A method for quantitatively detecting the concentration of Xe molecular probes based on small-angle pulse excitation

Abstract

The present invention discloses a method for quantitatively detecting the concentration of Xe molecular probe based on small-angle pulse excitation, comprising the following steps: preparing a Xe molecular probe solution with a concentration to be measured; introducing a hyperpolarized Xe mixed gas into the solution to be measured, stopping the gas introduction and standing for a time t 静置 ; setting the experimental parameters of the small-angle excitation pulse sequence and sampling to obtain a series of dissolved Xe signal intensities; based on the data obtained in the previous step, fitting the concentration ratio fr of the Xe molecule probe-bound Xe to the dissolved Xe in the solution to be measured according to the formula; calculating the concentration of the Xe molecular probe in the solution to be measured according to the formula. The present invention can reduce the influence of the Xe signal fluctuation during multiple gas introductions in the prior art on the quantitative accuracy of the Xe molecular probe concentration, and reduce the time required for sampling.

Description

Technical Field

[0001] The present invention belongs to the fields of magnetic resonance technology and analytical measurement, and particularly relates to a method for quantitatively detecting the concentration of Xe molecular probe based on small-angle pulse excitation, which is applicable to measuring the concentration of Xe molecular probe in solution. Background Art

[0002] In recent years, Xe molecular probes using Spin Exchange Optical Pumping (SEOP) technology and Chemical Exchange Saturation Transfer (CEST) technology have been widely used in the detection of biological macromolecules, metabolites, metal ions, etc. Thanks to the enhanced detection sensitivity brought by the spin exchange optical pumping technology and the chemical exchange saturation transfer technology, ultrasensitive detection of Xe molecular probes at the 10 -12 M concentration level has been achieved.

[0003] Currently, the concentration quantification methods for low-concentration Xe molecular probes mainly include: (1) fitting the CESTz spectral data of the Xe molecular probe [Analytical solution for the depolarization of hyperpolarized nuclei by chemical exchange saturation transfer between free and encapsulated xenon (HyperCEST). J. Chem. Phys., 2012, 136: 144106.]; (2) measuring the relationship between the CEST effect and the concentration at different saturation irradiation intensities or saturation irradiation times [Quantitative Chemical Exchange Saturation Transfer with Hyperpolarized Nuclei (qHyper-CEST): Sensing Xenon-Host Exchange Dynamics and Binding Affinities by NMR. J. Chem. Phys., 2014, 141: 194202.]; (3) measuring the linear relationship between the depolarization rate of dissolved Xe and the concentration of the Xe molecular probe [Quantitative biosensor detection by chemically exchanging hyperpolarized 129Xe. Phys. Chem. Chem. Phys., 2018, 20: 1800 - 1808; (4) Methods such as measuring the contrast effect during selective inversion pulse-long interval time repeated irradiation disclosed in CN111505039B are used to obtain the molecular probe concentration information in the solution to be measured [Molecular Concentration Determination Using Long-Interval Chemical Exchange Inversion Transfer (CEIT) NMR Spectroscopy. J. Phys. Chem. Lett., 2021, 12: 8652 - 8657]. However, due to the irrecoverability of the hyperpolarized Xe signal, it is usually necessary to perform multiple ventilations to update the Xe signal in the solution. On the one hand, this leads to a long required sampling time. On the other hand, the signal fluctuations of the multiple injections of the hyperpolarized Xe mixture gas will affect the magnitude of the dissolved Xe signal in the solution, thereby affecting the accuracy of the quantitative detection of the probe molecule concentration. Summary of the Invention

[0004] The object of the present invention is to overcome the disadvantages and deficiencies existing in the existing quantitative methods for Xe molecular probe concentration, and propose a quantitative detection method for Xe molecular probe concentration based on small-angle pulse excitation. This method samples through multiple small-angle pulse excitations, reducing the number of ventilations required during the scanning process of implementing the detection, so that this method has the characteristics of short sampling time and avoiding the influence of Xe signal fluctuations during multiple ventilations on concentration quantification.

[0005] The steps to achieve the object of the present invention are as follows:

[0006] A quantitative detection method for Xe molecular probe concentration based on small-angle pulse excitation includes the following steps:

[0007] Step 1, prepare a solution to be measured of Xe molecular probe, and measure the binding constant K between the Xe molecular probe and Xe;

[0008] Step 2, introduce hyperpolarized Xe mixture gas into the solution to be measured, and let it stand for t 静置 after;

[0009] Step 3, apply a small-angle excitation pulse sequence to the solution to be measured N times, and sample to obtain the dissolved Xe signal intensity S of the Xe molecular probe solution to be measured i , where i is the order of applying the small-angle excitation pulse sequence to the solution to be measured;

[0010] The small-angle excitation pulse sequence applied for the i-th time includes an 180° selective inversion pulse and an interval time t 间隔Pretreatment of the 180° radiofrequency pulse block and subsequent small-angle excitation pulse α. When the small-angle excitation pulse sequence is applied for the i-th time, the 180° radiofrequency pulse block is repeated n i times. The 180° pulse is a shaped pulse with a frequency offset of f1, and the small-angle excitation pulse α is a bp pulse used to excite the dissolved Xe signal for sampling detection. The degree α of the small-angle excitation pulse has a frequency offset of f2;

[0011] Step 4: Based on the dissolved Xe signal intensities S of the N Xe molecular probe solutions to be measured obtained successively in Step 3 i , according to the formula, fit the concentration ratio fr between the Xe bound to the Xe molecular probe and the dissolved Xe in the Xe molecular probe solution to be measured. S N is the integral value of the dissolved Xe signal in the Xe NMR spectrum when the 180° radiofrequency pulse block is repeated n i times during the excitation sampling of the N-th repetition of the small-angle excitation pulse sequence,

[0012]

[0013] t aq is the acquisition data time when the small-angle excitation pulse sequence is applied for sampling each time. T1 represents the longitudinal relaxation time of the dissolved Xe in the Xe molecular probe solution to be measured, and S is the constant value to be fitted;

[0014] Step 5: Calculate the concentration of the Xe molecular probe in the Xe molecular probe solution to be measured according to the following formula,

[0015] [Xe molecular probe] 待测 =(1 / K + [Xe]) * fr

[0016] [Xe] is the concentration of the dissolved Xe during the test of the Xe molecular probe solution to be measured. [Xe] = Xe partial pressure * Xe solubility. The Xe partial pressure is the product of the total pressure of the mixed gas used in Step 2 and the proportion of Xe in the mixed gas.

[0017] Furthermore, the Xe molecular probe in Step 1 is a carrier capable of reversibly binding Xe, including cryptophane, cucurbituril, zeolitic imidazolate framework, etc. and their derivatives.

[0018] Furthermore, the hyperpolarized Xe mixed gas in Step 2 is prepared by a hyperpolarization device based on spin exchange optical pumping technology. The ventilation time and the standing time t 静置 are set to 3 - 60 s and 0.1 - 5 s respectively.

[0019] Furthermore, the frequency offset f1 in Step 3 is the resonance frequency difference between Xe and the dissolved Xe after the Xe molecular probe binds Xe, and the frequency offset f2 is the resonance frequency of the dissolved Xe. The interval time is set to t间隔 ≥3*t 滞留 ,t 滞留 is the residence time of Xe in the Xe molecular probe. When the small-angle excitation pulse sequence is applied for the i-th time, the 180° RF pulse block is repeated n i times, where n is an even number not less than 2, and α is 3° - 20°.

[0020] Further, the Xe solubility in step 5 is obtained by looking up a table according to the temperature of the solution in step 3.

[0021] The present invention is a method for quantitatively detecting the concentration of a Xe molecular probe based on small-angle pulse excitation, which can directly measure the concentration of the Xe molecular probe under certain conditions. Compared with the existing methods for quantitatively detecting the concentration of a Xe molecular probe: this method only needs to introduce the hyperpolarized Xe mixed gas once to complete the subsequent scanning. On the one hand, it shortens the total time required for data acquisition, and on the other hand, it avoids the influence of the hyperpolarized Xe signal fluctuation caused by multiple gas injections required by other quantitative methods on the accuracy of concentration quantification. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a diagram of a small-angle excitation pulse sequence. First, introduce the hyperpolarized Xe mixed gas and let it stand for t 静置 to update the Xe signal in the solution. Secondly, perform n i times of 180° selective inversion pulses with a frequency offset of f1 and an interval time of t 间隔 for preprocessing. Subsequently, use small-angle pulses α with a frequency offset of f2 to excite the dissolved Xe signal and sample, and repeat the preprocessing and sampling processes N times according to the experimental settings;

[0023] Figure 2 is a schematic flow diagram of a method for quantitatively detecting the concentration of a Xe molecular probe based on small-angle pulse excitation according to the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] The present invention will be further described below with reference to the accompanying drawings. It should be understood that the embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0025] A method for quantitatively detecting the concentration of a Xe molecular probe based on small-angle pulse excitation. The small-angle excitation pulse sequence is as Figure 1 shown. First, introduce the hyperpolarized Xe mixed gas and let it stand for t 静置 to update the Xe signal in the solution. Secondly, perform n i times of 180° selective inversion pulses with a frequency offset of f1 and an interval time of t 间隔The pretreatment of the 180° RF pulse block is then performed using a small-angle pulse α with a frequency bias of f2 to excite and sample the dissolved Xe signal, and the pretreatment and sampling process is repeated N times according to the experimental settings.

[0026] A method for quantitative detection of Xe molecular probe concentration based on small-angle pulse excitation comprises the following steps:

[0027] Step 1, preparing a Xe molecular probe solution of a concentration to be measured;

[0028] A Xe molecular probe solution with a known concentration to be tested is prepared. The Xe molecular probe is a carrier that can reversibly bind to Xe, including Xe carrier molecules such as cryptanthide, cucurbituril, zeolite imidazolate skeleton and their derivatives.

[0029] Cryptophane includes a metallized cyclophane main molecule (Cryptophane cage) formed by connecting cyclotriveratrol and its enantiomers through three bridges as disclosed in the invention patent WO2012051323A2;

[0030] Cucurbituril is a glycoluril connected by a methylene bridge (-CH2-) A macrocyclic molecule made from monomers. The oxygen atoms are positioned along the edges of the band and tilted inward to form a partially enclosed cavity. Cucurbituril is usually written as cucurbituril [n] uril, where n is the number of units of glycoluril (and n can be, for example, 5, 6, 7, 8, and 10). Two common abbreviations are CB [n], or CB n for short. Cucurbituril is an effective molecular recognition host molecule and has a particularly high affinity for positively charged or cationic compounds. They have been identified as effective Hyper-CEST solutes (Kunth et al., 2015). EP3251702A1 discloses CB [6] uril, CB [7] uril, CB [8] uril, CB [9] uril and CB

[10] uril, all of which can be used as Xe molecular probes.

[0031] The zeolite imidazolate framework is a sheet-like network structure with micropores formed by connecting carboxylic acid MOF ligands and metal A ion ligands. Each metal A ion ligand is connected to a deprotonated carboxyl group in the six carboxylic acid MOF ligands. The inner diameter of the micropore is

[0032] Step 2: Pass the hyperpolarized Xe mixed gas through the solution to be tested and let it stand for t 静置 back;

[0033] The sample tube containing the solution to be tested is connected to the hyperpolarization device based on the spin exchange optical pump technology, and then placed in the magnetic resonance detection equipment. The hyperpolarized Xe mixed gas is passed through at a certain flow rate and pressure. After a period of ventilation, the ventilation is stopped and the tube is left to stand for t 静置 , ventilation time and rest time t静置 They are respectively set to 3 to 60 s and 0.1 to 5 s.

[0034] Step 3: Apply the small-angle excitation pulse sequence to the solution to be measured N times. After each application, sample to obtain the dissolved-state Xe signal intensity S of the Xe molecular probe solution to be measured i , where i is the order of applying the small-angle excitation pulse sequence to the solution to be measured;

[0035] The small-angle excitation pulse sequence applied for the i-th time includes preprocessing of a 180° radiofrequency pulse block composed of a 180° selective inversion pulse and an interval time t 间隔 and subsequent small-angle excitation pulse α. When the small-angle excitation pulse sequence is applied for the i-th time, the 180° radiofrequency pulse block is repeated n i times. The 180° pulse is a shaped pulse with a frequency offset of f1, and the small-angle excitation pulse α is a bp pulse used to excite the dissolved-state Xe signal for sampling detection. The degree α of the small-angle excitation pulse has a frequency offset of f2. Among them, the frequency offset f1 is the resonance frequency difference between Xe and dissolved-state Xe after Xe binds to the Xe molecular probe, and the frequency offset f2 is the resonance frequency of dissolved-state Xe. The interval time is set to t 间隔 ≥3*t 滞留 , t 滞留 is the residence time of Xe in the Xe molecular probe. When the small-angle excitation pulse sequence is applied for the i-th time, the number of repetitions n of the 180° radiofrequency pulse block i is an even number not less than 2, and α is 3° to 20°.

[0036] Step 4: Based on the N dissolved-state Xe signal intensities S of the Xe molecular probe solution to be measured obtained successively in Step 3 i , fit the concentration ratio fr between the Xe bound to the Xe molecular probe and the dissolved-state Xe in the Xe molecular probe solution to be measured according to the formula, where S N is the integral value of the dissolved-state Xe signal in the Xe NMR spectrum for the number of repetitions n of the 180° radiofrequency pulse block during the excitation sampling of the N-th repetition of the small-angle excitation pulse sequence i ,

[0037]

[0038] t aq is the acquisition data time for each application of the small-angle excitation pulse sequence for sampling. T1 represents the longitudinal relaxation time of the dissolved-state Xe in the Xe molecular probe solution to be measured. S is the constant value to be fitted, and is the unknown.

[0039] Step 5: Calculate the concentration of the Xe molecular probe in the Xe molecular probe solution to be measured according to the following formula

[0040] [Xe molecular probe]待测 =(1 / K + [Xe]) * fr

[0041] [Xe] is the dissolved Xe concentration during the test of the Xe molecular probe solution to be measured, [Xe] = Xe partial pressure * Xe solubility, and the Xe partial pressure is the product of the total pressure of the mixed gas used in step 2 and the proportion of Xe in the mixed gas; the Xe solubility is obtained by looking up the table according to the temperature of the solution in step 3.

[0042] Example 1

[0043] In this example, the cryptophane derivative CrA-(COOH)6 is used as the Xe molecular probe for quantitative detection of the concentration of the Xe molecular probe based on small-angle pulse excitation, and all magnetic resonance-related detection is completed on a Bruker 400M wide-bore spectrometer.

[0044] A method for quantitative detection of the concentration of CrA-(COOH)6 based on small-angle pulse excitation includes the following steps:

[0045] Step 1, prepare an aqueous solution to be measured of the Xe molecular probe with a concentration of 2.500 μM. The Xe molecular probe is CrA-(COOH)6, and the binding constant K of CrA-(COOH)6 with Xe is K = 3148.184 M as shown in the example of CN111505039B -1 ;

[0046] K = [Xe@molecular probe] / (([Xe molecular probe] - [Xe@molecular probe]) * [Xe])

[0047] where, [Xe] y is the dissolved Xe concentration in the solution = (Xe partial pressure * Xe solubility). The Xe partial pressure is the product of the total pressure of the mixed gas used during the test and the proportion of Xe in the mixed gas. The Xe solubility depends on the temperature of the solution during the test and can be obtained by looking up the table. [Xe molecular probe] is the concentration of the Xe molecular probe in the prepared solution, and [Xe@molecular probe] represents the concentration of the Xe molecular probe bound to Xe. [Xe@molecular probe] = I1 / I2 * [Xe] y , where I1 and I2 are the integral values of the concentration of the Xe molecular probe bound to Xe and the dissolved Xe concentration in the solution, respectively. The values of I1 and I2 are obtained by integrating the signal ranges of the Xe molecular probe bound to Xe and the dissolved Xe signal range in the 1D Xe NMR spectrum, respectively.

[0048] Step 2: After connecting the sample tube containing the CrA-(COOH)6 solution to be measured to the hyperpolarization device based on the spin-exchange optical pumping technique, place it in the nuclear magnetic resonance detection equipment. When the total pressure of the hyperpolarized Xe mixed gas (the volume ratio of Xe is 0.02) is 4.477 atm, ventilate the solution to be measured at a flow rate of 0.1 standard liters per minute for 60 s, stop ventilation and let it stand for a time t 静置 = 3 s, and prepare for sampling.

[0049] Step 3: Use a small-angle excitation pulse sequence for sampling. The small-angle excitation pulse sequence is applied to the solution to be measured N times. i is the order of applying the small-angle excitation pulse sequence to the solution to be measured. The small-angle excitation pulse sequence applied for the i-th time includes a preprocessing of an 180° selective inversion pulse block composed of an 180° selective inversion pulse and an interval time t 间隔 and subsequent excitation and sampling by a small-angle pulse α. When the small-angle excitation pulse sequence is applied for the i-th time, the 180° radiofrequency pulse block is repeated n i times. The 180° pulse is a shaped pulse with a frequency offset of f1. The small-angle excitation pulse α is a rectangular pulse used to excite the dissolved Xe signal for sampling detection. The degree α of the small-angle excitation pulse has a frequency offset of f2.

[0050] In this embodiment, the total number of samplings N excited by the small-angle excitation pulse sequence is 8. The repetition times n1 to n8 of the 180° radiofrequency pulse block before the i-th (i = 1 to 8) small-angle excitation samplings are 2, 4, 6, 8, 10, 12, 14, and 16 respectively. The small-angle excitation pulse α = 15°. The frequency offset f1 is the resonance frequency difference between CrA-(COOH)6-bound Xe and dissolved Xe, f1 = -14889.15 Hz. The frequency offset f2 = -14889.15 Hz is the resonance frequency of dissolved Xe. The interval time is t 间隔 = 0.124 s ≥ 3*t 滞留 t 滞留 is the residence time of Xe in the Xe molecular probe. The sample temperature is set to 298 K during the test. Integrate the dissolved Xe signals in the Xe NMR spectra obtained when the repetition times n1 to n8 of the 180° radiofrequency pulse block are 2, 4, 6, 8, 10, 12, 14, and 16 respectively during the i-th (i = 1 to 8) small-angle excitation pulse excitation samplings, and obtain the corresponding integral values S1 to S8 of the dissolved Xe signals as 9.725, 9.471, 8.655, 7.603, 6.515, 5.729, 4.940, and 4.279 respectively.

[0051] Step 4: Based on the data obtained in Step 3, according to the formula

[0052]

[0053] Calculate the concentration ratio fr between the Xe bound to CrA-(COOH)6 in the solution to be measured and the dissolved Xe in the solution to be measured by solving simultaneous equations, where S is the constant value to be fitted, and the data acquisition time t aq = 0.983 s for each application of the small-angle excitation pulse, and the longitudinal relaxation time T1 of the dissolved Xe in the Xe molecular probe solution to be measured is 121.7 s. Generally, repeating the application of the small-angle excitation pulse sequence to the solution to be measured at least 2 times is sufficient. By solving two equations simultaneously, fr can be obtained by fitting. If the application of the small-angle excitation pulse sequence to the solution to be measured is repeated more than 2 times, the obtained fr is more accurate.

[0054] Using Matlab fitting, fr = 0.003568 is obtained.

[0055] Step 5. Further, calculate the concentration of the Xe molecular probe in the Xe molecular probe solution to be measured according to the following formula

[0056] [Xe molecular probe] 待测 = (1 / K + [Xe]) * fr

[0057] The binding constant K between CrA-(COOH)6 and Xe is 3148.184 M -1 , [Xe] is the concentration of the dissolved Xe during the measurement of the Xe molecular probe solution to be measured, [Xe] = (Xe partial pressure * Xe solubility). The Xe partial pressure is the product of the total pressure of the mixed gas used in Step 3 and the proportion of Xe in the mixed gas. The total pressure of the hyperpolarized Xe mixed gas used in Step 3 is 4.477 atm, and the volume ratio of Xe in the mixed gas is 0.02. The Xe partial pressure in the mixed gas is 4.477 atm * 0.02 = 0.08954 atm. The temperature of the solution to be measured in Step 3 is 298 K. Looking up the table, the solubility of Xe in the solution at 298 K is 4.4 mM / atm. The concentration of the dissolved Xe in the solution to be measured [Xe] = 0.08954 atm * 4.4 mM / atm = 394 μM. Substitute into the formula

[0058] [Xe molecular probe] 待测 = (1 / K + [Xe]) * fr

[0059] The concentration of CrA-(COOH)6 in the solution to be measured is calculated to be 2.539 μM. The difference between the measured concentration of the Xe molecular probe in the solution and the prepared concentration is small. The quantitative detection method of the Xe molecular probe concentration based on small-angle pulse excitation can achieve the rapid measurement of the Xe molecular probe concentration in the solution to be measured.

[0060] It should be noted that the specific embodiments described in the present invention are only illustrative examples of the present invention. Those skilled in the art to which the present invention pertains can make various modifications or supplements to the described specific embodiments or use similar means for substitution, but will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. A method for quantitatively detecting the concentration of Xe molecular probe based on small-angle pulse excitation, comprising the following steps: Step 1: Prepare the test solution of the Xe molecular probe and measure the binding constant K between the Xe molecular probe and Xe; Step 2, introduce a hyperpolarized Xe mixed gas into the solution to be measured and let it stand for t 静置 after that; Step 3, apply the small-angle excitation pulse sequence to the solution to be measured N times, and sample to obtain the dissolved-state Xe signal intensity S of the Xe molecular probe solution to be measured after each application, where i is the order of applying the small-angle excitation pulse sequence to the solution to be measured; i ​ The small-angle excitation pulse sequence applied for the i-th time includes a 180° RF pulse block composed of a 180° selective inversion pulse and an interval time t 间隔 and a subsequent small-angle excitation pulse. When the small-angle excitation pulse sequence is applied for the i-th time, the 180° RF pulse block is repeated n i times. The 180° selective inversion pulse is a shaped pulse with a frequency offset of f1, and the small-angle excitation pulse is a bp pulse used to excite the dissolved Xe signal for sampling detection. The degree of the small-angle excitation pulse is α, and its frequency offset is f2. Among them, the frequency offset f1 is the resonance frequency difference between Xe and dissolved Xe after the Xe molecular probe binds to Xe, and the frequency offset f2 is the resonance frequency of dissolved Xe; Step 4, based on the dissolved Xe signal intensities S of the N Xe molecular probe solutions to be measured obtained successively in Step 3 i , according to the formula, fit the concentration ratio fr between the Xe bound to the Xe molecular probe and the dissolved Xe in the Xe molecular probe solution to be measured, S N is the integral value of the dissolved Xe signal in the Xe NMR spectrum when the 180° RF pulse block is repeated n i times during the excitation sampling of the Nth repeated small-angle excitation pulse sequence t aq It is the acquisition data time for each application of a small-angle excitation pulse sequence for sampling. T1 represents the longitudinal relaxation time of dissolved Xe in the Xe molecular probe solution to be measured, and S is the constant value to be fitted. Step 5: Calculate the concentration of the Xe molecular probe in the test Xe molecular probe solution according to the following formula, [Xe Molecular Probe] 待测 =(1 / K + [Xe]) * fr [Xe] is the concentration of dissolved Xe during the test of the test Xe molecular probe solution, [Xe] = Xe partial pressure * Xe solubility. The Xe partial pressure is the product of the total pressure of the mixed gas used in Step 2 and the proportion of Xe in the mixed gas.

2. The method according to claim 1, characterized in that: The Xe molecular probe described in Step 1 is a carrier capable of reversibly binding Xe, including cryptophane, cucurbituril, zeolitic imidazolate framework and their derivatives.

3. The method according to claim 1, characterized in that: The hyperpolarized Xe mixed gas described in Step 2 is prepared by a hyperpolarization device based on spin exchange optical pumping technology, and the gas injection time and the standing time t 静置 are respectively set to 3 to 60 s and 0.1 to 5 s.

4. The method according to claim 1, characterized in that: The interval time is set to t 间隔 ≥3*t 滞留 , t 滞留 is the residence time of Xe in the Xe molecular probe. When the small-angle excitation pulse sequence is applied for the i-th time, the 180° RF pulse block repeats n i times, which is an even number not less than 2, and α is 3° to 20°.

5. The method according to claim 1, characterized in that: The Xe solubility in Step 5 is obtained by looking up the table according to the temperature of the solution in Step 3.

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