CN114088693A - Simulated radioactivity18F-labeled cold labeling method and kit - Google Patents

Abstract

The invention discloses a simulated radioactivity¹⁸F-labeled cold labeling method and kit. The kit comprises a standard curve solution, an inorganic fluorine ion developing reagent, a standard fluorine labeled test solution and the like; the invention also discloses a simulated radioactivity using the kit, the ultraviolet detection method and the like¹⁸F-labelling cold labelling method. The invention has the advantages of fast detection response, high sensitivity, convenience, rapidness, low cost and the like; a flexible method is provided aiming at the current situation of insufficient supply of radioactive labeled nuclide, a safe and reliable research mode is provided for a large number of research and researches of radioactive experiments in the early stage, and the method is suitable for popularization and application.

Description

Simulated radioactivity18F-labeled cold labeling method and kit

Technical Field

The invention relates to the field of radioactive labeling and ion detection technology, in particular to a method for simulating radiationProperty of (2)¹⁸F-labeled cold labeling method; also relates to a method for simulating radioactivity¹⁸Kit for the cold labelling method of the F-label.

Background

Early diagnosis of tumors is a major hotspot in current medicine, Positron Emission Tomography (PET) can noninvasively and dynamically observe the distribution, metabolism and the like of labeled drugs in vivo from the outside, and the method has received great attention as a means for increasing popularization of biomedical research and clinical diagnosis, so that the early diagnosis of tumors is breakthrough depending on the development of PET tumor imaging agents.

¹⁸F has a positron efficiency, positron energy (0.649 MeV) and half-life (t) of nearly 100%_1/2109.8min), has lower radiation dose and shorter range to tissues, is the most commonly used positron nuclide, and is suitable for more complex organic synthesis positron emission drugs and PET clinical application. The currently most widely used positron-emitting radiopharmaceuticals are¹⁸F-deoxyglucose (C)¹⁸F-FDG), has been widely used in the research and clinical diagnosis of tumors, coronary heart diseases, and neuropsychiatric diseases.

In recent years, the development of marking methods and the possibility of automated production have led to a variety of approaches¹⁸The study of F radiolabels has become a hotspot in the study of PET drugs. But do not¹⁸The acquisition of F ions depends on the production of an accelerator, and the half-life of 109.8min is not suitable for over 4 hours of long-distance transportation, and has the defects of high safety and transportation cost, so that¹⁸The study of F radiolabels has certain limitations.

In addition to this, the present invention is,¹⁸the radiation protection of F ions is also an important factor, but because of the factors such as the safety quality of a laboratory, the radiation protection grade and the like, the requirements of experimental radiation protection cannot be met, and the research of the radioactive marker has certain limitations.

Disclosure of Invention

Based on the above deficiencies of the prior art, it is an object of the present invention to provide a method for simulating radioactivity¹⁸F markA cold labeling method and a kit. The method is based on the color development principle of fluoride ions and alizarin fluoroblue, can detect the concentration of the fluoride ions to be 10ng accurately by using an ultraviolet spectrophotometry, and further discloses a preparation method and a composition of a kit thereof.

Carrying out a labeling experiment by using a fluorine-containing salt, extracting a fluorine-labeled organic compound to an organic phase by using an organic solvent after the reaction is finished, extracting unreacted fluorine ions to a water phase, and detecting inorganic fluorine ions in the water phase; inorganic fluorine ions, alizarin fluorine blue and cerous nitrate form a bluish purple complex in a solution with the pH value of 4.5; with the difference of ion concentration, an ultraviolet-visible spectrophotometer can be used for calculating the corresponding concentration, and the marking efficiency of the crude product can be calculated.

The technical scheme of the invention is as follows:

one of the technical schemes provided by the invention is as follows: simulated radioactivity¹⁸A cold-marking method of F-marking, the method comprising:

(1) the labeling experiment was performed using a fluorine-containing salt;

(2) after the reaction is finished, extracting the fluorine-labeled organic compound to an organic phase by using an organic solvent, extracting unreacted fluorine ions to a water phase, and detecting inorganic fluorine ions in the water phase by using an alizarin fluoroblue solution and a cerous nitrate complexing solution.

In the labeling experiments, the concentration of the fluoride-containing salt used is preferably 10ng/mL to 50 ng/mL.

In some preferred embodiments, 0.1-1.0g/L alizarin fluoroblue solution is used as the detection reagent, e.g., 0.2 g/L.

In some preferred embodiments, the concentration of the cerous nitrate complexing solution employed is from 0.1 to 0.4g/L, such as 0.22 g/L.

In some preferred embodiments, the assay is performed at a pH of the assay environment of 3.5 to 5.0, e.g., 4.5.

In a preferred embodiment, the buffer for adjusting the pH is one or more of an acetate buffer, a citrate buffer and a phosphate buffer, such as an acetate buffer.

In some preferred embodiments, the organic phase is a chloroform solution.

In a preferred embodiment, the fluorine-containing salt is sodium fluoride or potassium fluoride.

Preferably, the complex formed in step (2) is detected using an ultraviolet-visible spectrophotometer or microplate reader in the present invention.

Preferably, the assay is performed using a standard curve solution prior to performing the assay, drawing a standard curve.

The preparation process of the standard curve solution is preferably as follows:

(1) taking 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL of sodium fluoride solution with the concentration of 1 mu g/mL, and respectively mixing with 10mL of alizarin fluorine blue;

(2) adding 5mL of acetic acid-sodium acetate buffer solution, 10mL of cerous nitrate solution and 25mL of pure water;

(3) respectively detecting the absorbance of the complex obtained by the 5 sodium fluoride solutions and alizarin fluoroblue;

(4) based on the resulting absorbance and 5 sodium fluoride concentrations: standard curves were prepared at 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL and 50 ng/mL.

The second technical scheme provided by the invention is as follows: simulated radioactivity¹⁸An F-labelled cold labelling kit comprising:

(1) 10mL of 1 mu g/mL sodium fluoride standard solution; the preparation process is as described above;

(2) 250mL of 0.1-1.0g/L, preferably 0.2g/L alizarin fluoro blue solution;

(3) 250mL of 0.1-0.4g/L, preferably 0.22g/L, cerous nitrate solution;

(4) 250mL of acetic acid-sodium acetate buffer solution with the pH value of 4.5;

(5) experimental cold labeling 1Ci sodium fluoride solution used: 100 mu L of the solution; wherein, the mass of the sodium fluoride is 24.53ng, and the concentration is as follows: 245.3 ng/mL;

(6) chloroform solution 500. mu.L.

It should be noted that: variations on the form of the kit of the present invention, such as an integral multiple expansion of the volume of the reagents, are within the scope of the present invention.

The invention has the beneficial effects that:

the invention can reach the lower limit of the detection of the concentration of the fluorine ions which can not be realized by the prior art, and is a few detection methods capable of detecting the concentration of the fluorine ions at the nanogram level. The invention also has the advantages of quick detection response, high sensitivity, simplicity, convenience, quickness, low cost and the like. The invention provides a flexible method aiming at the current situation of insufficient supply of the radioactive labeled nuclide, and provides a safe and reliable research mode for a large number of research and researches of radioactive experiments in the early stage.

Drawings

FIG. 1: a full wavelength spectrum.

FIG. 2: the full wavelength spectrum is background (maximum at wavelength 610 nm).

FIG. 3 a: and (5) linearly detecting data by using the reagent.

FIG. 3 b: reagent linearity results plot (R)²＝97.75％)。

FIG. 4 a: reagent precision assay data 1.

FIG. 4 b: reagent precision assay data 2.

FIG. 4 c: reagent precision assay data 3.

FIG. 5 a: reagent sensitivity detection data 1.

FIG. 5 b: reagent sensitivity detection data 2.

FIG. 5 c: reagent sensitivity detection data 3.

FIG. 6: and (5) detecting an experimental object diagram by using the reagent.

FIG. 7: heat-labeled TLC spectrum.

FIG. 8: the color rendering principle is schematically shown.

Detailed Description

In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the following examples. The described embodiments are intended only to illustrate and describe the best mode presently contemplated for the invention, and the scope of the invention is not intended to be limited in any way by the embodiments described herein. The experimental reagents and instruments in the experimental examples without further description are as follows:

1. reagents used for the experiment:

alizarin fluorine blue, cerous nitrate, sodium fluoride, sodium acetate, acetic acid, sodium hydroxide and deionized water. All the listed reagents are conventional reagents which are purchased from the market.

2. The apparatus used for the experiment:

enzyme-labeling instrument (MOLECULAR/i3x), analytical balance (Mettler/XPR 56), ultrapure water instrument (Millipor/IQ 7000), and pH detector (Mettler/FE 28).

3. Preparing a reagent:

1) acetic acid-sodium acetate buffer (pH 4.5): weighing 36.0 +/-0.1 g of sodium acetate, placing the sodium acetate in a beaker, adding about 50mL of water for dissolution, adding 50mL of glacial acetic acid, adding water to 300mL, detecting the pH value of 4.5 by a pH detector, transferring the total amount to a 500mL volumetric flask, diluting the volume to a scale by using ultrapure water, and shaking the volume uniformly for later use.

2) Sodium hydroxide solution (1.2%): weighing 1.2 +/-0.01 g of sodium hydroxide, placing the sodium hydroxide in a 100mL volumetric flask, diluting the sodium hydroxide to a scale mark with ultrapure water, and shaking up for later use.

3) Alizarin fluoro blue solution (0.2 g/L): weighing 0.1 +/-0.005 g of alizarin fluoro blue, putting the alizarin fluoro blue into a 500mL beaker, adding 100mL of water, and adjusting the pH to 4.5 by using an acetic acid-sodium acetate buffer solution with the pH of 4.5 and a sodium hydroxide solution; adding the solution into a 500mL volumetric flask, diluting with ultrapure water to a scale, shaking up, and storing in a dark place for later use.

4) Cerous nitrate solution (0.22 g/L): weighing 0.22 +/-0.005 g of cerous nitrate, placing the cerous nitrate in a 1000mL beaker, adding 100mL of pure water for dissolution, 0.05 +/-0.005 g of hydroxylamine hydrochloride, pouring the mixture into a 1000mL volumetric flask, adding ultrapure water for dilution until the scales are evenly shaken for later use.

5) Sodium fluoride standard solution (20 mg/mL): weighing 2 +/-0.005 g of sodium fluoride, placing the sodium fluoride in a 100mL volumetric flask, diluting the sodium fluoride to a scale with ultrapure water, shaking up and storing the sodium fluoride in the dark for later use.

4. Preparing a blank solution and a standard curve solution:

1) stock solution of sodium fluoride 1 (200. mu.g/mL): 1mL of sodium fluoride standard solution (20mg/mL) is transferred and placed in a 100mL volumetric flask, diluted to the scale with ultrapure water and shaken up for later use.

2) Sodium fluoride stock 2 solution (10 μ g/mL): 2.5mL of sodium fluoride stock 1 (200. mu.g/mL) was removed, placed in a 50mL volumetric flask, diluted to the mark with ultrapure water and shaken well for further use.

3) Stock sodium fluoride 3 solution (1. mu.g/mL): 5.0mL of sodium fluoride stock 2 (10. mu.g/mL) was removed, placed in a 50mL volumetric flask, diluted to the mark with ultrapure water and shaken well for use.

4) Blank solution: taking 10mL of alizarin fluoro blue, placing the alizarin fluoro blue into a 50mL volumetric flask, shaking up, taking 5mL of acetic acid-sodium acetate buffer solution, adding 10mL of cerous nitrate solution, shaking up, diluting with ultrapure water to a scale, shaking up, and placing for 0.5h in a dark place for later use.

5) Standard curve solution: taking 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL of the sodium fluoride stock solution 3, respectively placing the sodium fluoride stock solution in a 50mL volumetric flask, respectively taking 10mL of alizarin fluoroblue, placing the alizarin fluoroblue in the 50mL volumetric flask, shaking uniformly, taking 5mL of acetic acid-sodium acetate buffer solution, adding 10mL of cerous nitrate solution, shaking uniformly, diluting the solution to a scale with ultrapure water, shaking uniformly, and placing the solution for 0.5h in a dark place for later use (respectively serving as standard curve solutions with sodium fluoride concentrations of about 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL and 50 ng/mL).

5. Preparing a standard fluorine labeled test solution:

1)1Ci fluorine cold labeling standard reagent: stock solution 100. mu.L, (1 Ci)¹⁸F-NaF＝24.53ng ¹⁹F-NaF) with a concentration of 245.3ng/mL and a total amount of 24.53 ng.

2)0.3Ci fluorine Cold-labeled test solutions: and (3) putting 30 mu L (7.359ng) of stock solution into a 0.5mL centrifuge tube, respectively taking 0.1mL of alizarin fluoroblue and 0.05mL of acetic acid-sodium acetate buffer solution, adding 0.1mL of cerous nitrate solution, adding 210 mu L of pure water, shaking uniformly, keeping the mixture in the dark for 0.5h, and preparing 2 parts in parallel for later use (the actual concentration is 14.718 ng/mL).

Example 1:

verifying and measuring the absorption wavelength, linearity, stability, precision and sensitivity of the method; the experimental conditions are as follows: and (4) room temperature.

The method comprises the following steps: full wavelength spectrum

The experimental steps are as follows: the standard curve solutions at concentrations of 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL were pipetted into a 96-well plate and absorbance was measured at full wavelength according to the following validation protocol requirements. The results are shown in FIGS. 1 and 2.

The method 2 comprises the following steps: linearity

The experimental steps are as follows: standard curve solutions at concentrations of 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL were pipetted into a 96-well plate and absorbance values at a wavelength of 610nm were determined according to the following validation project requirements.

Referring to the following distribution diagram, 150 μ L of a standard curve solution (sodium fluoride solution) (Std1 to Std5) was added to each test well in duplicate wells in a 96-well plate; 10ng/mL of Std1, 20ng/mL of Std2, 30ng/mL of Std3, 40ng/mL of Std4 and 50ng/mL of Std 5.

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The detection wavelength was set at 610nm, and the absorbance value of each well of the 96-well plate was measured. And taking the average value of the absorbance of the blank solution as a background, taking the concentration of sodium fluoride in the solution of the standard curve as an abscissa and the absorbance value as an ordinate, and drawing a standard curve.

Acceptance criteria: r²≥95％。

The test data is shown in FIG. 3a, and the results are shown in detail in FIG. 3 b.

The method 3 comprises the following steps: stability of

To any three wells of a 96-well plate, 150. mu.L of a solution containing 60ng/mL of sodium fluoride was added, and the initial instrumental readings of the three wells were measured and recorded, and the instrumental readings of the three wells were recorded after 20 min.

Acceptance criteria: the standard deviation of the stability of the absorbance value of the sample is less than or equal to 0.05.

The method 4 comprises the following steps: precision degree

Adding 200 mu L of sodium fluoride solution containing 20ng/mL and 40ng/mL into a 96-well plate, repeatedly measuring for three times (within 20min, each time interval is not less than 1min), recording the value of the instrument, respectively calculating the average value and the standard deviation of the mean value of the absorbance values of the three wells with the same concentration for three times, and indicating the precision of the absorbance of the instrument by the variation coefficient of the experimental result.

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The coefficient of variation is standard deviation/average × 100%.

Acceptance criteria: the coefficient of variation is less than or equal to 10.0 percent.

The test data is shown in fig. 4 a-4 c.

The method 5 comprises the following steps: sensitivity of the probe

150 mu L of sodium fluoride solution with the content of 10ng/mL is added into any three wells of the 96-well enzyme label plate, the measurement is repeated three times (within 20min, the interval is not less than 1min each time), the indication value of the instrument is recorded, and the average value and the standard deviation of the absorbance values of the three wells are calculated.

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Acceptance criteria: the standard deviation is less than or equal to 0.05.

The test data is shown in fig. 5 a-5 c.

FIG. 6 shows a schematic diagram of the reagent assay of methods 2 to 5 described above.

Example 2:

the reagents used in the kit were formulated according to the method described in example 1.

1. The kit comprises the following components:

(1)1 mu g/mL of completely prepared sodium fluoride standard solution is 10 mL; used for making standard songs;

(2) alizarin fluoro blue solution (0.2 g/L): 250 mL;

(3) cerous nitrate solution (0.22 g/L): 250 mL;

(4) acetic acid-sodium acetate buffer (pH 4.5): 250 mL;

(5) experimental cold labeling 1Ci sodium fluoride solution used: 100 mu L of the solution; (the mass of the sodium fluoride is 24.53ng, and the concentration is 245.3 ng/mL);

(6) chloroform solution 500. mu.L.

The experiment verifies that: all reagents are prepared and stored at 4 ℃ for 1 month, can still be used for color reaction, and has no influence on the result, so the effective period of the kit is at least one month.

2. The using method comprises the following steps:

(1) standard curve solution: and (2) respectively placing 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL of the sodium fluoride solution with the concentration of 1 mu g/mL in a 50mL volumetric flask, respectively placing 10mL of alizarin fluoroblue in the 50mL volumetric flask, shaking up, then taking 5mL of acetic acid-sodium acetate buffer solution, adding 10mL of cerous nitrate solution, shaking up, diluting with ultrapure water to a scale, shaking up, and placing in the dark for 0.5h for later use. (as standard curve solutions with sodium fluoride concentrations of about 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, respectively);

(2) taking 1Ci of sodium fluoride cold-labeled solution for reaction, extracting by using chloroform after the reaction, separating out a water phase and an organic phase, leaving unreacted sodium fluoride in the water phase, separating out the water phase, controlling the volume of the water phase within the range of 10-250 mu L, and concentrating if the volume is too large;

(3) adding 100 μ L alizarin fluoroblue, 50 μ L acetic acid-sodium acetate buffer solution and 100 μ L cerous nitrate solution into the water phase, and supplementing the volume with pure water to 500 μ L;

(4) and (3) testing: calculating the mass of the sodium fluoride according to the detected fluorine ion concentration result; the corresponding radiolabelling rate can be found to be: 100% - (mass of sodium fluoride to be measured/24.53 ng)%.

Example 3:

tests were also performed to compare the binding efficiency of cold-labeled and hot-labeled chemistries.

Thermal marking using¹⁸F-BPA, Cold-labeled reference group¹⁹F-BPA; the method is used for judging the marking efficiency of the crude product, and further guiding the process optimization of the crude product.

The experimental method is that the experimental method,¹⁸after the F-BPA heat-labeled part is synthesized according to the scheme shown in the figure, the radioactive labeling detection is carried out by using TLC;¹⁹F-BPA is synthesized according to the same process, chloroform is used for extraction, and the aqueous phase is taken to detect the concentration of fluorine ions.

And (4) conclusion: the heat labeling efficiency was about 58% by radioactive detection TLC results; the cold marking result is 52% by detecting the concentration of fluorine ions; the results of the two methods are almost the same, and the method can be simulated by a cold marking method¹⁸F-BPA is subjected to heat marking, and a consistent marking conclusion is obtained. The hot-label TLC pattern is shown in FIG. 7. The principle of color development is schematically shown in FIG. 8.

Claims (10)

1. Simulated radioactivity¹⁸A cold marking method of F-marking, characterized in that the method comprises:

(1) the labeling experiment was performed using a fluorine-containing salt;

(2) after the reaction is finished, extracting the fluorine-labeled organic compound to an organic phase by using an organic solvent, extracting unreacted fluorine ions to a water phase, and detecting inorganic fluorine ions in the water phase by using an alizarin fluoroblue solution and a cerous nitrate complexing solution.

2. The cold-labelling method according to claim 1, wherein said fluoride-containing salt is present in a concentration of from 10ng/mL to 50ng/mL in said labelling assay.

3. The cold labelling method according to claim 1, wherein the concentration of alizarin fluoroblue is 0.1-1.0g/L, preferably 0.2 g/L;

and/or the concentration of the cerous nitrate complexing solution is 0.1-0.4g/L, preferably 0.22 g/L.

4. The cold labelling method according to claim 1, wherein said detection is carried out at a pH in the detection environment of 3.5 to 5.0, preferably 4.5.

5. The cold-labeling method of claim 4, wherein the buffer that adjusts the pH is one or more of an acetate buffer, a citrate buffer, and a phosphate buffer; preferably an acetate buffer.

6. The cold marking method of claim 1, wherein the organic phase is a chloroform solution.

7. The cold marking method of claim 1, wherein the fluorine-containing salt is sodium fluoride or potassium fluoride.

8. The cold labelling method as claimed in any of claims 1 to 7, wherein the complex formed in step (2) is detected using an ultraviolet-visible spectrophotometer or microplate reader.

9. The cold-marking method according to any one of claims 1 to 7, wherein a standard curve is drawn by performing measurement using a standard curve solution before the detection;

the preparation process of the standard curve solution is as follows:

(1) taking 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL of sodium fluoride solution with the concentration of 1 mu g/mL, and respectively mixing with 10mL of alizarin fluorine blue;

(2) adding 5mL of acetic acid-sodium acetate buffer solution, 10mL of cerous nitrate solution and 25mL of pure water;

(3) respectively detecting the absorbance of the complex obtained by the 5 sodium fluoride solutions and alizarin fluoroblue;

(4) based on the resulting absorbance and 5 sodium fluoride concentrations: standard curves were prepared at 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL and 50 ng/mL.

10. Simulated radioactivity¹⁸F-labelled cold-labelling kit, characterised in that it comprises:

(1) 10mL of 1 mu g/mL sodium fluoride standard solution; the formulation process is as defined in claim 9;

(2) 250mL of 0.1-1.0g/L, preferably 0.2g/L alizarin fluoro blue solution;

(3) 250mL of 0.1-0.4g/L, preferably 0.22g/L, cerous nitrate solution;

(4) 250mL of acetic acid-sodium acetate buffer solution with the pH value of 4.5;

(5) experimental cold labeling 1Ci sodium fluoride solution used: 100 mu L of the solution; wherein, the mass of the sodium fluoride is 24.53ng, and the concentration is as follows: 245.3 ng/mL;

(6) chloroform solution 500. mu.L.

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