阅读说明：本技术 基于饱和能量非均匀分布的Xe分子探针浓度快速定量测量方法 (Xe molecular probe concentration rapid quantitative measurement method based on saturation energy non-uniform distribution ) 是由 周欣 蒋卫平 娄昕 于 2020-04-30 设计创作，主要内容包括：本发明公开了基于饱和能量非均匀分布(Saturation Energy Nonuniform Distributed,SEND)的Xe分子探针浓度快速定量测量新方法,包括以下步骤：配制已知浓度Xe分子探针溶液,测量Xe分子探针与Xe的结合常数K；配制待测浓度Xe分子探针溶液,分别进行频率偏置为Δω<Sub>rf</Sub>和-Δω<Sub>rf</Sub>的SEND脉冲序列实验,采样获得待测溶液溶解态Xe信号强度S1和S2；计算SEND对比效果SENDR；根据公式计算待测溶液中Xe分子探针浓度。本方法采用饱和能量非均匀分布方法,可以快速测量溶液中Xe分子探针的浓度。(The invention discloses a new method for rapidly and quantitatively measuring the Xe molecular probe concentration based on the Nonuniform distribution of Saturated Energy (SEND), which comprises the following steps: preparing a Xe molecular probe solution with a known concentration, and measuring the binding constant K of the Xe molecular probe and Xe; preparing Xe molecular probe solution with concentration to be measured, and respectively carrying out frequency bias to be delta omega rf And- Δ ω rf The SEND pulse sequence experiment, sampling to obtain the dissolved Xe signal intensity S1 and S2 of the solution to be measured; calculating SEND contrast effect SENDR; and calculating the concentration of the Xe molecular probe in the solution to be detected according to a formula. The method adopts a saturated energy non-uniform distribution method, and can quickly measure the concentration of the Xe molecular probe in the solution.)

1. The Xe molecular probe concentration rapid quantitative measurement method based on saturation energy non-uniform distribution comprises the following steps:

step 1, preparing a Xe molecular probe solution with a known concentration, and measuring the binding constant K of the Xe molecular probe and Xe;

step 2, preparing a Xe molecular probe solution to be detected, and setting the frequency bias to be delta omega_rfThe SEND pulse sequence is applied to the Xe molecular probe solution to be detected, and sampling is carried out to obtain the dissolved Xe signal strength S1 of the Xe molecular probe solution to be detected;

step 3, setting the frequency offset as-delta omega_rfThe SEND pulse sequence is applied to the Xe molecular probe solution to be detected, and sampling is carried out to obtain the dissolved Xe signal strength S2 of the Xe molecular probe solution to be detected;

step 4, calculating contrast effect SENDR according to the formula SENDR of S1/S2;

step 5, calculating the Xe molecular probe concentration in the Xe molecular probe solution to be measured according to the following formula

[ Xe molecular probe to be measured]＝(1/K+[Xe])*(1-SENDR^(1/N))/2

Wherein [ Xe ] is the dissolved Xe concentration in 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 step 3 and the proportion of Xe in the mixed gas, the Xe solubility depends on the temperature of the solution at the time of the test, obtained by looking up a table, the SENDR is the contrast effect, and N is the number of repetitions of a 180 ° rf pulse block in the SEND pulse sequence.

2. The method of claim 1, wherein: in step 1, the bonding constant K of the Xe molecular probe to Xe is measured by using a 1D Xe NMR spectroscopy method, but is not limited to this method, and the calculation formula of the bonding constant K is

K ═ Xe @ molecular probe]/([ molecular Probe)]- [ Xe @ molecular Probe])*[Xe]_y)

Wherein [ Xe molecular probe]Is the solution of step 1Xe molecular probe concentration in liquid, [ Xe @ molecular probe]Indicates the concentration of Xe molecular probe binding to Xe, [ Xe @ molecular probe]＝I1/I2*[Xe]_yI1 and I2 are the integrated value of the Xe molecular probe concentration bound to Xe in solution and the integrated value of the dissolved Xe concentration, respectively, and the values of I1 and I2 are obtained by integrating the Xe molecular probe signal range bound to Xe and the dissolved Xe signal range in 1D Xe NMR spectrum, respectively, [ Xe]_yFor dissolved Xe concentration in Xe molecular Probe solution in step 1, [ Xe]_yThe Xe solubility is obtained by table lookup, depending on the temperature of the solution in step 1, by multiplying the total pressure of the mixed gas used in step 1 by the proportion of Xe in the mixed gas (Xe partial pressure × Xe solubility).

3. The method of claim 1, wherein: in step 2 and step 3, the SEND pulse sequence comprises pretreatment of 180 DEG radio frequency pulse block composed of 180 DEG pulse and interval time t, and subsequent 90 DEG bp pulse with frequency offset of f2, the 180 DEG radio frequency pulse block is repeated for N times, the 90 DEG bp pulse is used for exciting dissolved Xe signal for sampling detection, wherein, the 180 DEG pulse is a shape pulse with frequency offset of f1, and f1 in step 2 and step 3 is respectively set to be delta omega_rfAnd- Δ ω_rf，Δω_rfShows the frequency difference between Xe molecular probe signal combined with Xe and dissolved Xe signal, and is used for realizing the selective operation of 180 DEG pulse to the Xe molecular probe signal combined with Xe, -delta omega_rfThe frequency difference between the symmetrical position of the Xe molecular probe signal combined with Xe relative to the dissolved Xe signal and the dissolved Xe signal is shown, the frequency difference is used for obtaining the influence of external interference factors of the Xe molecular probe signal combined with Xe on the dissolved Xe signal, the interval time t is more than or equal to 3 times of residence time, and the residence time is the residence time of Xe in the Xe molecular probe so as to ensure that the Xe molecular probe signal combined with Xe and the dissolved Xe signal are fully exchanged.

4. The method of claim 1, wherein: s1 and S2 set the RF pulse frequency offset f1 of 180 degrees in step 2 and step 3 to delta omega_rfAnd- Δ ω_rfIn the method, the SEND pulse sequence is respectively applied to the Xe molecular probe solution to be measured by using the signal intensity of the dissolved Xe obtained by the SEND pulse sequence to obtain a 1D Xe NMR spectrum, the dissolved Xe signal range in the 1D Xe NMR spectrum is integrated, and the integrated intensity of the dissolved Xe signal of the solution to be measured is measured and recorded.

5. The method of claim 1, wherein: in step 4, if the contrast effect SENDR obtained in step 4 is greater than 0.95, the repetition number N of the 180 ° radio frequency pulse block is increased, and if the contrast effect SENDR obtained in step 4 is less than 0.05, the repetition number N of the 180 ° radio frequency pulse block is decreased, and then step 2 and step 3 are repeated until the SENDR is not greater than 0.95 and not less than 0.05.

Technical Field

The invention belongs to the field of magnetic resonance technology and analytical measurement, and particularly relates to a novel method for rapidly and quantitatively measuring the concentration of a Xe molecular probe based on non-uniform distribution of saturated energy.

Background

In recent years, researchers at home and abroad have developed many different Xe molecular probes such as psammophyte, nanoemulsion, cucurbituril, gas capsule, protein, and the like. These molecular probes, especially the probes, have been widely used for the detection of biological macromolecules (such as proteins, nucleic acids, enzymes, etc.), metabolites (thiols), metal ions (zinc, mercury, lead, cadmium, etc.), and micro-environmental parameters (temperature or pH). By combining Spin Exchange Optical Pumping (SEOP) hyperpolarized Xe technology and Chemical Exchange Saturation Transfer (CEST) technology, the detection limit of the Xe molecular probe can reach pM magnitude. However, the method for rapidly and quantitatively detecting the low-concentration Xe molecular probe is still lacking.

The existing quantitative method of Xe molecular probe mainly comprises the following steps: the method comprises the steps of fitting CEST z spectrum data of the Xe molecular probe, measuring the relation between different saturation irradiation intensities or saturation irradiation time and the CEST effect, and measuring the linear relation between the depolarization rate of dissolved Xe and the concentration of the Xe molecular probe during saturation irradiation to obtain the concentration of the molecular probe and kinetic information, but the methods have the defects of long sampling consumption time, complex data processing steps and the like.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the existing quantitative method, and provides a novel method for rapidly and quantitatively measuring the concentration of a Xe molecular probe based on non-uniform distribution of saturated energy.

The invention aims to realize the following steps:

the Xe molecular probe concentration rapid quantitative measurement method based on the non-uniform distribution SEND of saturation energy comprises the following steps:

step 1, preparing a Xe molecular probe solution with a known concentration, and measuring the binding constant K of the Xe molecular probe and Xe;

step 2, preparing a Xe molecular probe solution to be detected, and setting the frequency bias to be delta omega_rfThe SEND pulse sequence is applied to the Xe molecular probe solution to be detected, and sampling is carried out to obtain the dissolved Xe signal strength S1 of the Xe molecular probe solution to be detected;

step 3, setting the frequency offset as-delta omega_rfApplying the SEND pulse sequence to Xe molecular probe solution to be measured, and samplingObtaining the dissolved Xe signal intensity S2 of the Xe molecular probe solution to be detected;

step 4, calculating contrast effect SENDR according to the formula SENDR of S1/S2;

step 5, calculating the Xe molecular probe concentration in the Xe molecular probe solution to be measured according to the following formula

[ Xe molecular probe to be measured]＝(1/K+[Xe])*(1-SENDR^(1/N))/2

Wherein [ Xe ] is the dissolved Xe concentration in 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 step 3 and the proportion of Xe in the mixed gas, the Xe solubility depends on the temperature of the solution at the time of the test, obtained by looking up a table, the SENDR is the contrast effect, and N is the number of repetitions of a 180 ° rf pulse block in the SEND pulse sequence.

Further, in step 1, the binding constant K of the Xe molecular probe to Xe is measured by using a 1D Xe NMR spectroscopy method but is not limited to this method, and the calculation formula of the binding constant K is

K ═ Xe @ molecular probe]/([ molecular Probe)]- [ Xe @ molecular Probe])*[Xe]_y)

Wherein [ Xe molecular probe][ Xe @ molecular Probe ] concentration of Xe molecular Probe in the solution of step 1]Indicates the concentration of Xe molecular probe binding to Xe, [ Xe @ molecular probe]＝I1/I2*[Xe]_yI1 and I2 are the integrated value of the Xe molecular probe concentration bound to Xe in solution and the integrated value of the dissolved Xe concentration, respectively, and the values of I1 and I2 are obtained by integrating the Xe molecular probe signal range bound to Xe and the dissolved Xe signal range in 1D Xe NMR spectrum, respectively, [ Xe]_yFor dissolved Xe concentration in Xe molecular Probe solution in step 1, [ Xe]_yThe Xe solubility is obtained by table lookup, depending on the temperature of the solution in step 1, by multiplying the total pressure of the mixed gas used in step 1 by the proportion of Xe in the mixed gas (Xe partial pressure × Xe solubility).

Further, in step 2 and step 3, the SEND pulse sequence includes a pre-treatment of a 180 DEG RF pulse block consisting of 180 DEG pulses and a separation time t, and a subsequent 90 DEG bp pulse with a frequency offset of f2, 180 DEG RF pulseRepeating the pulse block for N times, wherein the 90-degree bp pulse is used for exciting the dissolved Xe signal for sampling detection, the 180-degree pulse is a shape pulse with the frequency offset of f1, and f1 is respectively set to be delta omega in the step 2 and the step 3_rfAnd- Δ ω_rf，Δω_rfShows the frequency difference between Xe molecular probe signal combined with Xe and dissolved Xe signal, and is used for realizing the selective operation of 180 DEG pulse to the Xe molecular probe signal combined with Xe, -delta omega_rfThe frequency difference between the symmetrical position of the Xe molecular probe signal combined with Xe relative to the dissolved Xe signal and the dissolved Xe signal is shown, the frequency difference is used for obtaining the influence of external interference factors of the Xe molecular probe signal combined with Xe on the dissolved Xe signal, the interval time t is more than or equal to 3 times of residence time, and the residence time is the residence time of Xe in the Xe molecular probe so as to ensure that the Xe molecular probe signal combined with Xe and the dissolved Xe signal are fully exchanged.

Further, S1 and S2 are set to Δ ω for the 180 ° rf pulse frequency offset f1 in step 2 and step 3, respectively_rfAnd- Δ ω_rfIn the method, the SEND pulse sequence is respectively applied to the Xe molecular probe solution to be measured by using the signal intensity of the dissolved Xe obtained by the SEND pulse sequence to obtain a 1D Xe NMR spectrum, the dissolved Xe signal range in the 1D Xe NMR spectrum is integrated, and the integrated intensity of the dissolved Xe signal of the solution to be measured is measured and recorded.

Further, in step 4, if the contrast effect SENDR >0.95 obtained in step 4, the repetition number N of the 180 ° rf pulse block is increased, and if the contrast effect SENDR <0.05 obtained in step 4, the repetition number N of the 180 ° rf pulse block is decreased, and then step 2 and step 3 are repeated until the SENDR is not greater than 0.95 and not less than 0.05.

The invention relates to a new method for rapidly and quantitatively measuring the concentration of an Xe molecular probe based on the nonuniform distribution of saturated energy, which can directly measure the concentration of the Xe molecular probe under certain conditions, compared with the prior art, ① the method only needs to measure that the Xe molecular probes respectively have delta omega at 180 DEG radio frequency pulse frequency offset_rfAnd- Δ ω_rfThe time consumption is shorter when the signal intensity of dissolved Xe, namely the concentration of Xe molecular probe can be quantified by 2 times of scanning, ② the method uses a 180 DEG radio frequency pulse block,③ combining with MRI method, it can quickly and quantitatively measure Xe molecular probe concentration distribution or measure multiple sample Xe molecular probe concentrations at the same time.

Drawings

FIG. 1 is a SEND pulse sequence diagram, which is first pre-processed by a 180 DEG RF pulse block composed of 180 DEG pulses and intervals t, wherein the 180 DEG pulses are shape pulses (in the figure, but not limited to Gaussian pulses), the frequency offset is f1, the 180 DEG RF pulse block is repeated for N times, and then 90 DEG bp pulses with the frequency offset of f2 are applied to excite the dissolved Xe signal for sampling detection;

FIG. 2 shows 298K at 4.477atm, 9.32. mu.M CrA- (COOH)₆1D Xe NMR spectra of the solution;

fig. 3 shows 298K, 4.477atm, 180 ° rf pulse block repetition number N of 200, interval time t of 150ms, and saturation pulse frequency offset Δ ω_rf-14889Hz (blue) and- Δ ω_rf14889Hz (Red), 2.50. mu.M CrA- (COOH)₆1D Xe NMR superposition spectra of the solution;

FIG. 4 shows 2.50. mu.M CrA- (COOH)₆Comparing the measured concentration with the real concentration of the solution when the repetition times of the 180-degree radio frequency pulse block are respectively 25, 50, 100 and 200;

FIG. 5 is a schematic flow chart of the method for rapidly and quantitatively measuring the concentration of the Xe molecular probe based on the nonuniform distribution of saturation energy.

Detailed Description

The present invention will be further described with reference to the accompanying drawings, wherein the embodiments are described herein for illustrative and explanatory purposes only and are not limiting of the invention.

Based on a Xe molecular probe concentration rapid quantitative measurement method with non-uniform distribution of saturation energy, a SEND pulse sequence is shown in FIG. 1, and firstly, preprocessing is carried out on a 180 DEG radio frequency pulse block consisting of 180 DEG pulses and interval time t, wherein the 180 DEG pulses are shape pulses (in FIG. 1, the shape pulses are not limited to Gaussian pulses), the frequency of the shape pulses is f1, the interval time is t, the repetition time of the 180 DEG radio frequency pulse block is N, and then 90 DEG bp pulses with the frequency of f2 are applied to excite a dissolved Xe signal for sampling detection.

The Xe molecular probe concentration rapid quantitative measurement method based on saturation energy non-uniform distribution comprises the following steps:

step 1, preparing a Xe molecular probe solution with a known concentration, and measuring the binding constant K of the Xe molecular probe and Xe;

preparing Xe molecular probe solution with known concentration, measuring the bonding constant K of the Xe molecular probe and Xe under the condition of certain solution temperature and mixed gas pressure by using a 1D Xe NMR spectrum method but not limited to the method, and calculating the bonding constant by the formula

K ═ Xe @ molecular probe]/([ Xe molecular Probe)]- [ Xe @ molecular Probe])*[Xe]_y)

Wherein [ Xe]_y[ Xe molecular probe ] obtained by table lookup, as a dissolved Xe concentration in a solution (Xe partial pressure-Xe solubility), which is the product of the total pressure of a mixed gas used in a test and the proportion of Xe in the mixed gas, and the Xe solubility depends on the temperature of the solution in the test]To formulate the Xe molecular Probe concentration in the solution, [ Xe @ molecular Probe]Indicates the concentration of Xe molecular probe binding to Xe, [ Xe @ molecular probe]＝I1/I2*[Xe]_yI1 and I2 are the integrated value of Xe molecular probe concentration bound to Xe in solution and the integrated value of dissolved Xe concentration, respectively, and the values of I1 and I2 are obtained by integrating the Xe molecular probe signal range bound to Xe and the dissolved Xe signal range in 1D Xe NMR spectrum, respectively.

Step 2, preparing a Xe molecular probe solution to be detected, and setting the frequency bias to be delta omega_rfThe SEND pulse sequence is applied to the Xe molecular probe solution to be detected, and sampling is carried out to obtain the dissolved Xe signal strength S1 of the Xe molecular probe solution to be detected;

in step 2, the frequency is biased to Δ ω_rfThe SEND pulse sequence is applied to a Xe molecular probe solution to be measured, the SEND pulse sequence is as shown in figure 1, firstly, the pretreatment of a 180 DEG radio frequency pulse block consisting of 180 DEG pulses and interval time t is carried out, the 180 DEG radio frequency pulse block is repeated for N times, and then 90 DEG bp pulses with the frequency bias of f2 are applied to excite a dissolved Xe signalCarrying out sampling detection, and setting relevant parameters of the SEND pulse sequence: the 180 pulse is a Gaussian pulse but is not limited to a Gaussian pulse shape pulse with a frequency offset of f1 of value Δ ω_rfThe frequency difference between the Xe molecular probe signal combined with Xe and the dissolved Xe signal is used to realize the selective operation of the Xe molecular probe signal combined with Xe by a 180 DEG radio frequency pulse block; the interval time t is more than or equal to 3 times of residence time (the residence time is the residence time of Xe in the Xe molecular probe) so as to ensure that the Xe molecular probe signal combined with Xe and the dissolved Xe signal are fully exchanged; the 180 deg. rf pulse block repetition number is set to N. The SEND experiment is carried out, a 1D Xe NMR spectrum of the Xe molecular probe solution to be measured is obtained, the dissolved Xe signal range in the 1D Xe NMR spectrum is integrated, the dissolved Xe signal strength S1 of the Xe molecular probe solution to be measured is measured and recorded, and S1 shows that the dissolved Xe signal is influenced by different factors including the Xe molecular probe concentration.

Step 3, setting the frequency offset as-delta omega_rfThe SEND pulse sequence is applied to the Xe molecular probe solution to be detected, and sampling is carried out to obtain the dissolved Xe signal strength S2 of the Xe molecular probe solution to be detected;

in step 3, the same SEND pulse sequence as that in step 2 is adopted for experiment, and in the setting of relevant parameters of the SEND pulse sequence, except that the frequency offset of 180-degree pulses in a 180-degree radio frequency pulse block is different, the other parameters are consistent with the parameter setting in step 2, and the frequency offset f1 of the 180-degree pulses has the value-delta omega_rfThe Xe molecular probe signal combined with Xe is symmetrical relative to the dissolved Xe signal, and the frequency difference is between the dissolved Xe signal and the Xe molecular probe signal, so that the influence of other factors besides the Xe molecular probe signal combined with Xe on the dissolved Xe signal is obtained. Performing SEND experiment under the same solution temperature and mixed gas pressure conditions as step 2 to obtain a 1D Xe NMR spectrum of the Xe molecular probe solution to be measured, integrating the dissolved Xe signal range in the 1D Xe NMR spectrum, measuring and recording the dissolved Xe signal strength S2 of the Xe molecular probe solution to be measured, wherein S2 shows that the dissolved Xe signal is influenced by other factors (namely relaxation and the like) except the Xe molecular probe concentration.

Step 4, calculating contrast effect SENDR according to the formula SENDR of S1/S2;

in step 4, according to the formulaThe formula calculates the contrast effect SENDR as S1/S2, S1 and S2 respectively set the saturation irradiation frequency offset to delta omega in step 2 and step 3_rfAnd- Δ ω_rfThe signal intensity of dissolved Xe is in the range of 0.05 to 0.95, if SENDR is more than 0.95, the repetition number N of 180-degree radio frequency pulse blocks is properly increased, if SENDR is less than 0.05, the repetition number N of 180-degree radio frequency pulse blocks is properly decreased, and then the step 2 and the step 3 are repeated;

step 5, calculating the Xe molecular probe concentration in the Xe molecular probe solution to be measured according to the following formula

In step 5, the Xe molecular probe concentration calculation formula of the Xe molecular probe solution to be measured is

[ Xe molecular probe to be measured]＝(1/K+[Xe])*(1-SENDR^(1/N))/2

Wherein K is the bonding constant between the Xe molecular probe obtained in the step 1 and Xe, [ Xe ] is the dissolved Xe concentration in the Xe molecular probe solution to be measured in the steps 2 and 3, the calculation formula is [ Xe ] (Xe partial pressure × Xe solubility), the size of which is related to the test conditions (i.e. total pressure of the mixed gas, proportion of Xe in the mixed gas, and temperature of the solution to be tested) used by the solution to be tested in step 2 and step 3, the Xe partial pressure is the product of the total pressure of the mixed gas and the proportion of Xe in the mixed gas, the Xe solubility depends on the temperature of the solution to be tested, and looking up a table to obtain SENDR, wherein SENDR is the comparison effect obtained by calculation according to the measurement results of the step 2 and the step 3 in the step 4, N is the repetition number of the 180-degree radio frequency pulse block used in the step 2 and the step 3, and the Xe molecular probe concentration in the solution to be measured is obtained by substituting K, [ Xe ], SENDR and N into a formula.