permeating the sample, the magnetic field preferably being static; Radiating, by means of the at least one antenna element, radio frequency or microwave pulses for influencing the nuclear spin moments in the biological sample and/or influencing the detection spin moments in the substrate, and Acquiring, by means of the sensor, an NMR signal from the sample.
  • the acquisition step of the method exploits the fact that the detection spin moments can serve as magnetic field sensors in the sense that nuclear spin moments of the sample influence the detection spin moment, preferably by means of dipolar interactions, said influencing being detectable or measurable.
  • placing the sample in the proximity of the sensor means that the sample is placed within the detection region of at least part of the detection spin moments of the detection area so that at least part of the nuclear spin moments of the sample influence least this part of the detection spin moments sufficiently to be detectable or measurable.
  • the sample can be any substance of interest. In particular, as explained below, it can be a biological substance or a metabolite of a biological cell.
  • polarization is defined according to the standard definition: the number of nuclear spins in the preferred direction minus the number in the opposite direction, divided by the total number of nuclear spins.
  • parahydrogen is the isomeric form of molecular hydrogen, H2, in which the nuclear spins of the two protons of the hydrogen molecule are aligned in an antiparallel fashion. This aspect of the invention exploits the fact that with parahydrogen, a high polarisation can be achieved.
  • the problem is solved by a method for analysing a sample, the sample comprising a metabolite that can be converted by one or more biological cell(s), a derivative that results from the metabolisation of a metabolite by the biological cell(s), and/or a biological material, by NMR spectroscopy according to claim 2.
  • the method comprising the steps of: Providing a sensor, a magnet and at least one antenna element, wherein the sensor comprises a substrate with a multiplicity of detection spin moments in a detection area;
  • a biological material is a material that can interact with with a cell.
  • the biological material can be internalised into the cell, in other embodiments, the biological material interacts with the cell surface.
  • the biological material is endogenous to living tissues, such as amino acids, proteins, metabolites, RNA, DNA and others.
  • the biological material can be a nanoparticle with hyperpolarized nuclear spins attached to a molecule which interacts with the cell, for example a protein that binds to a receptor on the cell membrane.
  • the term“cell” is meant to encompass, beyond conventional biological cells, all kinds of biological tissues and biological organisms, microorganisms and infectious agents such as viruses, bacteria, protozoans, prions, viroids, and fungi.
  • Selectively polarised in the context of the present invention, means that the at least part of the metabolite or the biological material is submitted to a different polarising than at least part of the other material in the proximity of the sensor, in particular preferably than at least part of the biological cell(s).
  • the at least part of the metabolite or the biological material can be distinguished from the at least part of the other material in the proximity of the sensor, in particular from the at least part of the biological cell(s).
  • An example for a selective polarising is the polarising of the at least part of the metabolite or the biological material to a higher degree than the other material in the proximity of the sensor, for example a polarising of the at least part of the metabolite or the biological material to at least 0.1 %, more preferably at least 0.3 %, more preferably at least 1 %.
  • the at least part of the metabolite or the biological material is polarised and after polarisation added to the cell(s) in order to allow it to interact with the cell(s), for example being metabolised by the cell(s).
  • Selectively isotopically labelled in the context of the present invention, means that in the at least part of the metabolite or the biological material the abundance of at least one isotope the nuclear spin moments of which can affect the NMR signal is different to the abundance in at least part of the other material in the proximity of the sensor, in particular preferably from at least part of the biological cell(s).
  • An example for a selectively isotopic labelling is an enriching of an isotope, for example 13 C, in the at least part of the metabolite or the biological material above natural abundance, while such enriching is not or only to a lesser degree performed in the other material in the proximity of the sensor, in particular the biological cell(s).
  • the metabolite, its derivative or the biological material can be selectively detected.
  • the metabolism of the cells can be studied. From this, for example, tissue characteristics of the sample can be derived.
  • a derivative of a metabolite is a product that results from the biological cell’s metabolisation of the metabolite.
  • the problem is solved by a device for analysing a sample by NMR spectroscopy according to claim 14.
  • the device comprises a magnet, at least one antenna element and a sensor, wherein the sensor comprises a substrate with a multiplicity of detection spin moments in a detection area characterised in that the device further comprises a controller configured to carry out the above method.
  • a controller is a unit which is configured, by means of programming and/or circuitry, for carrying out the generating, radiating and acquiring steps of the method described above.
  • the controller comprises a microcontroller with a processor and a data memory, programmed in a way to perform these steps.
  • a device for analysing a sample by NMR spectroscopy comprises a magnet, at least one antenna element and a sensor, wherein the sensor comprises a substrate with a multiplicity of detection spin moments in the detection area characterised in that the device further comprises bioreactor.
  • a bioreactor is an apparatus for keeping the biological cells of the sample viable, ie, for ensuring that the cells are in a state in which they can metabolise the metabolite.
  • the invention can advantageously be used for detecting characteristics of biological tissue by sensing the hyperpolarized NMR signal from metabolites of biological cells, tissue or organisms, in particular by analysing the conversion of a polarized metabolite into a derivative.
  • the hyperpolarized nuclear spin in the metabolite or biological molecule can be of any species with a nuclear spin, including but not limited to 13 C, 15 N, 1 H, 19 F, 31 P, and 129 Xe.
  • the nuclear spins polarised are low gamma nuclear spins, such as 13 C nuclear spins in particular.
  • 13 C nuclear spins have a much larger chemical shift splitting for the molecules of interest than 1 H nuclear spins, providing the necessary information for distinguishing the signal originating from a metabolite from the signal originating from its derivative(s), therefore enabling the quantification of the rate of conversion.
  • 13 C nuclear spins have a significantly longer relaxation time than 1 H nuclear spins for the molecules of interest (eg, for pyruvate the 13 C relaxation time is on the order of 30 to 60 seconds, while 1 H relaxation is on the order of 1 to 3 seconds).
  • the polarization has to be orders of magnitude higher than that described in the state of the art when combined with detection via electron spin moments, such as NV sensing.
  • the polarization of the nuclear spin preferably is at least 0.1%, more preferably at least 1 %, more preferably at least 10 %.
  • the invention can make use of the fact that such polarization levels can be achieved when using polarization methods developed for large volume NMR, usually for MR imaging.
  • At least part of the sample preferably the metabolite or biological molecule, is hyperpolarised.
  • “Hyperpolarisation” means polarisation well above the thermal equilibrium.
  • the degree of polarisation is above 1 %, more preferably above 10 %, more preferably above 30 %.
  • the metabolite or biological molecule is isotopically altered such that the nuclear spin to be polarised has a higher than natural abundance over other isotopes of that same element, which do not contain the desired nuclear spin in high concentrations.
  • the metabolite or biological molecule has both a higher concentration of the target nuclear spin as well as a higher signal from the hyperpolarisation. This therefore increases further the selectivity of the detected signal.
  • 13 C enrichment the case of 13 C enrichment
  • hyperpolarised metabolite or biological molecule preferably has at least 10 % 13 C enrichment at the desired carbon position, more preferably at least 50 %, more preferably at least 90 %, more preferably at least 99 % 13 C carbon enrichment.
  • the polarization preferably the hyperpolarisation, of at least part of the sample, preferably the metabolite, is performed by means of parahydrogen.
  • parahydrogen advantageously, very high polarization can be achieved.
  • the method of polarisation is selected form a variety of different polarization sources which do not rely on electron spin polarization, including parahydrogen induced polarization (PHIP), parahydrogen induced polarization by sidearm hydrogenation (PHIP-SAH), Signal amplification by reversible exchange (SABRE) and SABRE-RELAY.
  • PHIP parahydrogen induced polarization
  • PHIP-SAH parahydrogen induced polarization by sidearm hydrogenation
  • SABRE Signal amplification by reversible exchange
  • SABRE-RELAY SABRE-RELAY
  • the hyperpolarisation is performed by transferring the polarisation of hyprepolarised electron spins, for example via spin exchange optical pumping (SEOP) or dynamic nuclear polarization (DNP) including dissolution DNP or optical methods.
  • SEOP spin exchange optical pumping
  • DNP dynamic nuclear polarization
  • a preferred method comprises analysing a sample, the sample comprising a metabolite that can be converted by a biological cell, a derivative that results from the metabolisation of a metabolite by a biological cell and/or a biological material of the cell, by NMR spectroscopy.
  • an NMR signal from the hyperpolarised metabolites and/or its derivative or the biological material is acquired.
  • the preferred substrate is a solid or multiple solids, preferably a diamond or multiple diamonds, preferably one or multiple diamond chip(s) or microdiamond(s).
  • the preferred diamond(s) is/are isotopically purified with regard the carbon atoms.
  • the presence of 13 C in the diamond lattice of the diamond(s) or the detection area(s) can be reduced to below the concentration of 3 M for natural abundance of 13 C.
  • the substrate has a 13 C concentration below that of natural abundance in diamond ( ⁇ 3 M), more preferably less than 300 mM, more preferably less than 30 mM, more preferably less than 3 mM.
  • the detection spin moments are located in a detection area of the substrate.
  • the detection spin moments and thus the detection area are arranged suitably near the surface of the substrate, in particular at a distance of a few nanometres to micrometres from the surface which the sample is in contact with or which is nearest to the sample.
  • This surface area of the detection area is in the following referred to as the“sensing area”.
  • the sensing area is at least 1 micrometres squared, ie, (1 pm) 2 , more preferably at least (10 pm) 2 , more preferably at least (50 pm) 2 in size.
  • the sample preferably is placed in proximity to the detector, more preferably the sensing area, preferably closer than 10 millimetres (mm), more preferably closer than 1 mm, more preferably closer than 100 micrometres (pm).
  • the detection spin moments are present below the substrate surface and below the sensing area in a layer that forms the detection area, the layer having a thickness of at least 1 pm, more preferably at least 10 pm, and potentially even 100 pm.
  • the invention also encompasses embodiments in which the substrate contains several detection areas and/or sensing areas or spots of a single detection area or sensing area where NMR spectroscopy investigation of tissue can be performed. Potentially NMR spectroscopy of sample in the different spots can be performed in parallel.
  • the desired concentration of detection spin moments is preferably of a density of at least 10 parts per billion (ppb), more preferably at least 100 ppb, more preferably at least 1 parts per million (ppm), more preferably at least 10 ppm.
  • the detection spin moments are electron spin moments, more preferably electron spin moments of colour centres of the substrate.
  • A“colour centre” is a point defect in a crystal lattice, in which a vacancy is filled by one or more electrons. The defect can absorb light, preferably light in the visible range.
  • a particularly preferred colour centre is a nitrogen-vacancy centre (NV centre). In a NV centre, a nitrogen atom
  • An NV centre has a spin-1 electron spin moment having a ground state with zero field splitting of 2.87 GHz between a non-magnetic state ("0") and the associated magnetic states ("+1", "-1").
  • the NV centre can be polarized into the non-magnetic ground state ("0”) by irradiating it with green light, for example from a laser.
  • the states can be manipulated by means of radio-frequency pulses in the microwave and/or radiofrequency range.
  • the step of acquiring the NMR signal comprises illuminating the detection spin moments, preferably the NV centres, in the substrate, preferably periodically. Illumination may serve initialization, readout of the detection spin moments, preferably the NV centres, and auxiliary purposes la, the detection spin moments can be illuminated to be initialised into a defined state.
  • NV centres for example, their spin moments can be polarized into the non-magnetic state ("0”) by means of irradiating green light.
  • NV centres for example, their spin moments can be manipulated by microwave and/or radiofrequency fields during periods without illumination.
  • a layer is introduced between the detection spin moments in the substrate and the sample under investigation to block the light that is necessary for initialization, readout of the detection spin moments, preferably the NV centres, and auxiliary purposes.
  • a layer can include for example a metal layer, plasmonic structures, dielectric reflectors or absorbers.
  • the illumination of the substrate, in particular a diamond, contacting the detection spin moments, in particular NV centres, can be performed such that the laser light is totally internal reflected, however, the evanescent field will leak out into the direction of the sample under investigation and can lead to unwanted optical excitations.
  • a thick sacrificial layer eg dielectric material with sufficiently lower refractive index than diamond
  • the spin states of the detection spin moments preferably the detection spin states of NV centres, are read out optically, via the very established Optically Detected Magnetic Resonance (ODMR) detection.
  • ODMR Optically Detected Magnetic Resonance
  • NV centres In the case of NV centres, this exploits the fact that upon excitation NV centres emit light in the red wavelength range, wherein the number of photons is dependent on the spin state of the electron spin moment before the irradiation. In other words, the state of the electron spin moment of the NV centre is optically readable by detecting the emitted photons, providing a particularly simple detection of the signal of the transverse magnetization.
  • a photon detector a CCD sensor can be used, for example.
  • detection electrodes are placed on the substrate for electrical readout of the spin states of the detection spin moments, preferably the NV centres’ spin states, as demonstrated, for example, by Hrubesch, Florian M, et al in "Efficient
  • intermediate energy level electrons can be excited to move into the conduction band.
  • PDMR Photoelectrically Detected Magnetic Resonance
  • a high SNR can be reached in the sensing areas.
  • the spin states of the detection spin moments are read out magnetically by means of one or more Electron Paramagnetic Resonance (EPR) coil(s).
  • EPR Electron Paramagnetic Resonance
  • This method exploits the fact that very small EPR coils can be manufactured , thereby maximizing the signal from the NV center ensemble in the detection area.
  • An advantage of EPR detection of NV states is that due to the weak coupling of the NV centers to the detectors, the EPR readout is non destructive.
  • NV measurements typically include sequences with multiple refocusing echos, including XY-N, KDD-N, where after each such echo an EPR signal can be detected.
  • multiple readouts can be performed during the measurement sequence using EPR readout, preferably more than 10, more preferably more than 100, more preferably more than 1000.
  • a preferred hyperpolarized metabolite is selected from the group comprising [1- 13 C]pyruvate, [2- 13 C]pyruvate, [1- 13 C]acetate, [1- 13 C]alanine, 13 C urea/[ 13 C, 15 N 2 ]urea, perdeuterated [U- 13 C 6 ]glucose, [ 13 C]bicarbonate and [1 ,4- 13 C2]fumarate.
  • other hyperpolarised metabolites may be used.
  • a preferred sample comprises at least one biological cell.
  • the number of cells comprised in the sample is smaller than 100 000, preferably smaller than 30 000, preferably smaller than 10 000.
  • a particularly preferred number of cells is two thousands and below, more preferably two hundreds and below, more preferably ten and below, more preferably down to even an individual cell.
  • the cells can metabolise the metabolite, more preferably yielding one or more derivatives of the metabolite, and/or the cells can internalise the biological material.
  • a preferred device comprises a bioreactor for keeping the biological cell or cells of the sample viable, ie, for ensuring that the cell(s) are in a state in which they can metabolise the metabolite during - and, preferably, also following - the NMR spectroscopy. Accordingly, in a preferred method according to the invention, the step of placing the sample in the proximity of the sensor comprises placing the sample in the bioreactor.
  • the bioreactor allows altering the solution in which the cell(s) is/are kept (including physiological solutions, the addition of drugs and metabolites).
  • the bioreactor allows altering the temperature and pressure of the cell(s) in solution (including physiological temperatures and pressures).
  • the preferred bioreactor allows adjusting optical illumination of the cell(s) in solution (includes changing the wavelength and intensity of light, suppressing any illumination).
  • the cell(s) is/are exposed to the hyperpolarized biological material or metabolite, and via the sensor the signal from the hyperpolarized biological material, metabolite and/or its derivatives.
  • the biological material, the metabolite or a substance of interest is polarized before coming into contact with the rest of the sample, for example the biological cell(s) investigated according to the invention.
  • this is achieved by injecting the polarised biological material, metabolite or substance of interest into the bioreactor in which the rest of the sample, for example the biological cell(s) is/are already present.
  • a preferred method according to the invention comprises a step of placing a part of sample that is not submitted to polarising of its nuclear spins in the proximity of the sensor, this step occurring before the step of placing at least a part of the sample that has been submitted to polarisation in the proximity of the sensor.
  • the polarization is performed in close proximity to the sensor. It is an achievable advantage of this embodiment of the invention that the polarization does not significantly decay before coming into contact with the biological tissue to be investigated.
  • the metabolite or biological material is polarized after coming into contact with the rest of the sample.
  • the metabolite can still be selectively polarized even in these conditions.
  • selectivity can be achieved when using PHIP, where the 13 C polarization depends on the feasibility of hydrogenating the molecule, as well as on the specific J coupling between the two 1 H nuclei resulting from the hydrogenation and the 13 C nuclear spin.
  • the sample is a tissue containing cancerous cells, such as from cell culture or from biopsies. It is an achievable advantage of this embodiment of the invention that using the invention, a real-time analysis of the metabolic flux in this tissue can be obtained.
  • a hyperpolarised metabolite is used, preferably as a probe.
  • hyperpolarized [1- 13 C]pyruvate is injected into the tissue.
  • the detected pyruvate to lactate conversion ratio has a strong correlation to the aggressiveness of the tumour. Therefore, advantageously, the detected pyruvate to lactate conversion ratio can be used for tumour characterization and diagnostics and/or treatment assessment.
  • tumour biopsies are characterised and of potential treatments for the tumour are assessed. It is an achievable advantage of this embodiment of the invention that a treatment assessment for the biopsy sampled from the patient enables to target the treatment individually to the patient, a main hallmark of precision medicine.
  • biopsies are typically only a few millimetres in diameter and contain ⁇ 100,000 cancer cells, with current NMR sensitivities the entire biopsy will have to be used in the hyperpolarized NMR analysis, and even that might not be sufficient for a sufficiently high SNR from the hyperpolarized metabolite derivatives.
  • slices of the tumour containing much fewer cells are sufficient for the NMR analysis. Thereby, it is achievable that different tumour slices for investigations of various drugs are use, potentially in parallel. It is also achievable that other parts of the tumour can be used for diagnostics such as histology or DNA sequencing.
  • the preferred signal-to-noise ratio (SNR) of the hyperpolarized metabolite and/or its derivatives, for example the lactate signal, is larger than 3, preferably larger than 10, more preferably larger than 30 more preferably larger than 100.
  • the invention in particular the setup of a sensor for the detection of an NMR signal from a substrate in its proximity in combination with an external polarizer polarizing molecules of choice, can be used for other relevant applications.
  • An important application is the monitoring of a chemical reaction, where molecules involved in the reaction are polarized in the external polarizer, and the chemical reaction is performed in the substrate in proximity to the sensor, such that the hyperpolarized NMR signal detected by the sensor monitors and provides insight on the chemical reaction.
  • Figure 1 Configuration of a diamond sensor for polarized biological material or metabolite
  • Figure 2 Process of acquiring polarized NMR signal via the diamond sensor for
  • Figure 3 Depiction of parallel NMR investigation with the diamond sensor from several biological samples in parallel;
  • Figure 4 Illustration of the two different readout schemes - optical and electrical readout
  • Figure 5 Alternative configuration of diamond sensor for detection of chemical reactions using polarized molecules.
  • the invention can be implemented, mutatis mutandis, along the lines of the method and device disclosed in Smits, Janis et al in "Two-dimensional nuclear magnetic resonance spectroscopy with a microfluidic diamond quantum sensor”, arXiv preprint arXiv:
  • a sensor comprising a substrate of isotopically purified diamond is provided.
  • the substrate has dimensions of for example approximately 100 pm x 100 pm x 30 pm.
  • NV centres at a high concentration of approximately 1 ppm are provided forming the sensor’s detection area.
  • the top surface of the substrate thus constitutes the sensor’s sensing area.
  • a thinner substrate for example with dimensions of 100 pm x100 pm x 10 pm, is completely filled with NV centres of the above concentration to form a detection area.
  • the substrate comprises or consists of multiple microdiamonds, for example with a diameter of around 10 pm, filled with NV centres of the above concentration and forming the sensor’s detection area.
  • the advantage of such microdiamonds includes, in particular, that they can be obtained at low cost and lend themselves to be use as a single use substrate, ie, as consumables. This, advantageously, can dispense with the need of cleaning the substrate after use.
  • a microwave (MW) and/ or radio frequency (RF) antenna provided for radiating radio frequency pulses for influencing the nuclear spin moments in the biological sample and/or influencing the detection spin moments in the substrate.
  • a laser and a CCD sensor are provided for optical excitation and detection.
  • a bioreactor is integrated on the surface of the substrate in that the bottom of the bioreactor is formed by the surface of the substrate that constitutes the sensing area.
  • the bioreactor may include different parts to enable cell metabolism such as a feeding pump and effluent outlet, agitation system to ensure homogenous conditions for better transport of nutrients and oxygen and a thermal jacket to alter and stabilize the temperature of the cell environment.
  • biological cell are provided, for example a cancer cell line such as HeLa cells.
  • Biological material, a substrate for the cells or other chemical substances of interest can be polarised in a polariser, preferably a hyperpolariser, that is preferably external to the device for analysing the sample.
  • the preferred polariser can polarise low gamma nuclei such as 13 C nuclei.
  • the polarised biological material, metabolite or other substances of interest can then be placed near the sensing area of the substrate for analysis, for example in the bioreactor.
  • a suitable polariser is the one describe in Coffey, Aaron M., et al. Open-source automated parahydrogen hyperpolarizer for molecular imaging using 13 C metabolic contrast agents.” Analytical chemistry 88.16 (2016): 8279-8288. As described in the manuscript, using a PASADENA process in a custom-built PHIP polarizer, implementing rf pulses for the polarisation transfer, results in a high 13 C succinate polarization after hydrogenation and polarisation transfer. The parts of this publication that concern the construction of the hyperpolarisation device are herewith incorporated into the present disclosure by reference.
  • Polarization by PH IP as described above has the advantage that the metabolite is selectively polarized, even in the presence of other molecules, as the hyperpolarization of the 13 C is dependent on a hydrogenation reaction and the specific couplings between the 1 H and 13 C nuclei in the molecule.
  • the analysis can comprise three consecutive steps.
  • the sample for example the biological cells, unpolarised and without a polarised substrate biological material or substance of interest
  • the sensor preferably into the bioreactor of Figure 1.
  • the hyperpolarised material for example biological material, a metabolite of the cells or a substance of interest is added to, preferably injected into, the sample in the bioreactor.
  • the NMR signal of the sample including the polarised substance such as the polarised substance of interest or its chemical reaction product, the biological material, or, after metabolisation of the metabolite, the polarised derivative is detected.
  • NMR spectroscopy of sample in the different spots can be performed in parallel.
  • This example exploits the fact that, in contrast to inductive NMR detection methods, cross speak can be avoided by the present method of NMR spectroscopy by means of detection spin moments, in particular those of NV centres.
  • the detection spin moments in particular those of NV centres, are read out optically, as already discussed in the context of Figure 1.
  • the spin states are read out by means of electrodes placed on opposite sides of the samples in sensing area.
  • the example of Figure 5 is a variation of the one in Figure 5.
  • a chemical reaction where a substance of interest in the form of molecules involved in the reaction are polarized in the external polarizer and then these molecules or their reaction products are monitored by means of NMR spectroscopy according to the present invention.
  • the chemical reaction is performed in the substrate in proximity to the diamond sensor, such that the hyperpolarized NMR signal detected by the diamond sensor monitors and provides insight into the chemical reaction.

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Abstract

L'invention concerne un procédé d'analyse d'un échantillon par spectroscopie RMN, le procédé comprenant les étapes consistant : à fournir un aimant, au moins un élément d'antenne et un capteur, le capteur comprenant un substrat doté d'une multiplicité de moments de spin de détection dans une zone de détection ; à soumettre au moins une partie de l'échantillon à une polarisation de ses spins nucléaires ; à placer au moins la partie de l'échantillon qui a été soumise à une polarisation de ses spins nucléaires à proximité du capteur ; à générer, au moyen d'un aimant, un champ magnétique traversant l'échantillon ; à effectuer une exposition, au moyen de l'élément ou des éléments d'antenne, à des impulsions de radiofréquences ou de micro-ondes pour influencer les moments de spin nucléaire dans l'échantillon biologique et/ou influencer les moments de spin de détection dans le substrat ; et à acquérir, au moyen du capteur, un signal RMN à partir de l'échantillon. L'échantillon peut comprendre un métabolite qui peut être converti par une ou plusieurs cellules biologiques, un dérivé qui résulte de la métabolisation d'un métabolite par la ou les cellules biologiques, et/ou un matériau biologique. Dans certains modes de réalisation, la polarisation est une polarisation sélective et/ou marquée sélectivement de manière isotopique. La polarisation d'au moins une partie des spins nucléaires de l'échantillon peut se produire au moyen d'une polarisation induite par parahydrogène.
PCT/EP2020/060086 2019-04-08 2020-04-08 Système d'évaluation de molécules hyperpolarisées dans un échantillon biologique WO2020208103A1 (fr)

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