Method for detecting host molecules binding to xenon, and measuring device for the detection of host molecules binding to xenon

Abstract

The invention relates to a method for detecting xenon-binding host molecules (12), comprising the steps of: bringing the host molecules (12) into contact with a carrier (24); bringing the host molecules (12) into contact with xenon from a gas phase (26) so that host molecules (12) and xenon form a host-molecule-xenon complex (20); irradiating the host-molecule-xenon complexes (20) with electromagnetic depolarization radiation in order to at least partially selectively depolarize xenon in the host-molecule-xenon complex (20); and detecting the host molecules (12) on the basis of a change in nuclear magnetic polarization of xenon in the gas phase (26).

Description

[0001] Physikalisch-Technische Bundesanstalt Attorney's file: Braunschweig and Berlin 0454-0330 PCT-1 Bundesallee 100 38116 Braunschweig Date: March 17, 2025 Method for detecting xenon-binding host molecules and measuring device for detecting xenon-binding host molecules State of the art. Xenon is a noble gas whose stable isotope 129 Xe has a nuclear spin of 1 / 2 and a natural abundance of 26.4%. The solubility of xenon in water is relatively high. At atmospheric pressure of 0.1 MPa and a temperature of 298 K, its concentration in water at thermal equilibrium is about 4.8 mM, compared to its concentration of 40.8 mM in the gas phase. The NMR detection sensitivity is relatively low compared to the sensitivity of the 1H nucleus is relatively low at about 0.0056. It can be increased by several orders of magnitude by hyperpolarizing the xenon nuclear spins using suitable methods (see, for example, Navon et al., 1996; Pines et al., 2004). If xenon binds reversibly to a host molecule in a solution containing dissolved xenon, this host molecule can be indirectly detected with high sensitivity in the CEST (chemical exchange saturation transfer) experiment via an attenuation of the signal of the dissolved free xenon. A prerequisite for this is that the dissolved free xenon exchanges with the bound xenon sufficiently quickly, and that the resonance frequency of the bound xenon is sufficiently different from that of the free, dissolved xenon. Xenon binding takes place at cavity-like structures of the host molecule, whether naturally occurring or synthesized.Among synthetic xenon binding molecules, cryptophanes and cucurbiturils have proven particularly effective, offering sufficient affinity combined with favorable xenon exchange rates. They can be used in the CEST experiment together with hyperpolarized xenon. 129Xe can be detected in the subnanomolar range. If they are covalently bound to another molecule, this molecule can also be detected with high sensitivity. Numerous application examples have now been published (for a review, see, for example, Jayapaul and Schröder, 2020). Solution experiments are unsuitable for high-throughput applications, as each individual experiment requires a long wait until gaseous xenon PL / PL - PD003 / 20.10 is sufficiently dissolved and the solution is saturated with xenon. This can take a very long time. The fastest method for xenon saturation is to pass xenon gas through the solution. This method is typically used with hyperpolarized xenon. Gas saturation times of approximately 20 s per individual spectrum are required. A typical z-spectrum is generated from approximately 100 individual spectra. This increases the total measurement time by approximately 33 minutes.In addition, passing the gas leads to gas bubbles and can cause the denaturation of protein samples over time. Insoluble samples or samples that decompose in the solvent cannot generally be examined using this method. US 2010 / 0160173 A1 discloses the use of nuclear magnetic resonance measurement to detect interactions in the form of passive relaxation and diffusion processes between an immobilized sample and target molecules in a fluid. Aim of the invention: The invention relates to a novel method for detecting xenon-binding host molecules and their complexes. A second aspect relates to the invention of a concentration measuring device for quantifying xenon-binding host molecules. This also includes the theoretical description of the acquired measurement data, which supports quantification.A host molecule is understood, in particular, to be a molecule that can temporarily bind xenon atoms and that allows chemical exchange saturation transfer (CEST). In this exchange saturation transfer, the state of the molecule bound to the host molecule is changed. 129 Xe. The change in state occurs, for example, through relaxation decay, radiofrequency irradiation, or other manipulations of the nuclear spin polarization. However, the free 129 Xe, which is bound in exchange with the 129 Xe stands. Both pools of 129Xenon atoms are coupled by reversible complex formation, so that the detection of free xenon provides information about the presence (semiquantitatively) and / or concentration (quantitatively) of the host molecule. If the polarization of the host-bound xenon is reduced, for example, by frequency-selective radiofrequency irradiation, a decrease in the intensity of the free xenon signal is observed. The attenuation of the free xenon signal upon irradiation at the resonance frequency (RF) of the xenon-host molecule complex semiquantitatively indicates the presence of the host molecules in the solution. Measuring the decay rate enables the quantitative determination of the concentration of the host molecules (Mitschang et al., 2016, 2021). Such a method is known from DE 102021121238 A1. In this process, a solution of host molecules is brought into contact with hyperpolarized xenon,so that a host molecule-xenon complex is formed. By irradiating the sample with depolarizing radiation, the decay curve can be determined, which describes the decreasing polarization. From this decay curve, the concentration of the host molecules can be determined. For each different xenon concentration in the sample, the host molecule concentration can be determined without reference by irradiating the sample with depolarizing radiation of different amplitudes close to the resonance frequency of the xenon bound to the host molecule. In other words, only the measurement data from the NMR (nuclear magnetic resonance) measurement are sufficient to determine the host molecule concentration. For high-throughput applications, all of these methods have the disadvantage that the required introduction of the (hyperpolarized) xenon into the solution, i.e., the saturation of the solution with (hyperpolarized) xenon,only after a relatively long residence time or, alternatively, with significant bubble formation. The invention is based on the aim of reducing the above-described experimental disadvantages and limitations of the current state-of-the-art methods. The invention solves the problem by a method according to claim 1 and a measuring device according to claim 9. Preferred embodiments are specified in the subclaims. The invention solves the problem in particular by a novel method for detecting xenon-binding host molecules by determining the CEST effect by measuring the depolarization of xenon in the gas phase and applying the sample containing the host molecule to a solid support. The process can be described by the following steps: (a) Bringing the host molecules into contact with a support, in particular a solid support. With the feature that the host molecules are brought into contact with a support,In particular, it is understood that the host molecules of the type are brought into interaction with the carrier in such a way that they do not detach from the carrier during the course of the experiment. The carrier can, for example, be solid, gelatinous, or pasty. It is advantageous if the carrier contains fiber material or has a fiber-containing region, whereby the host molecules can be brought into contact with the fiber material. For example, the carrier can consist of hydrophilic material, for example cellulose and / or plant fibers. According to a preferred embodiment, the host molecule is dissolved or suspended in a solvent. This solvent is then brought into contact with the carrier. For example, the solution of solvent and host molecule is introduced into the carrier, for example, absorbed by the carrier, or otherwise brought into contact with it,that the solution does not move away from the support during the measurement. Alternatively or additionally, the host molecules are directly bonded to the support. For example, the host molecules can be applied to the support in powder form. Alternatively or additionally, the host molecules can first be applied to the support as a solution and then dried, leaving only the host molecules.which are bound to the support. Alternatively, the host molecules can be directly covalently or non-covalently bound to the support or fixed to the support via additional auxiliary molecules (e.g., antibodies). According to one embodiment, the xenon-binding host molecules are applied to the support in the solvent as a thin film on the surface. Alternatively, the solution of solvent and host molecules forms one or more drops on the support. According to an alternative embodiment, the xenon-binding host molecules in the solvent are absorbed into the solid support or incorporated into a solid support with one or more cavities. In particular, the xenon-binding host molecules are applied to the support in the solvent in such a way that rotating the support by 180° around a horizontal axis does not result inthat the host molecules move away from the carrier. During the measurement itself, the host molecules can be surrounded by the solvent, or alternatively, the solvent can be removed after application, e.g., by drying. (b) Bringing the host molecules into contact with xenon from a gas phase, so that host molecules of the sample form a host molecule-xenon complex with xenon. Bringing the host molecules into contact with xenon from a gas phase is understood to mean, in particular, any process in which xenon, which is present in gaseous form, is brought into spatial proximity with the host molecules such that the host molecule-xenon complex is formed. This can occur, for example, bythat gaseous xenon is guided past the carrier and initially dissolves in the solvent and diffuses to the host molecule. Alternatively, the gaseous xenon can diffuse directly to the host molecule. (c) Irradiating the host molecule-xenon complexes with electromagnetic depolarizing radiation to at least partially selectively depolarize xenon in the host molecule-xenon complex. In the NMR experiment, the depolarization frequency corresponds to the host molecule resonance frequency or contains frequency components at the host molecule resonance frequency in the given external magnetic field.at which the depolarization of the xenon in the host molecule-xenon complex is strongest. Preferably, electromagnetic depolarizing radiation with a fixed frequency is used. Alternatively, electromagnetic depolarizing radiation with a variable frequency is used. According to one alternative, depolarizing radiation with a constant amplitude is used. Alternatively, depolarizing radiation with a time-varying amplitude is used. Preferably, the electromagnetic depolarizing radiation is continuous or pulsed to cause RF saturation. By irradiating the sample at or near the resonance frequency of the xenon in the host molecule-xenon complex (depolarizing radiation), the spin system of the bound xenon is saturated (RF saturation). As a result, the magnetization of the bound xenon remains in a state of low magnetization, particularly with continuous or repeated RF irradiation.so that the relaxation of the bound xenon is specifically inhibited, while other xenon pools remain undisturbed. The depolarization of the bound xenon occurs selectively through RF saturation, while other pools remain unaffected and follow their natural relaxation processes. (d) Detection of the host molecules based on a nuclear magnetic polarization change of xenon in the gas phase. The detection of the host molecule-xenon complex is carried out indirectly by measuring changes in the nuclear magnetic polarization of xenon atoms in the gas phase that are in exchange with the xenon atoms bound in the host complex. The xenon atoms in the complex with the host molecule experience a depolarization induced by the depolarizing radiation—a polarization change that is transferred to the xenon gas when they pass into the gas phase. A specific signal reduction is thus achieved, regardless of the relaxation processes prevailing there.which is indicative of the presence of the host molecules. In accordance with the patent application, any suitable method can be used to measure nuclear polarization in the gas phase (e.g., SQIDS and optical detection methods). However, nuclear magnetic resonance detection is preferred, which is discussed in more detail below. In the simplest case, the sample is surrounded by xenon gas and is spectroscoped together with it. In principle, however, the detection of xenon gas depolarization can also take place spatially separated from steps (a) to (c), since the xenon atoms can diffuse away from the sample or flow away following a pressure difference. The maximum distance depends on the relaxation time of the xenon, the flow velocity of the gas, and the strength of the residual magnetic field. The distance can be small, e.g., 5 cm (within the NMR spectrometer), but also up to 10 m. Data from experiments are available here,which guide hyperpolarized xenon into the NMR spectrometer. Such an experimental setup is potentially less complex, as it is no longer necessary to ensure that the carrier is positioned in close proximity to the device measuring the polarization change. The measurement of the depolarization of the xenon gas must be correlated with the properties of the depolarizing radiation in order to obtain the qualitative information (existence of the host molecule in the sample) or the quantitative information (concentration of the host molecule in the sample) (see (c)). In NMR detection, the pulse sequence used,The associated NMR-relevant measurement parameters and the evaluation method are of great importance. The NMR pulse sequences used for the patent application itself are state-of-the-art and described extensively in the literature. An example of the CEST experiment and the recording of the z-spectrum can be seen in the figures. The simplest experiment consists of recording two NMR spectra with resonant irradiation at the host molecule resonance frequency and off-resonant irradiation at the same distance from the resonance frequency of the xenon gas. If the xenon signal with resonant irradiation is significantly weaker than with off-resonant irradiation, the existence of the host molecules is considered proven. If, in parallel, a sample with a defined host molecule concentration is measured and both sets are compared,From this, the concentration in the sample with an unknown concentration of the host molecule can be determined. More precise information can be obtained from a data fit with a suitable model function. The corresponding fit function is shown below. The data used for this purpose is either the depolarization during resonant irradiation of a fixed frequency but varied duration (recording a decay curve) or the depolarization with a fixed irradiation duration but varied frequency (recording a z-spectrum). According to a second aspect, the invention solves the problem by a measuring device, in particular a detection device and / or concentration measuring device, for detecting xenon-binding host molecules, comprising (i) a sample holder configured to receive a sample of carrier-bound host molecules, (ii) a xenon source arranged to generate a gas phase in contact with the sample, (iii) a radio-frequency transmitter,which is designed to automatically irradiate the sample with electromagnetic depolarization radiation for selectively depolarizing xenon in the host molecule-xenon complex, (iv) a depolarization change measuring device for measuring a polarization change of xenon in the gas phase, and (v) a computing unit designed to automatically carry out a method comprising the steps of: (a) controlling the depolarization change measuring device to detect a polarization change of xenon in the gas phase, and (b) detecting the host molecules based on the polarization change. Preferably, the measuring device is an NMR spectrometer having a computing unit designed to automatically carry out a method comprising the steps of (i) controlling the magnetic resonance measuring unit so that it detects depolarization radiation of a specific frequency (f, D), duration and amplitude, (ii) measuring the nuclear magnetic resonance signal strength (S) of xenon gas, and (iii) calculating the host molecule concentration (C W) from the signal strength (S). The advantage of the inventive approach is that the measurement time can generally be significantly reduced. The reason for this is that the complex gas saturation of the solution with hyperpolarized xenon is eliminated. While in prior art methods the hyperpolarized xenon was detected in the sample solution, for the invention it is sufficient to record the polarization change of the xenon in the gas phase. This allows the use of smaller samples or quantities of host molecules in most cases. As a rule, the invention can also be carried out with simpler apparatus, since the polarization change of the xenon in the gas phase can also be detected spatially distanced from the, in particular solid, carrier. In the context of the present description, the detection of xenon-binding host molecules is understood to mean that the existence of such host molecules is detected by means of the method.This is preferably achieved by determining the concentration of the host molecules or at least a number of host molecules. However, it is also possible to simply determine whether the number of host molecules is above a predetermined detection threshold. Detecting the host molecules preferably comprises quantitatively determining a host molecule concentration. According to one embodiment, the host molecule concentration is determined from the nuclear magnetic polarization change. The nuclear magnetic polarization change can also be referred to as an NMR polarization change. To achieve high sensitivities, the xenon is preferably hyperpolarized. The intensity changes of the gas signal upon irradiation with depolarizing radiation can be described as follows, and the corresponding parameters can be derived from the data fit.Due to the large interface, a concentration equilibrium quickly forms between xenon Xeliq, dissolved in sample film 22, and xenon Xegas 18.3 in the gas phase 26; sample 22 is saturated with xenon almost instantly. Similarly, an equilibrium forms between dissolved xenon Xeliq and host-bound xenon Xehost in sample film 22. However, the exchange rates are significantly lower than in the case of a phase change due to molecular interaction with the host molecule. Thus, there are three different pools (states) of xenon in the gas phase (^^). ^^^ , 18.3), freely solved in the film (^^ ^^^ , 18.1) and in the film bound to the host (^^ ^^^^, 18.2) that are constantly and successively in exchange equilibrium with each other^^^ ↔ ^^^ ↔ ^^^The kinetic equilibrium also remains in the NMR experiment, even if the magnetic moments are manipulated and a magnetization exchange between the pools is induced according to the kinetic equilibrium rates. The high particle currents across the large interface between the gas phase 26 and the sample film 22 cause an instantaneous equilibrium distribution of the magnetization between the phases. Upon RF saturation of host-bound xenon in the sample film 22, the NMR signals of xenon in the gas and xenon dissolved in the film are correspondingly attenuated. According to one embodiment, non-hyperpolarized xenon is used. In this case, the host molecule concentration in the solvent is preferably determined from the nuclear magnetic resonance signal strength using the following formulas: C^ = Where: R1 is the longitudinal relaxation rate of xenon in the gas phase without electromagnetic irradiation, R1^ is the longitudinal relaxation rate of xenon in the gas phase under irradiation of depolarizing radiation, Δ< ; = ^-=*> − -=*?@^< the squared distance of the angular frequency -=*> of the depolarization frequency fD from the Larmor frequency -AB?C of the host-bound xenon,D = for the nutation angular frequency of the xenon and die Exit rate for the dissolution of the host molecule-xenon complexes, M0 the nuclear magnetic output signal of xenon in the gas phase and H = H ^MN O6^MN P with k for the formation rate of the xenon-host coQR6S complexes, TUUs is the solubility of xenon in the solvent surrounding the xenon-binding host molecules, G is the ratio of gas to liquid volume in the effective volume of electromagnetic radiation, R is the gas constant, T is the absolute temperature, K is the affinity constant for xenon binding to the host molecule in the solvent environment, and p is the partial pressure of xenon in the gas phase. The derivation of this formula can be found below. The feature that the host molecule concentration is determined using the given formulas is understood in particular to mean that mathematical operations are performed that have the same result as the given formulas. It is not necessary that the formulas be used explicitly as stated; any calculation that leads to the same result as these formulas is a calculation using the formulas. According to another embodiment, hyperpolarized xenon is used and the host molecule concentration Cw in a solvent environment, the nuclear magnetic resonance signal strength S of hyperpolarized xenon in the gas phase is determined using the following formulas: YZ = ^1 + ]^_^ STUU\ in combination with S ∝ 2? (Eq. 2). It is possible, but not necessary, that only one type of host molecule is used. According to one embodiment, two or more different host molecules and / or at least one host molecule that forms at least two host molecule-xenon complexes with different host molecule frequencies are brought into contact with the carrier. If no hyperpolarized xenon is used, the host molecule concentration C w in a solvent environment from the nuclear magnetic resonance signal strength S preferably determined using the following formulas: T[,b = S ^ ^ in combinationTU \ 1 + ]^ ^ _ n U,b b q e ? " l B m- e 6∑ B n,k"fk hij,km- - n,k 9on,k(Eq. 3) where the index i counts the different host molecule types and states. If hyperpolarized xenon is used in this case, the host molecule concentrations C i w in solvent environment from the nuclear magnetic resonance signal strength S Spreferably determined using the following formulas: CT[,b^̀ = S ^1 + ]^ ^ _^ in TUU,b\b combination (Eq. 4) where the index i counts the different host molecule types and states. According to one embodiment of the method, the host molecules are applied directly to the support so that they have direct contact with the gas phase. In particular, the host molecules are then not surrounded by solvent. In this case, the host molecule concentration is determined from the nuclear magnetic resonance signal strength S of the xenon Sin in the gas phase, preferably using the formula T[,b = S ^1 + ]^ _ / tu^ in TUU,b\b combination and the respective expressions of the signal strength S from equations 1 to 4. The host molecule preferably contains a protein. In particular, the host molecule is an antibody or an antigen, DNA, RNA, or a fragment thereof. It is advantageous if the host molecule is composed of a biomolecule and a xenon binding molecule. The xenon binding molecule is preferably a cryptophane or a cucurbituril. In other words, the xenon binding molecule is from the organic compound classes of cryptophanes or cucurbiturils. The partition coefficient—the ratio of the Xe concentrations in liquid and in gas at equilibrium—is given by the product of the constants sRT (s xenon solubility, R gas constant, T absolute temperature). The formulas in Eqs. 2 and 4 for the signal intensity of hyperpolarized xenon gas result from the exchange between the pools of gaseous xenon dissolved in solvent on the support and xenon bound to the support as follows.Since the magnetization is the sum of the magnetic moments of all xenon atoms in the respective pool, in addition to the concentration, the volumes of the gas phase and liquid phase, more precisely the gas volume Vg and film volume Vl effective in the NMR measurement (spatial excitation and detection profile of the RF coil), must be taken into account. Accordingly, the magnetization density (proportional to the intensity of the NMR signal) in the gas phase can be calculated as v. ^ (Magnetization / Vg) in equilibrium with the sample film 22 but in exchange with the magnetization density of host-bound xenon v ^ (Magnetization / Vl) can be described by a simple 2-pool equation of motion w ^ wx v = − tu 1^ H^Ky + ^tu v^ + H^II y + ^tu v^(Eq.5) with G=Vg / Vl and H ^K or H ^IIfor the rate coefficient for the formation or dissolution of complexes between xenon and the host molecule in the sample film. For verification, a sample film 22 that is so greatly expanded that it fills the entire effective volume of the sample chamber can be considered – i.e., for G=0, which corresponds to the situation in conventional NMR on liquid samples. Then w / wx ^tuv^ =−H^K ^tuv^ + H^IIv^ and with v^ = ^tuv^ the equation of motion becomes (Eq. 6) which describes the magnetization exchange of freely dissolved and host-bound xenon in conventional saturation transfer experiments in the liquid phase (HyperCEST). The formal similarity allows the known solutions from Eq. 6 for the attenuation of the measurement signal from the detected pool under RF saturation of the exchanged pool in the conventional HyperCEST experiment to be transferred to the present situation according to Eq. 5 by substituting the rate H^K by H^K^tu / ^y + ^tu^ and the rate H^II by H^II / ^y + ^tu^. Accordingly, the nuclear magnetic resonance signal strength S of the NMR signal of hyperpolarized xenon in the gas phase, under resonant RF saturation of the duration t of the host-bound hyperpolarized xenon in the sample film, is (Eq. 7) The amplitude factor v^L ∝ _^3 / ^1 + ^_^ is proportional to the product of xenon partial pressure p (gas concentration) and spin polarization according to P0 / (1+^p), where the parameter ^ indicates the degree of hyperpolarization generated by optically pumped spin exchange (spin-exchange optical pumping, SEOP) as a function of the partial pressure and P0 is the maximum achievable polarization. The decay rate is the sum of a saturation transfer rate induced by exchange with irradiated host-bound xenon and the decay rate R1^(p,^^), which describes longitudinal relaxation and direct depolarization by irradiation (thus the known decay rate only under spin-locking t ^_, { ^ = t ^^^<^ + t ^ < 4GG^ " < G ^^ ^ where ^ = xD^ ^{" / ^{e^ − with F G for the RF amplitude, F for the RF angular frequency, which corresponds to the depolarization frequency 2^fD, and F 3^the Larmor frequency of xenon in the gas phase). In many cases, R1^(p,^^) is practically independent of the xenon partial pressure and often also of the irradiation amplitude (R1^ is then the longitudinal relaxation rate R1), or R1^(p,^^) can be completely neglected compared to the saturation transfer rate due to the difference in size. The saturation transfer rate induced by exchange with irradiated host-bound xenon can be written (Eq.8) with the entry rate H ^K ^_^ or the exit rate H ^II ^_^ for the formation or dissolution of the host molecule-xenon complex in the sample film in kinetic equilibrium.The term ^ _, refers to the only partial RF of host-bound xenon when the residence time of the xenon in the complex (1 / koff) at a given irradiation amplitude of the depolarizing radiation (F G) is too small to achieve complete magnetization quenching. In addition to a dissociative exchange (rate coefficient H4), a degenerate exchange (rate coefficient k) proportional to the xenon concentration in the sample film (sp) can also be decisive for the exit rate, as has already been shown for small host molecules, H^II^_^ = H4 + H^_. The entry rate is given for kinetic equilibrium in Eq. 5 and for the equilibrium densities in the two phases v^ = v^ / ^tu, v^ = ^_ using the affinity constants K for the xenon binding to the host molecule in the sample film and the concentration of host molecules Y` in the sample film as H^K^_^ = H^II^_^]Y` / ^1 + ]^_^. Using these explicit formulas, the kinetic parameters, in particular the concentration of host molecules in the sample film, can be quantitatively determined by modeling measurement data according to Eqs. 1 to 4.An alternative experimental approach involves RF saturation of host-bound xenon at a constant irradiation time t but varying the carrier frequency of the RF irradiation (z-spectrum). The intensity of the NMR signal of hyperpolarized xenon in the gas phase is then given by

[0009] depending , the squared distance of the RF frequency FM^^ from the Larmor frequency F3^ of the host-bound xenon. In the z-spectrum, the plot of the signal intensity as a function of^^^, a resonance around ^^ ≈ 0 can be seen as a depression, the depth of which is characterized by the saturation transfer rate hlBBij ^z, FG^ and the width by D^ =. The intensity of the gas signal according to Eqs. 7 and 9 assumes a transverse relaxation rate R2 of the host-bound xenon that is negligible compared to the escape rate H^II^_^. According to an analytical approximate solution of the Bloch-McConnell equation for RF saturation (Zaiss et al.), relaxation effects can be taken into account in the quantitative description by using the expression ^^^_, {"^ = F< < in Eq. 8 and thus also 7 and 9. <G / ^FG + H^II + H^IIt<^ und in Gl. 9 zusätzlich D^ =verwendet werden. The formulas in Eqs. 1 and 3 for the signal intensity of non-hyperpolarized—i.e., thermal—xenon gas are now calculated as follows. NMR measurements for saturation transfer can also be performed on xenon without hyperpolarization. The nuclear moments of the xenon atoms are then polarized by the Zeeman field, a static magnetic field in the range of a few Tesla field strengths, according to Curie's law (Boltzmann distribution). This is referred to as "thermal" polarization because of the small excess of magnetic energy over thermal energy in the sample; it lies several orders of magnitude below hyperpolarization. Therefore, in addition to the depolarization caused by RF irradiation, the ongoing effort to restore thermal polarization must also be considered, a process that occurs at a rate R1.From the well-known description of saturation transfer experiments on thermally polarized hydrogen atoms, the intensity of the NMR signal of thermally polarized xenon in the gas phase under RF saturation of host-bound xenon in the sample film in the z-spectrumc^z, FG, r, {e^^} results. (Eq. 10) and for ^h = 0 the expression for the case of exclusively resonant irradiation FM^ = F3^. The amplitude M0 is precisely the initial magnetization due to thermal polarization. For h Equation 8 applies, and if necessary, the substitutions above can be used to expand to include relaxation effects of host-bound xenon. The direct RF saturation of xenon in the gas phase can be modeled as follows: With RF irradiation near the Larmor frequency of xenon in the gas phase, with sufficient RF amplitude, the saturation of the signal is reached at a high rate, compared to which the exchange dynamics with the sample film and even more so within the film with the host molecule are negligible. This is where the well-known expression of the general solution of the Bloch equation for a 1-spin ensemble under RF irradiation comes into play. It results from Equation 10 for thermal xenon in the limit of a vanishing exchange rate. (hlBB ij ^z, FG^ = ?) to (Eq.11) with the decay rate under spin-locking < < = t< ^^^ ^ + tG ^^^ ^ decomposed into^ = t − t with tf < <^ G < < for the transverse relaxation rate in the xenon gas, D^ = FG and ^< <^ = ^{e^ − {?^^ for the distance square of the RF frequency FM^ from the Larmor frequency F 3^ ^of xenon in the gas phase. For hyperpolarized xenon, neglecting thermal polarization, the relationship between simpler (Eq. 12) Finally, the RF saturation transfer from xenon freely dissolved in solvent (sample film) on the support to the xenon gas can be described using the following formula for the signal intensity. Due to the large interface between gas and sample film discussed above, high exchange rates occur here, so that the exchange with the host molecules in the film can be neglected. Approximately two-way exchange occurs ^^^ ↔ ^^^ with rates H^K and H^^^. (Eq. 13) where G again represents the ratio of gas to film volume. In kinetic equilibrium, with the concentration ratio sRT, the rate becomes H^K = H^^^y^tu. Formally analogous to the two-way exchange in Eq. 10, the thermal gas signal in exchange with the liquid film is given by c^_, FG, r, {e^^ + q e" ? e D< ^ "f + ^^ D< + ^< ^ ^ (Eq.14) with the decay rate of the gas signal only under spin-locking tG^^_, FG^, ^^ = und ^< ^ = ^{ < e^ − {?^ ^ für the square of the RF frequency F M^ from the Larmor frequency F 3^ ^of the xenon released in the film. For ^^^_, {"^ and D^ the substitutions discussed above can be made to take into account the transverse relaxation of the xenon dissolved in the film. For hyperpolarized xenon, neglecting thermal polarization compared to hyperpolarization, it is again easier (Eq. 15) Overall, the xenon signal in the gas phase can be described as follows: direct RF saturation, RF saturation of the xenon freely dissolved in the solvent on the support, and RF saturation of the xenon bound to the host on the support. When recording the Z-spectrum, the frequency of the RF irradiation F M^ continuously across the resonance points F ^^ , und F 3^ The formulas derived above correspond to the two-way exchange of the xenon freely dissolved in the film with the gas phase (^^^ ↔ ^^^) and – virtually – the xenon bound in the film with the gas phase (^^^ ↔ ^^^). The exchange of these pools only with the gas phase but not with each other allows the combination of these exchange processes with the direct saturation of the gas signal according to (Eq. 16)where B; = k«¬¬WX is set according to Eq. 10. For hyperpolarized xenon, neglecting thermal polarization compared to hyperpolarization, the simpler (Eq. 17) The given models for the xenon signal in the gas phase express the dependence on the molecular and spectroscopic parameters; a proportionality factor for absolute determination is missing. It is the product of an instrumentation-dependent factor A (detection electronics and signal amplification) and a projection cos < Θdue to the related pulse sequence with Θ = für denInclination angle of the spin-lock axis with the Z-axis (orientation of the effective field under RF irradiation for the xenon gas). A first factor, cos Θ, represents the projection of the initial magnetization along the Z-axis onto the spin-lock axis at the beginning of RF irradiation, and a second factor, cos Θ, represents the projection of the gas magnetization along the spin-lock axis back onto the Z-axis at the end of RF irradiation, which is subsequently detected as transverse magnetization by a read pulse. For the fit of the resonances around ω 3; and ω 3» in the z-spectrum, i.e. the exchange with host-bound or film-dissolved xenon, the projection cos <Θ can be disregarded, since the term is virtually constant around the resonance and thus is included in the global amplitude or, if <" ≪ ^$® − ?º^ holds, practically cos Θ = 1. On the other hand, the projection cos<Θ is to be preceded as a factor in the model functions in Eqs. 8, 9 and 13, 14 for the fit only of the gas resonance or the entire z-spectrum, since it is close to the gas resonance ω 3½ the line shape dominates. In a measuring device according to the invention, the depolarization change measuring device is preferably a magnetic resonance measuring unit. Preferably, the measuring device is an NMR spectrometer having a computing unit configured to automatically carry out a method comprising the steps of (iv) controlling the magnetic resonance measuring unit so that it emits depolarization radiation of a specific frequency (fD), duration, and amplitude, (v) measuring the nuclear magnetic resonance signal strength (S), and (vi) calculating the host molecule concentration (C W) from the signal strength (S). The invention is explained in more detail below with reference to the attached drawings. Figure 1 shows a schematic view of a sample holder with a crypthophan A monoacetate sample for carrying out the method according to the invention. Figure 2 shows a schematic view of a measuring device according to the invention for carrying out a method according to the invention and Figure 3 shows a CEST pulse sequence. Figure 4 shows measurement data generated according to Figures 1, 2 and 3. Figure 1 shows a sample holder of a measuring device 10 according to the invention (see Figure 2) for detecting schematically drawn, xenon-binding host molecules 12.i. The xenon-binding host molecules 12.i consist of a molecule to be detected, in particular a biomolecule 14.i, which is bound to a xenon-binding molecule 16.i. The xenon-binding molecule 16.i is, for example, a cucurbituril. The host molecule 12.i can form a host molecule-xenon complex 20.i with xenon atoms 18.i. Thus, bound xenon atoms 18.i are labeled Xehost in the drawing. The host molecules 12.i are located in a sample 22, in this case in the form of a sample film formed on a carrier 24. In this way, the host molecules 12.i are brought into contact with the carrier 24. The sample 22 consists of a solution containing a solvent, the host molecules 12.i, and dissolved xenon Xeliq. It is also possible that biomolecules 14 not bound to the xenon binding molecule 16 and unbound xenon binding molecule 16 are present in the solution. In addition, free xenon atoms 18 (Xe. liq ) contained in the film 22. On its side facing away from the carrier 24, the sample film 22 is in a gas exchange equilibrium with a gas phase 26, the xenon gas Xe gas 18.3. In this way, the host molecules are coated with xenon Xe gasfrom the gas phase 26, so that the host molecule-xenon complex 20 is formed. In Figure 1, the sample 22 is shown arranged on the carrier 24. However, it is also possible for the sample 22 to be held in the carrier 24. The carrier 24 can be formed from absorbent material or, for example, comprise a paper strip. Such a carrier can, for example, be folded or rolled and inserted into a sample holder 28 of the measuring device 10. Figure 1 shows the sample holder 28, which is provided with an inlet nozzle 29a and an outlet nozzle 29b for xenon gas, for example as an NMR sample tube that can be filled with the carrier with applied host molecules. Figure 2 shows a schematic view of the measuring device 10, which has, in addition to the sample holder 28, a xenon source 30 which releases xenon gas into the sample holder 28 so that the gas phase 26 (see Figure 1) is built up.A magnetic field is applied to the sample 22 by means of a magnet 32, in particular a superconducting one. The magnetic field strength B is typically between 0.5 and 14 Tesla, for example 1.5 Tesla. The sample 22 can be irradiated with electromagnetic depolarizing radiation by means of a radio-frequency transmitter 34. This causes an at least partially selective depolarization of the xenon Xehost in the host molecule-xenon complex 20. A nuclear magnetic resonance signal of a specific strength S is detected by means of an antenna 36, ​​which is connected to a computing unit 38. The antenna 36 and the computing unit 38 form a polarization change measuring device 40. The computing unit 38 calculates the host molecule concentration (C) from the signals received by the antenna 36. W).20. The measuring device 10 can be based on a high-field spectrometer, for example, a Bruker Avance Neo 600 MHz spectrometer (Bruker Biospin, Karlsruhe), or a low-field or compact spectrometer, for example, a Bruker Fourier 80. Figure 3 shows a typical CEST pulse sequence using hyperpolarized xenon, with the introduction of hyperpolarized xenon into the sample chamber, followed by a rest phase to dampen possible turbulence and RF irradiation for RF saturation, as well as the final generation and recording of the NMR measurement signal. The frequency of the depolarizing radiation (blue) is varied from experiment to experiment at a fixed duration (recording a z-spectrum) or, alternatively, the duration of the depolarizing radiation is varied at a fixed frequency close to the Larmor frequency 2πf 0Wof the host-bound xenon varies (recording a decay curve). Since with thermal xenon the gas does not flow through the sample vessel but is enclosed within it, the two phases marked in orange can be summarized as waiting times for sufficient spin-lattice relaxation back to thermal equilibrium. Figure 4 shows the z-spectra obtained according to the measurement schemes in Figs. 1 to 3. The measurement on hyperpolarized xenon is shown in Fig. 4(A). It was recorded on a compact spectrometer at a field strength of 1.88 T (Bruker Fourier 80). The sample, a rolled-up paper strip (1.8 x 4.0 cm) moistened with 4 mM CB6-enriched buffer solution 2), was recorded in a conventional NMR tube with a 5 mm outer diameter, which was connected via inlet and outlet nozzles to the gas flow from a xenon hyperpolarization device. The polarization was approximately 10%; signal accumulation was unnecessary due to the exceptional sensitivity. RF saturation lasted 40 s at an RF amplitude of 2^30 Hz (xenon nutation frequency). For each data point, hyperpolarized xenon was injected into the sample well for 10 s before irradiating to RF saturation, followed by a 10 s wait for the sample to settle. The total measurement time was 30 min. For the high-field measurement with thermal xenon (Fig. 4B), the sample well was an NMR sample tube with a 5 mm outer diameter filled with a paper strip (30 mm x 40 mm) as a carrier with applied host molecules. Cryptophane A monoacetate (CrA-ma) was chosen as the xenon binding molecule. The paper strips were coated with 0.5 mL of 10.The paper strips were soaked in [4 mM CrA-ma in DMSO (dimethyl sulfoxide)] and dried. The paper strips were then rolled up, transferred to sample well 28, and repeatedly gassed with xenon (0.1 MPa). Further experimental parameters for the measurements included the number of signal accumulations performed (NS=4), the spectral width (the recording frequency range) of 65,789.477 Hz, the repetition time of 51 seconds, the RF saturation pulse duration of 50 seconds at 0.02 W, corresponding to a 90-second pulse of 25.5 microseconds, and a magnetic field amplitude of 3,085 Hz (xenon nutation frequency). The depolarizing radiation in the measurements on a Bruker Avance Neo 600 MHz spectrometer had a frequency fD of 165.97 MHz, which corresponds to a host molecule frequency fW of CrA-ma at which the depolarization of the xenon in the host molecule-xenon complex 20 was greatest. The measurement temperature was 25°C.The z-spectra show signals that can be assigned to three different fractions of xenon: xenon in the gas phase, xenon bound to the xenon binding molecule (CrA-ma, CB6) on the solid support, and xenon free in a solvent environment. Accordingly, the data were fitted with model functions so that numerical values ​​for the parameters can in principle be obtained in further evaluation. Figure 4a shows the z-spectrum of hyperpolarized xenon gas on a compact NMR spectrometer with cucurbituril as the host molecule, which was applied in aqueous solution to a paper strip. (Black) Fit with Equation 17 through three RF saturation resonances. Figure 4b shows the z-spectrum of thermal xenon on a high-field NMR spectrometer with crypthophan as the host molecule, which was applied and dried to a paper strip. (Red) Fit with Equation 16 through three RF saturation resonances.The RF frequency in both spectra is referenced to the Larmor frequency of xenon gas at 0 Hz. References J, Jayapaul, L. Schröder. Molecular Sensing with Host Systems for Hyperpolarized 129Xe. Molecules 2020, 25, 4627 L. Mitschang, W. Kiliian, S. Korchak (2016) NMR method for quantitatively determining an analyte in a liquid sample using a hyperpolarized gas. EP 3330729 A1 L. Mitschang, W. Kiliian, S. Korchak (2021) Method for quantitatively determining a host molecule concentration (Ctot) of host molecules and concentration measuring device. DPMA 102021121238. G. Navon, Y.-Q. Song, T. Rõõm, S. Appelt, RE Taylor, A. Pines. Enhancement of Solution NMR and MRI with Laser-Polarized Xenon. Science 1996, 271, 1848–1851. A. Pines, David E. Wemmer, M. Spence, S. Rubin (2004) Functionalized active-nucleus complex sensor. US 2004 / 0062715 A1 M. Zaiss, M. Schnurr, P.Bachert (2012) Analytical solution of the depolarization of hyperpolarized nuclei by chemical saturation transfer between free and encapsulated xenon (HyperCEST), J. Chem. Phys. 136, 144106 (2012). Reference numeral 10 Measuring device i Counting index 12 Host molecule t Duration of radiofrequency saturation 14 Biomolecule Xeliq Dissolved xenon 16 Xenon binding molecule Xehost Host-bound xenon 18 Xenon atom Xegas enon in the gas phase C. w Host molecule concentration 20 Host molecule-xenon complex 22 Sample, sample film 24 Carrier 26 Gas phase 28 Sample holder 30 Xenon source 32 Magnet 34 High-frequency transmitter 36 Antenna 38 Computing unit 40 Depolarization change measuring device

Claims

Physikalisch-Technische Bundesanstalt Attorney's file: Braunschweig and Berlin 0454-0330 PCT-1 Bundesallee 100 38116 Braunschweig Date: March 17, 2024 Patent claims 1. Method for detecting xenon-binding host molecules (12) with the steps: (a) bringing the host molecules (12) into contact with a carrier (24), (b) bringing the host molecules (12) into contact with xenon from a gas phase (26) so that host molecules (12) form a host molecule-xenon complex (20) with xenon, (c) irradiating the host molecule-xenon complexes (20) with electromagnetic depolarization radiation for at least partially selectively depolarizing xenon in the host molecule-xenon complex (20), and (d) Detecting the host molecules (12) based on a nuclear magnetic polarization change of xenon in the gas phase (26).

2. The method according to claim 1, characterized in that the host molecules (12) are (a) dissolved or suspended in a solvent and / or (b) directly bonded to the carrier (24).PL / PL - PD002 / 20.

10. - 2- 3. Method according to one of the preceding claims, characterized in that (a) detecting the host molecules (12) comprises quantitatively determining a host molecule concentration (C W ) of host molecules (12) and (b) the host molecule concentration (C W) is determined from the nuclear magnetic polarization change. (c) Wherein the measurement of the nuclear magnetic polarization change is carried out by measuring a change in a nuclear magnetic resonance signal strength (S) of xenon in the gas phase (26) and / or by measuring a change in the magnetic field strength of the xenon in the gas phase (26).

4. Method according to one of the preceding claims, characterized in that the measurement of the polarization change comprises the following steps: (a) measuring the nuclear magnetic resonance signal strength (S) of xenon in the gas phase (26) for depolarizing radiation of different durations, in particular with a predetermined bandwidth, and a fixed depolarization frequency (fD) corresponding to a host molecule frequency (fW) at which the depolarization of the xenon in the host molecule-xenon complex (20) is greatest, so that a decay curve is obtained, and determining the host molecule concentration (C W) from the decay curve and / or (b) measuring the nuclear magnetic resonance signal strength (S) of the nuclear magnetic signal of xenon in the gas phase (26) for depolarization radiation of different depolarization frequencies, but well-defined bandwidth and fixed duration, so that the depolarization is obtained as a function of the depolarization frequency, and determining the host molecule concentration (C W ) from the polarization change as a function of the depolarization frequency.

5. Method according to one of the preceding claims, characterized by the steps (a) guiding the xenon from the gas phase (26) away from the carrier (24) and (b) detecting the polarization change of the xenon in the gas phase (26) at a distance from the carrier (24). - 3- 6. Method according to one of the preceding claims, characterized in that the xenon-binding host molecules (12) in the solvent are (a) applied to the carrier (24) as a thin film and / or (b) absorbed into the carrier (24).

7. Method according to one of the preceding claims, characterized in that (a) non-hyperpolarized xenon is used and (b) the host molecule concentration (C W ) in solvent environment is determined from the nuclear magnetic resonance signal strength (S) using the formula where R1 is the longitudinal relaxation rate of xenon in the gas phase (26) without electromagnetic irradiation, R1^ is the longitudinal relaxation rate of xenon in the gas phase (26) under irradiation of depolarizing radiation, Δ) ( = ^*^+ − ^*^#,^^ is the squared distance of the angular frequency^*^+ of the depolarization frequency fD from the Larmor angular frequency^*^#, of the host-bound xenon, -( = . / ) 0 is / 0 for the nutation frequency of the Xenonsu nd 1 455 is the exit rate for the dissolution of the host molecule-xenon complexes (20), M0 is the nuclear magnetic output signal of xenon in the gas phase (26) and is 126 for the formation rate of the xenon-host complexes, s for the solubility of xenon in the solvent in the environment of the xenon-binding host molecules (12), G for the ratio of gas to liquid volume in the effective volume of the electromagnetic radiation, R for the gas constants, - 4- T is the absolute temperature, K is the affinity constant for the xenon binding to the host molecule in the solvent environment, and p is the partial pressure of xenon in the gas phase (26).

8. Method according to one of the preceding claims, characterized in that (a) hyperpolarized xenon is used and the host molecule concentration C win a solvent environment from the nuclear magnetic resonance signal strength S of hyperpolarized xenon in the gas phase (26) is determined using the following formulas: m it (b) two or more different host molecules (12) and / or at least one host molecule forming at least two host molecule-xenon complexes (20) with different host molecule frequencies (fW1, fW2) are brought into contact with the carrier (24) and no hyperpolarized xenon is used and the host molecule concentration C w in a solvent environment is determined from the nuclear magnetic resonance signal strength S using the following formulas: K ombination Q R,S Y<VWX Z? ? ><[\]],^ and - 5- wherein the index i counts the different host molecule types and states, or (c) two or more different host molecules (12) and / or at least one host molecule forming at least two host molecule-xenon complexes (20) with different host molecule frequencies (fW1, fW2) are brought into contact with the carrier (24), hyperpolarized xenon is used and the host molecule concentration C w in a solvent environment is determined from the nuclear magnetic resonance signal strength S using the following formulas: K ombination t k VWX Z? mi TUU > QR,S = kQR,SY <VWX Z? ? und ><[\]],^ ^^^ ^ ^C $∑ ^,c ^ ∝ @ c ^^^,c A B ^^ ^ <9> I%J ^,c G ^,c,wherein the index i counts the different host molecule types and states, or (d) the host molecules (12) are applied directly to the carrier (24) and thus have direct contact with the gas phase (26) and the host molecule concentration is determined from the nuclear magnetic resonance signal strength S of the xenon in the gas phase (26) using the formula K ombination the respective expressions of the Signal strength S is determined.

9. Measuring device (10) for detecting xenon-binding host molecules (12), comprising (i) a sample holder (28) designed to receive a sample of carrier-bound host molecules (12), (ii) a xenon source (30) arranged to generate a gas phase (26) in contact with the sample, (iii) a radio-frequency transmitter (34) designed to automatically irradiate the sample with electromagnetic depolarization radiation - 6- for selectively depolarizing xenon in the host molecule-xenon complex (20), (iv) a depolarization change measuring device (40) for measuring a polarization change of xenon in the gas phase (26), and (v) a computing unit (38) designed to automatically carry out a method comprising the steps of: (a) controlling the depolarization change measuring device (40) to detect a polarization change of xenon in the gas phase (26), and (b) detecting the host molecules (12) based on the polarization change. 10.Measuring device (10) according to claim 9, characterized in that (a) the depolarization change measuring device (40) is a magnetic resonance measuring unit and (b) the computing unit (38) is designed to automatically carry out a method comprising the steps of (i) controlling the magnetic resonance measuring unit so that it emits depolarization radiation of a specific frequency (fD), duration and amplitude, (ii) measuring the nuclear magnetic resonance signal strength (S), and (iii) calculating the host molecule concentration (C. W ) from the signal strength (S).

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