Lymphocyte preservation solution, method of preparation and use in sample pretreatment

Abstract

The application relates to the technical field of medicines, in particular to a lymphocyte preservation solution, a preparation method and application in sample pretreatment, and the lymphocyte preservation solution is prepared from the following components in parts by mass: Tris-HCl buffer 90-100 parts, trehalose 10-15 parts, trichostatin A 0.01-0.03 parts, nuclease inhibitor 0.3-0.5 parts and the like raw materials. Through the synergistic design of a composite buffer system and functionalized nanomaterials, combined with multi-stage active protection technology, cell metabolism regulation and precise isolation of external interference factors are realized. The application improves the processing efficiency of complex samples, prolongs the preservation time of key biomarkers, and enhances the adaptability in extreme environments. The integration of the innovative preparation process and the intelligent quality control module provides reliable support for immune diagnosis and treatment, rare cell research and remote medical treatment, and has wide clinical application and scientific research transformation prospects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a lymphocyte preservation solution, its preparation method, and its application in sample pretreatment. Background Technology

[0002] With the development of biological and medical technologies, scientific research and clinical diagnosis have gradually risen to the cellular and gene levels. As the core carrier of immune response, the preservation quality of lymphocytes directly determines the reliability of subsequent analysis. Existing lymphocyte preservation solutions are usually a composite solution based on anticoagulants such as heparin and EDTA, supplemented with buffer salts, glucose and a small amount of protein stabilizers. By regulating osmotic pressure and inhibiting metabolism at low temperature, short-term cell activity can be maintained.

[0003] In existing technologies, lymphocyte preservation solutions cannot effectively stabilize key biomolecules such as nucleic acids, epigenetic modifications, and exosomes within lymphocytes, leading to distortion of high-precision detection data such as single-cell sequencing and spatial transcriptomics. Furthermore, passively inhibiting metabolism through low temperature cannot block the activity of key enzymes, resulting in rapid cell apoptosis. In addition, when faced with special samples such as high bilirubin or chylous blood, traditional hemolytic agents and density gradient methods are difficult to balance the efficiency of red blood cell lysis with the integrity of lymphocyte membranes.

[0004] Therefore, based on the relevant technologies mentioned above, there is an urgent need to develop a lymphocyte preservation solution and its application in sample pretreatment. Summary of the Invention

[0005] In view of this, the purpose of this invention is to propose a lymphocyte preservation solution and its application in sample pretreatment, so as to solve the problems of poor stability, lack of molecular integrity and poor technical adaptability in the prior art.

[0006] To achieve the above objectives, the present invention provides a lymphocyte preservation solution and its application in sample pretreatment.

[0007] A lymphocyte preservation solution comprises the following components in parts by weight: 90-100 parts Tris-HCl buffer, 10-15 parts trehalose, 0.01-0.03 parts tromethorphan A, 0.3-0.5 parts nuclease inhibitor, 1-2 parts hemoglobin-MnO2 nanoparticles, 1-1.2 parts modified lactate oxidase immobilized microspheres, 2-3 parts modified hydrogel microspheres, 3-5 parts polyethylene glycol-polylactic acid copolymer, and 6-8 parts poloxamer 407.

[0008] Preferably, the preparation steps of the Tris-HCl buffer are as follows:

[0009] Add tris(hydroxymethyl)aminomethane to ultrapure water a, heat to 2-4℃, stir for 15-20 min at 200-300 rpm, add 36%wt concentrated hydrochloric acid, adjust the pH to 7.38-7.42, add ultrapure water b, stir for 4-6 min, filter through a 0.22μm PES membrane to obtain Tris-HCl buffer.

[0010] Preferably, the mass ratio of tris(hydroxymethyl)aminomethane, ultrapure water a, and ultrapure water b is 1:(130-135):(30-35).

[0011] By adjusting the pH of the Tris-HCl buffer to approximate the physiological environment, the stability of the cell membrane lipid bilayer structure can be maintained, avoiding changes in membrane permeability or cell swelling or shrinkage caused by pH fluctuations. At the same time, it has a stronger dynamic buffering capacity, which can reduce acidification damage caused by lactic acid accumulation.

[0012] Preferably, the preparation steps of the nuclease inhibitor are as follows:

[0013] Step A1: Add 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid to ultrapure water, heat to 20-25℃, stir for 15-20 min at 400-500 rpm, add 5% wt sodium hydroxide solution, adjust pH to 7-7.2, filter through a 0.22 μm filter membrane to obtain HEPES basic buffer.

[0014] Step A2: Add sodium alginate to glycerol, heat to 50-70℃, stir for 20-25 min at 600-800 rpm, add HEPES basic buffer, cool to 2-4℃, stir for 25-35 min at 200-300 rpm to obtain HEPES buffer.

[0015] Step A3: Add the lyophilized RNasin powder to HEPES buffer, heat to 2-4℃, stir for 8-12 min at 200-300 rpm, add dithiothreitol, stir for 8-12 min, add vanadate-ribose complex, heat to 4-6℃, sonicate for 3-5 min at 30-40 kHz and 40-50 W to obtain the nuclease inhibitor.

[0016] Preferably, the mass ratio of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid to ultrapure water in step A1 is 1:(38-42).

[0017] The mass ratio of sodium alginate, glycerol and HEPES basic buffer in step A2 is 1:(4-6):(35-45).

[0018] The mass ratio of RNasin powder, HEPES buffer, dithiothreitol and ribose vanadate complex in step A3 is 1:(900-1000):(0.04-0.06):(0.15-0.25).

[0019] A pH-responsive sustained-release system is formed by using sodium alginate and glycerol as a carrier, and then encapsulating RNasin through ion cross-linking. This system gradually releases nuclease inhibitors into the sample, thereby inhibiting RNA degradation efficiency.

[0020] Preferably, the preparation steps of the hemoglobin-MnO2 nanoparticles are as follows:

[0021] Step B1: Add 0.1 mol / L potassium permanganate solution to 0.3 mol / L HCl solution, stir for 8-12 min at 400-600 rpm, heat to 110-130℃, react for 5-7 h, and cool to 25-30℃ to obtain MnO2 nanosheet suspension.

[0022] Step B2: Under a nitrogen atmosphere, add lyophilized hemoglobin to deoxy PBS solution, heat to 4-6℃, stir for 3-5 min, add trehalose and ethylenediaminetetraacetic acid, let stand for 50-70 min, and obtain pretreatment solution;

[0023] Step B3: Under an anaerobic environment, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added to the pretreatment solution, heated to 2-4℃, activated for 20-40 min, MnO2 nanosheet suspension was added, ultrasonic treatment was performed for 3-5 min at 40-50 W, and the reaction was stirred for 10-14 h at 100-150 rpm to obtain hemoglobin-MnO2 nanoparticles.

[0024] The mass ratio of potassium permanganate solution to HCl solution in step B1 is 1:(2.8-3.2).

[0025] In step B2, the mass ratio of hemoglobin, deoxyPBS solution, trehalose, and EDTA is 1:(45-50):(0.08-0.09):(0.01-0.02).

[0026] In step B3, the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, pretreatment solution and MnO2 nanosheet suspension is (8-12):(12-14):(90-100):1.

[0027] By cross-linking between carboxyl and amino groups, hemoglobin is stably anchored on the surface of MnO2 nanosheets, increasing the retention of oxygen-regulating active sites. At the same time, hemoglobin binds to excess oxygen, which can inhibit oxidation reactions. Furthermore, the MnO2 nanosheets catalyze the decomposition of H2O2, which can remove reactive oxygen species, thereby protecting the mitochondrial membrane potential.

[0028] Preferably, the preparation steps of the modified lactate oxidase immobilized microspheres are as follows:

[0029] Step C1: Under a nitrogen atmosphere, N-isopropylacrylamide, acrylic acid, and crosslinking agent N,N'-methylenebisacrylamide are added to deionized water and stirred for 10-15 min. Then, ammonium persulfate and tetramethylethylenediamine are added as initiators. The temperature is raised to 30-40℃, the rotation speed is 150-250 rpm, and the reaction is carried out for 1.5-2.5 h. After filtration, the particle size is 150-250 nm, and hydrogel microspheres are obtained.

[0030] Step C2: Add 2-(N-morpholino)ethanesulfonic acid to ultrapure water, add sodium hydroxide to adjust the pH to 4.5-5.5, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir for 8-12 min, add hydrogel microspheres, heat to 2-4℃, shake for 0.8-1.2 h to obtain activated microspheres;

[0031] Step C3: Add lactate oxidase and luciferase to the activated microspheres, heat to 2-4℃, shake for 10-14h to complete the reaction, add serum albumin, heat to 20-30℃, react for 20-40min, then add PBS solution containing 5%wt trehalose-gelatin, cool to 2-4℃, and soak for 1-3h to obtain modified lactate oxidase immobilized microspheres;

[0032] The mass ratio of N-isopropylacrylamide, acrylic acid, crosslinking agent N,N'-methylenebisacrylamide, ammonium persulfate, and tetramethylethylenediamine is (8-9):1:(0.1-0.2):(0.01-0.03):(0.015-0.016).

[0033] The mass ratio of 2-(N-morpholino)ethanesulfonic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide to hydrogel microspheres is 1:(0.2-0.4):(0.3-0.4):(9-10).

[0034] The mass ratio of lactate oxidase, luciferase, serum albumin, activated microspheres to PBS solution containing 5% wt trehalose-gelatin is (1.8-2.2):1:(0.4-0.6):(9-10):(18-20).

[0035] Preferably, the modified sensitive hydrogel microspheres are prepared as follows:

[0036] Step D1: Under a nitrogen atmosphere, N-isopropylacrylamide, acrylic acid, sodium dodecyl sulfate, and crosslinking agent N,N'-methylenebisacrylamide were added to deionized water. The mixture was heated to 20-30°C, stirred for 20-40 min at 300-500 rpm, and then ammonium persulfate and tetramethylethylenediamine were added. The mixture was heated to 60-80°C and reacted for 5-7 h. After the reaction was complete, the mixture was cooled to 20-30°C, centrifuged for 10-20 min, and then freeze-dried to obtain porous microspheres.

[0037] Step D2: Add porous microspheres to 2-morpholine ethanesulfonic acid buffer, cool to -10 to 0°C, sonicate for 2 to 4 minutes at 90 to 110 W, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, heat to 20 to 30°C, and shake for 20 to 40 minutes to obtain an activated microsphere suspension;

[0038] Step D3: Add phosphatidylserine affinity peptide to the activated microsphere suspension, add 0.1 mol / L sodium hydroxide solution, adjust the pH to 6.8-7.2, heat to 30-40℃, shake for 3-5 h, after the reaction is complete, add ethanolamine, cool to 20-25℃, shake for 20-40 min, wash, and obtain modified sensitive hydrogel microspheres;

[0039] The mass ratio of N-isopropylacrylamide, acrylic acid, sodium dodecyl sulfate, crosslinking agent N,N'-methylenebisacrylamide, ammonium sulfate, and tetramethylethylenediamine is (12-16):1:(0.08-0.12):(0.25-0.35):(0.15-0.25):(0.015-0.025).

[0040] The mass ratio of the porous microspheres, 2-morpholine ethanesulfonic acid buffer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:(8.5-9.0):(0.025-0.035):(0.015-0.025).

[0041] The mass ratio of the phosphatidylserine affinity peptide, the activated microsphere suspension, and ethanolamine is 1:(170-190):(18-20).

[0042] By utilizing phosphatidylserine for specific expression on the exosome membrane surface, and then improving exosome recovery rate through peptide affinity binding;

[0043] A method for preparing a lymphocyte preservation solution, comprising the following steps:

[0044] Step S1: Add trehalose to Tris-HCl buffer, heat to 2-4℃, stir for 20-40 min at 200-400 rpm, add poloxamer 407 and polyethylene glycol-polylactic acid copolymer, heat to 30-50℃, stir for 15-25 min at 100-200 rpm to obtain a mixed solution;

[0045] Step S2: Under a nitrogen atmosphere, add trogostatin A and nuclease inhibitor to the mixed solution, stir for 10-20 min, add hemoglobin-MnO2 nanoparticles, sonicate for 3-7 min at 40-60 W, then add modified lactate oxidase immobilized microspheres and modified sensitive hydrogel microspheres, stir for 1-3 min at 1800-2200 rpm, filter through a 0.22 μm filter membrane to obtain lymphocyte preservation solution.

[0046] Preferably, an application of a lymphocyte preservation solution sample pretreatment is used for immunotherapy monitoring, rare cell analysis, and sampling in remote areas.

[0047] The beneficial effects of this invention are:

[0048] This invention provides a lymphocyte preservation solution and its application in sample pretreatment. Through the synergistic design of a composite buffer system and functionalized nanomaterials, combined with multi-stage activity protection technology, it achieves precise isolation of cell metabolic regulation from external interference factors. Compared with existing technologies, this invention improves the processing efficiency of complex samples, extends the preservation time of key biomarkers, and enhances adaptability to extreme environments. Its innovative preparation process and intelligent quality control module integration provide reliable support for immunodiagnosis, rare cell research, and telemedicine, and has broad prospects for clinical application and scientific research translation. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0050] Example 1: The preparation steps of Tris-HCl buffer are as follows:

[0051] Add 10g of tris(hydroxymethyl)aminomethane to 1300g of ultrapure water (a), heat to 2°C, stir for 20min at 200rpm, add 36%wt concentrated hydrochloric acid, adjust the pH to 7.38-7.42, add 300g of ultrapure water (b), stir for 6min, filter through a 0.22μm PES membrane to obtain Tris-HCl buffer.

[0052] Example 2: The preparation steps of Tris-HCl buffer are as follows:

[0053] Add 10g of tris(hydroxymethyl)aminomethane to 1320g of ultrapure water a, heat to 3℃, stir for 17min at 250rpm, add 36%wt concentrated hydrochloric acid, adjust the pH to 7.38-7.42, add 320g of ultrapure water b, stir for 5min, filter through a 0.22μm PES membrane to obtain Tris-HCl buffer.

[0054] Example 3: The preparation steps of Tris-HCl buffer are as follows:

[0055] Add 10g of tris(hydroxymethyl)aminomethane to 1350g of ultrapure water (a), heat to 4°C, stir for 15min at 300rpm, add 36%wt concentrated hydrochloric acid, adjust the pH to 7.38-7.42, add 350g of ultrapure water (b), stir for 4min, filter through a 0.22μm PES membrane to obtain Tris-HCl buffer.

[0056] Example 4: The preparation steps of the nuclease inhibitor are as follows:

[0057] S1: Add 10g of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid to 380g of ultrapure water, heat to 20℃, stir for 20min at 400rpm, add 5%wt sodium hydroxide solution, adjust pH to 7-7.2, filter through a 0.22μm filter membrane to obtain HEPES basic buffer.

[0058] S2: Add 10g of sodium alginate to 40g of glycerol, heat to 50℃, stir for 25min at 600rpm, add 350g of HEPES basic buffer, cool to 2℃, stir for 35min at 200rpm to obtain HEPES buffer.

[0059] S3: Add 1g of lyophilized RNasin powder to 900g of HEPES buffer, heat to 2℃, stir for 12min at 200rpm, add 0.04g of dithiothreitol, stir for 8min, add 0.15g of vanadate-ribose complex, heat to 6℃, sonicate for 3min at 40kHz and 40W to obtain the nuclease inhibitor.

[0060] Example 5: The preparation steps of the nuclease inhibitor are as follows:

[0061] S1: Add 10g of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid to 400g of ultrapure water, heat to 22℃, stir for 17min at 450rpm, add 5%wt sodium hydroxide solution, adjust pH to 7-7.2, filter through a 0.22μm filter membrane to obtain HEPES basic buffer.

[0062] S2: Add 10g of sodium alginate to 50g of glycerol, heat to 60℃, stir for 23min at 700rpm, add 400g of HEPES basic buffer, cool to 3℃, stir for 30min at 250rpm to obtain HEPES buffer.

[0063] S3: Add 1g of lyophilized RNasin powder to 950g of HEPES buffer, heat to 3℃, stir for 10min at 250rpm, add 0.05g of dithiothreitol, stir for 10min, add 0.2g of vanadate-ribose complex, heat to 5℃, sonicate for 4min at 35kHz and 45W to obtain the nuclease inhibitor.

[0064] Example 6: The preparation steps of the nuclease inhibitor are as follows:

[0065] S1: Add 10g of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid to 420g of ultrapure water, heat to 25℃, stir for 15min at 500rpm, add 5%wt sodium hydroxide solution, adjust pH to 7-7.2, filter through a 0.22μm filter membrane to obtain HEPES basic buffer.

[0066] S2: Add 10g of sodium alginate to 60g of glycerol, heat to 70℃, stir for 20min at 600rpm, add 450g of HEPES basic buffer, cool to 2℃, stir for 35min at 200rpm to obtain HEPES buffer.

[0067] S3: Add 1g of lyophilized RNasin powder to 1000g of HEPES buffer, heat to 4℃, stir for 12min at 200rpm, add 0.06g of dithiothreitol, stir for 8min, add 0.25g of vanadate-ribose complex, heat to 6℃, sonicate for 3min at 40kHz and 40W to obtain the nuclease inhibitor.

[0068] Example 7: The preparation steps of hemoglobin-MnO2 nanoparticles are as follows:

[0069] S1: Add 10g of 0.1mol / L potassium permanganate solution to 28g of 0.3mol / L HCl solution, stir for 8min, rotate at 600rpm, heat to 110℃, react for 7h, and cool to 25℃ to obtain MnO2 nanosheet suspension.

[0070] S2: Under a nitrogen atmosphere, 10g of lyophilized hemoglobin was added to 450g of deoxyPBS solution, heated to 4℃, stirred for 5min, 0.8g of trehalose and 0.1g of ethylenediaminetetraacetic acid were added, and the mixture was allowed to stand for 50min to obtain the pretreatment solution.

[0071] S3: Under an anaerobic environment, 8g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 12g of N-hydroxysuccinimide were added to 90g of pretreatment solution, heated to 2℃, activated for 40min, 1g of MnO2 nanosheet suspension was added, ultrasonic treatment was performed for 3min at 40W power, and the reaction was stirred for 10h at 150rpm to obtain hemoglobin-MnO2 nanoparticles.

[0072] Example 8: The preparation steps of hemoglobin-MnO2 nanoparticles are as follows:

[0073] S1: Add 10g of 0.1mol / L potassium permanganate solution to 30g of 0.3mol / L HCl solution, stir for 10min, rotate at 500rpm, heat to 120℃, react for 6h, and cool to 27℃ to obtain MnO2 nanosheet suspension.

[0074] S2: Under a nitrogen atmosphere, 10g of lyophilized hemoglobin was added to 450g of deoxyPBS solution, heated to 5°C, stirred for 4min, 0.85g of trehalose and 0.15g of ethylenediaminetetraacetic acid were added, and the mixture was allowed to stand for 60min to obtain the pretreatment solution.

[0075] S3: Under an anaerobic environment, 10g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 13g of N-hydroxysuccinimide were added to 95g of pretreatment solution, heated to 3℃, activated for 30min, 1g of MnO2 nanosheet suspension was added, ultrasonic treatment was performed for 4min at 45W power, and the reaction was stirred for 12h at 130rpm to obtain hemoglobin-MnO2 nanoparticles.

[0076] Example 9: The preparation steps of hemoglobin-MnO2 nanoparticles are as follows:

[0077] S1: Add 10g of 0.1mol / L potassium permanganate solution to 32g of 0.3mol / L HCl solution, stir for 12min, rotate at 400rpm, heat to 130℃, react for 5h, and cool to 30℃ to obtain MnO2 nanosheet suspension.

[0078] S2: Under a nitrogen atmosphere, 10g of lyophilized hemoglobin was added to 500g of deoxyPBS solution, heated to 6°C, stirred for 3min, 0.9g of trehalose and 0.2g of ethylenediaminetetraacetic acid were added, and the mixture was allowed to stand for 70min to obtain the pretreatment solution.

[0079] S3: Under an anaerobic environment, 12g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 14g of N-hydroxysuccinimide were added to 100g of pretreatment solution, heated to 4℃, activated for 20min, 1g of MnO2 nanosheet suspension was added, ultrasonic treatment was performed for 5min at 40W power, and the reaction was stirred for 14h at 100rpm to obtain hemoglobin-MnO2 nanoparticles.

[0080] Example 10: The preparation steps of modified lactate oxidase immobilized microspheres are as follows:

[0081] S1: Under a nitrogen atmosphere, 80g of N-isopropylacrylamide, 10g of acrylic acid and 1g of crosslinking agent N,N'-methylenebisacrylamide were added to 100mL of deionized water and stirred for 10min. Then, 0.1g of initiator ammonium persulfate and 0.15g of tetramethylethylenediamine were added. The temperature was raised to 30℃ and the reaction was carried out at 250rpm for 1.5h. After filtration, the particle size was 150-250nm, and hydrogel microspheres were obtained.

[0082] S2: Add 10g of 2-(N-morpholino)ethanesulfonic acid to 100mL of ultrapure water, add sodium hydroxide to adjust the pH to 4.5-5.5, add 2g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 3g of N-hydroxysuccinimide, stir for 8min, add 90g of hydrogel microspheres, heat to 4℃, shake for 0.8h to obtain activated microspheres;

[0083] S3: Add 18g of lactate oxidase and 10g of luciferase to 90g of activated microspheres, heat to 2℃, shake for 14h, and the reaction is complete. Add 4g of serum albumin, heat to 20℃, react for 40min, then add 180g of PBS solution containing 5%wt trehalose-gelatin, cool to 2℃, and soak for 3h to obtain modified lactate oxidase immobilized microspheres.

[0084] Example 11: The preparation steps of modified lactate oxidase immobilized microspheres are as follows:

[0085] S1: Under a nitrogen atmosphere, 85g of N-isopropylacrylamide, 10g of acrylic acid and 1.5g of crosslinking agent N,N'-methylenebisacrylamide were added to 100mL of deionized water and stirred for 12min. Then, 0.2g of initiator ammonium persulfate and 0.155g of tetramethylethylenediamine were added. The temperature was raised to 35℃ and the reaction was carried out at 200rpm for 2h. After filtration, the particle size was 150-250nm, and hydrogel microspheres were obtained.

[0086] S2: Add 10g of 2-(N-morpholino)ethanesulfonic acid to 100mL of ultrapure water, add sodium hydroxide to adjust the pH to 4.5-5.5, add 3g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 3.5g of N-hydroxysuccinimide, stir for 10min, add 95g of hydrogel microspheres, heat to 3℃, shake for 1h to obtain activated microspheres;

[0087] S3: Add 20g of lactate oxidase and 10g of luciferase to 95g of activated microspheres, heat to 3℃, shake for 12h, and the reaction is complete. Add 5g of serum albumin, heat to 25℃, react for 30min, then add 190g of PBS solution containing 5%wt trehalose-gelatin, cool to 3℃, and soak for 2h to obtain modified lactate oxidase immobilized microspheres.

[0088] Example 12: The preparation steps of modified lactate oxidase immobilized microspheres are as follows:

[0089] S1: Under a nitrogen atmosphere, 90g of N-isopropylacrylamide, 10g of acrylic acid and 2g of crosslinking agent N,N'-methylenebisacrylamide were added to 100mL of deionized water and stirred for 15min. Then, 0.3g of initiator ammonium persulfate and 0.16g of tetramethylethylenediamine were added. The temperature was raised to 30℃ and the reaction was carried out at 250rpm for 1.5h. After filtration, the particle size was 150-250nm, and hydrogel microspheres were obtained.

[0090] S2: Add 10g of 2-(N-morpholino)ethanesulfonic acid to 100mL of ultrapure water, add sodium hydroxide to adjust the pH to 4.5-5.5, add 4g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 4g of N-hydroxysuccinimide, stir for 12min, add 100g of hydrogel microspheres, heat to 4℃, shake for 0.8h to obtain activated microspheres;

[0091] S3: Add 22g of lactate oxidase and 10g of luciferase to 100g of activated microspheres, heat to 4℃, shake for 10h, and the reaction is complete. Add 6g of serum albumin, heat to 20℃, react for 40min, then add 200g of PBS solution containing 5%wt trehalose-gelatin, cool to 2℃, and soak for 3h to obtain modified lactate oxidase immobilized microspheres.

[0092] Example 13: The preparation method of modified sensitive hydrogel microspheres is as follows:

[0093] S1: Under a nitrogen atmosphere, 120g of N-isopropylacrylamide, 10g of acrylic acid, 0.8g of sodium dodecyl sulfate and 2.5g of crosslinking agent N,N'-methylenebisacrylamide were added to 200mL of deionized water, heated to 20℃, stirred for 40min at 300rpm, 1.5g of ammonium persulfate and 0.15g of tetramethylethylenediamine were added, heated to 60℃, and reacted for 7h. After the reaction was completed, the temperature was lowered to 20℃, centrifuged for 20min, and freeze-dried to obtain porous microspheres.

[0094] S2: Add 10g of porous microspheres to 85g of 2-morpholine ethanesulfonic acid buffer, cool to -10℃, sonicate for 4min at 90W, add 0.25g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.15g of N-hydroxysuccinimide, heat to 20℃, and shake for 40min to obtain an activated microsphere suspension;

[0095] S3: Add 1g of phosphatidylserine affinity peptide to 170g of activated microsphere suspension, add 0.1mol / L sodium hydroxide solution, adjust pH to 6.8-7.2, heat to 30℃, shake for 5h, after the reaction is complete, add 18g of ethanolamine, cool to 20℃, shake for 40min, wash, and obtain modified sensitive hydrogel microspheres.

[0096] Example 14: The preparation method of modified sensitive hydrogel microspheres is as follows:

[0097] S1: Under a nitrogen atmosphere, 140g of N-isopropylacrylamide, 10g of acrylic acid, 1g of sodium dodecyl sulfate and 3g of crosslinking agent N,N'-methylenebisacrylamide were added to 200mL of deionized water, heated to 25℃, stirred for 30min at 400rpm, 2g of ammonium persulfate and 0.2g of tetramethylethylenediamine were added, heated to 70℃, and reacted for 6h. After the reaction was completed, the temperature was lowered to 25℃, centrifuged for 15min, and freeze-dried to obtain porous microspheres.

[0098] S2: Add 10g of porous microspheres to 87g of 2-morpholine ethanesulfonic acid buffer, cool to -5℃, sonicate for 3min at 100W, add 0.3g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.2g of N-hydroxysuccinimide, heat to 25℃, and shake for 30min to obtain an activated microsphere suspension;

[0099] S3: Add 1g of phosphatidylserine affinity peptide to 180g of activated microsphere suspension, add 0.1mol / L sodium hydroxide solution, adjust pH to 6.8-7.2, heat to 35℃, shake for 4h, after the reaction is complete, add 190g of ethanolamine, cool to 22℃, shake for 30min, wash, and obtain modified sensitive hydrogel microspheres.

[0100] Example 15: The preparation method of modified sensitive hydrogel microspheres is as follows:

[0101] S1: Under a nitrogen atmosphere, 160g of N-isopropylacrylamide, 10g of acrylic acid, 1.2g of sodium dodecyl sulfate and 3.5g of crosslinking agent N,N'-methylenebisacrylamide were added to 200mL of deionized water, heated to 30℃, stirred for 20min at 500rpm, 2.5g of ammonium persulfate and 0.25g of tetramethylethylenediamine were added, heated to 80℃, and reacted for 5h. After the reaction was completed, the temperature was lowered to 30℃, centrifuged for 10min, and freeze-dried to obtain porous microspheres.

[0102] S2: Add 10g of porous microspheres to 90g of 2-morpholine ethanesulfonic acid buffer, cool to -0℃, sonicate for 2min at 110W, add 0.35g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.25g of N-hydroxysuccinimide, heat to 30℃, and shake for 20min to obtain an activated microsphere suspension;

[0103] S3: Add 1g of phosphatidylserine affinity peptide to 190g of activated microsphere suspension, add 0.1mol / L sodium hydroxide solution, adjust pH to 6.8-7.2, heat to 40℃, shake for 3h, after the reaction is complete, add 20g of ethanolamine, cool to 25℃, shake for 20min, wash, and obtain modified sensitive hydrogel microspheres.

[0104] Example 16: A method for preparing a lymphocyte preservation solution is as follows:

[0105] S1: Add 10g of trehalose to 90g of Tris-HCl buffer, heat to 2℃, stir for 40min at 200rpm, add 6g of poloxamer 407 and 3g of polyethylene glycol-polylactic acid copolymer, heat to 30℃, stir for 25min at 100rpm to obtain a mixed solution.

[0106] S2: Under a nitrogen atmosphere, 0.01g of trachomatis A and 0.3g of nuclease inhibitor were added to 100g of a mixed solution and stirred for 10min. Then, 10g of hemoglobin-MnO2 nanoparticles were added, and the mixture was sonicated for 7min at 40W. Next, 1g of modified lactate oxidase immobilized microspheres and 2g of modified hydrogel microspheres were added, and the mixture was stirred for 1min at 2200rpm. The mixture was then filtered through a 0.22μm filter membrane to obtain the lymphocyte preservation solution.

[0107] Example 17: A method for preparing a lymphocyte preservation solution is as follows:

[0108] S1: Add 12g of trehalose to 95g of Tris-HCl buffer, heat to 3℃, stir for 30min at 300rpm, add 7g of poloxamer 407 and 4g of polyethylene glycol-polylactic acid copolymer, heat to 40℃, stir for 20min at 150rpm to obtain a mixed solution.

[0109] S2: Under a nitrogen atmosphere, 0.02 g of trogopterin A and 0.4 g of nuclease inhibitor were added to 100 g of mixed solution and stirred for 15 min. Then, 1.5 g of hemoglobin-MnO2 nanoparticles were added, and the mixture was sonicated for 5 min at 50 W. Next, 1.1 g of modified lactate oxidase immobilized microspheres and 2.5 g of modified hydrogel microspheres were added, and the mixture was stirred for 2 min at 2000 rpm. The mixture was then filtered through a 0.22 μm filter membrane to obtain the lymphocyte preservation solution.

[0110] Example 18: A method for preparing a lymphocyte preservation solution is as follows:

[0111] S1: Add 15g of trehalose to 100g of Tris-HCl buffer, heat to 4℃, stir for 20min at 400rpm, add 8g of poloxamer 407 and 5g of polyethylene glycol-polylactic acid copolymer, heat to 30℃, stir for 25min at 100rpm to obtain a mixed solution.

[0112] S2: Under a nitrogen atmosphere, 0.03 g of trogopterin A and 0.5 g of nuclease inhibitor were added to 100 g of mixed solution and stirred for 20 min. Then, 2 g of hemoglobin-MnO2 nanoparticles were added, and the mixture was sonicated for 3 min at 60 W. Next, 1.2 g of modified lactate oxidase immobilized microspheres and 3 g of modified hydrogel microspheres were added and stirred for 3 min at 1800 rpm. The mixture was then filtered through a 0.22 μm filter membrane to obtain the lymphocyte preservation solution.

[0113] Comparative Example 1:

[0114] Compared with Example 16, this comparative example only replaces "hemoglobin-MnO2 nanoparticles" with "Fe3O4 nanoparticles". All other steps and parameters are the same, and will not be repeated here. The final lymphocyte preservation solution is obtained.

[0115] Comparative Example 2:

[0116] This comparative example differs from Example 16 only in that the "nuclease inhibitor" is replaced with "free RNasin". All other steps and parameters are the same, and will not be repeated here. The final lymphocyte preservation solution was obtained.

[0117] Comparative Example 3:

[0118] This comparative example differs from Example 16 only in that "poloxam 407" is replaced with "polyethylene glycol 6000". All other steps and parameters are the same, and will not be repeated here. The final lymphocyte preservation solution is obtained.

[0119] Comparative Example 4:

[0120] Compared with Example 16, this comparative example omits step D3 in the preparation of modified sensitive hydrogel microspheres. All other steps and parameters are the same, and will not be repeated here. The final lymphocyte preservation solution is obtained.

[0121] Comparative Example 5:

[0122] Compared with Example 16, this comparative example only changed the preparation process of hemoglobin-MnO2 nanoparticles to physical mixing. All other steps and parameters are the same, and will not be repeated here. The final lymphocyte preservation solution was obtained.

[0123] Preparation of test samples

[0124] Venous blood was collected using vacuum blood collection tubes containing lithium heparin anticoagulant. The blood was mixed with the preservation solutions of Examples 16-18 and Comparative Examples 1-5, with a blood:preservation solution ratio of 1:2. The blood collection tubes were gently inverted 8-10 times to complete the mixing. The mixture was then placed at 4°C and allowed to stand for 15 minutes. A stabilizer was added, and the temperature was raised to 37°C and incubated for 5 minutes. The temperature was then lowered to 4°C and centrifuged for 25 minutes. The intermediate lymphocyte layer was collected, and 0.1% magnetic titanium dioxide nanoparticles were added. The mixture was vortexed for 30 seconds, and the particles adsorbed with bilirubin were separated using a magnetic rack. Finally, CD45 and CD3 dual-labeled thermosensitive magnetic beads were added, and the temperature was raised to 37°C and incubated for 10 minutes. The temperature was then lowered to 4°C and magnetically separated to obtain a preservation solution containing lymphocytes.

[0125] Performance testing:

[0126] 72-hour cell viability assay:

[0127] Following the ISO 10993-5 guidelines for cytotoxicity assessment, flow cytometry was used for detection. 1 mL of whole blood samples treated with the preservation solutions of Examples 16-18 and Comparative Examples 1-5 were taken respectively, diluted to 5 mL with PBS solution, centrifuged for 10 min, the supernatant was discarded, and the samples were resuspended in 1 mL of RPMI-1640 medium. 5 μL of Annexin V-FITC and 5 μL of propidium iodide were added, and the samples were incubated for 15 min.

[0128] RNA integrity count:

[0129] 1. Total RNA was extracted from lymphocytes using the Qiagen RNeasy Mini Kit, following the Publication G2938-90034 testing standard.

[0130] 2. Take 1 μL of RNA samples from Examples 16-18 and Comparative Examples 1-5 respectively and load them into an Agilent RNA 6000 Nano chip for detection.

[0131] Exosome recovery rate (CD63+) assay:

[0132] The samples from Examples 16-18 and Comparative Examples 1-5 were respectively added with CD63-PE antibody and incubated at 4°C for 30 min. Using a NanoFCM N30E nanoflow cytometer, the detection channels were set to SSC 488 nm and PE 561 nm, with a threshold of 100 nm particles. The recovery rate of CD63-PE positive particles was calculated using the following formula:

[0133]

[0134] Table 1

[0135]

[0136] Determination of chylous blood treatment time:

[0137] The test was conducted using a Hach-2100N turbidimeter, following the CLSI-EP07-A3 interference test guideline.

[0138] 1. Add soybean oil and Tween-80 to physiological saline at a mass ratio of 9:1, heat to 30-40℃, emulsify for 5 minutes to obtain an oil-water emulsion;

[0139] 2. Add healthy human EDTA anticoagulated plasma to an oil-water emulsion at a volume ratio of 4:1, vortex for 2 minutes to obtain a chylous blood model;

[0140] 3. Take 10 mL of the preservation solution from Examples 16-18 and Comparative Examples 1-5 respectively, mix it with 5 mL of chylous blood model, heat to 4°C, let stand for 15 min, add 0.1% magnetic titanium dioxide nanoparticles, vortex for 30 s, centrifuge for 15 min, take the supernatant and test.

[0141] Sample stability testing

[0142] 1. Collect peripheral blood samples from 10 healthy volunteers, 2 mL each. Take 40 mL each of the preservation solutions from Examples 16-18 and Comparative Examples 1-5, mix them thoroughly to obtain whole blood samples.

[0143] 2. Take 100 μL of whole blood sample, add CD3-FITC, CD4-APC, and CD8-PE antibodies, incubate in the dark for 20 min, add 2 mL of erythrocyte lysis buffer, vortex to mix, lyse at room temperature in the dark for 10 min, centrifuge for 5 min, wash with PDS, and use Beckman-Coulter...

[0144] CytoFLEX-LX flow cytometer assay, repeated 3 times;

[0145] 3. CV value calculation formula:

[0146]

[0147] In the formula, σ---standard deviation, ;

[0148] μ --- average value .

[0149] Table 2

[0150]

[0151] Data Analysis:

[0152] As can be seen from Tables 1-2, the lymphocyte preservation solution prepared by this invention has excellent cell viability, RNA integrity, exosome recovery rate, and shorter chylous blood treatment time.

[0153] In Comparative Example 1, replacing hemoglobin-MnO2 nanoparticles with Fe3O4 nanoparticles led to the accumulation of H2O2 within the cells. Cellular hypoxia reduced the metabolic rate, accelerating apoptosis. Simultaneously, H2O2 reacted with mitochondrial membrane phosphate, causing a decrease in membrane potential, triggering the opening of the mitochondrial permeability transition pore, releasing cytochrome C, activating the Caspase cascade, and ultimately inducing apoptosis. Excessive H2O2 also oxidized exosome membrane lipids, damaging their structural integrity and leading to the loss of surface markers such as CD63. This is because hemoglobin has a high oxygen affinity, binding and slowly releasing oxygen to maintain oxygen homeostasis in the cellular microenvironment, preventing metabolic disorders caused by hypoxia or hyperxia. Potassium permanganate catalyzes the decomposition of H2O2 into H2O and O2, reducing the accumulation of reactive oxygen species and protecting the integrity of the mitochondrial membrane potential. The two work synergistically to form a dynamic balance, inhibiting cellular oxidative stress.

[0154] Comparative Example 2, due to the use of free RNasin instead of nuclease inhibitors, resulted in a significant decrease in RNA integrity and exosome recovery rate. Furthermore, free RNasin could not form a sustained-release system via the sodium alginate-glycerol carrier, leading to short-lasting and uneven nuclease inhibition and rapid RNA degradation. This was because free RNasin dissolved rapidly in the preservation solution, initially reaching excessively high concentrations that caused local toxicity or ineffective binding. Subsequent degradation and inactivation prevented sustained inhibition of RNase. In contrast, sodium alginate and glycerol formed an ionic cross-linking network, gradually releasing RNasin through pH response, maintaining its long-term effective concentration. Ammonium disulfate, as a reducing agent, protected the active site of RNasin; and the vanadate-ribose complex enhanced the blocking of RNase through competitive inhibition.

[0155] Comparative Example 3, due to the replacement of poloxamer 407 with polyethylene glycol 6000, could not dynamically adjust the viscosity according to temperature, resulting in low erythrocyte lysis efficiency and the inability to form micelles or gels, making it difficult to optimize the physical environment for cell separation. At the same time, the static high viscosity of polyethylene glycol 6000 hindered the diffusion of the lysing agent, leading to incomplete erythrocyte lysis. This is because poloxamer 407 is a triblock copolymer with excellent temperature responsiveness. At low temperatures, it promotes the penetration of erythrocyte lysing agent, and the increased viscosity after heating can accelerate lymphocyte sedimentation and reduce centrifugation time. In addition, poloxamer 407 can form a physical barrier to reduce mechanical damage and maintain the integrity of the lymphocyte membrane.

[0156] Comparative Example 4, by omitting step D3 in the preparation of the modified sensitive hydrogel microspheres, resulted in a reduced exosome recovery rate. Furthermore, the lack of phosphatidylserine-specific binding ability prevented exosomes from efficiently anchoring to the microsphere surface, leading to a significant decrease in recovery rate. This is because phosphatidylserine is a component of the cell membrane endothelial cells, but during apoptosis and exosome formation, it everts to the membrane surface, becoming a marker molecule for exosomes. Introducing a phosphatidylserine affinity peptide into the modified sensitive hydrogel microspheres aims to achieve targeted capture of exosomes through the specific binding of this peptide to phosphatidylserine, thereby significantly improving binding efficiency.

[0157] In Comparative Example 5, the physical mixing of hemoglobin and MnO2 nanoparticles resulted in the inability to stably immobilize hemoglobin, leading to the easy deactivation of oxygen-binding sites. Simultaneously, the catalytic activity of MnO2 decreased, resulting in insufficient reactive oxygen species scavenging efficiency. This is because simple physical mixing cannot form a stable bond, allowing hemoglobin to easily detach from the MnO2 surface, resulting in insufficient exposure of oxygen-binding sites and an inability to effectively regulate oxygen concentration. Furthermore, fluctuations in pH, ionic strength, and temperature in the preservation solution accelerate hemoglobin denaturation, further reducing its function. In addition, physical mixing causes a loose interface between hemoglobin and MnO2, hindering the synergistic effect of the catalytic reaction. Unfixed hemoglobin may cover the catalytically active sites on the MnO2 surface, reducing the H2O2 decomposition efficiency.

[0158] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

[0159] This invention is intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A lymphocyte preservation solution, characterized in that, By weight, it consists of the following components: 90-100 parts Tris-HCl buffer, 10-15 parts trehalose, 0.01-0.03 parts qugu styrax A, 0.3-0.5 parts nuclease inhibitor, 1-2 parts hemoglobin-MnO2 nanoparticles, 1-1.2 parts modified lactate oxidase immobilized microspheres, 2-3 parts modified sensitive hydrogel microspheres, 3-5 parts polyethylene glycol-polylactic acid copolymer, and 6-8 parts poloxamer 407.

2. The lymphocyte preservation solution according to claim 1, characterized in that, The preparation steps for the Tris-HCl buffer are as follows: Add tris(hydroxymethyl)aminomethane to ultrapure water a, heat to 2-4℃, stir for 15-20 min at 200-300 rpm, add 36%wt concentrated hydrochloric acid, adjust the pH to 7.38-7.42, add ultrapure water b, stir for 4-6 min, filter through a 0.22μm PES membrane to obtain Tris-HCl buffer.

3. The lymphocyte preservation solution according to claim 2, characterized in that, The mass ratio of the trihydroxymethylaminomethane, ultrapure water a to ultrapure water b is 1:(130-135):(30-35).

4. The lymphocyte preservation solution according to claim 1, characterized in that, The preparation steps of the nuclease inhibitor are as follows: Step A1: Add 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid to ultrapure water, heat to 20-25℃, stir for 15-20 min at 400-500 rpm, add 5% wt sodium hydroxide solution, adjust pH to 7-7.2, filter through a 0.22 μm filter membrane to obtain HEPES basic buffer. Step A2: Add sodium alginate to glycerol, heat to 50-70℃, stir for 20-25 min at 600-800 rpm, add HEPES basic buffer, cool to 2-4℃, stir for 25-35 min at 200-300 rpm to obtain HEPES buffer. Step A3: Add the lyophilized RNasin powder to HEPES buffer, heat to 2-4℃, stir for 8-12 min at 200-300 rpm, add dithiothreitol, stir for 8-12 min, add vanadate-ribose complex, heat to 4-6℃, sonicate for 3-5 min at 30-40 kHz and 40-50 W to obtain the nuclease inhibitor.

5. The lymphocyte preservation solution according to claim 4, characterized in that, The mass ratio of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid to ultrapure water in step A1 is 1:(38-42). The mass ratio of sodium alginate, glycerol and HEPES basic buffer in step A2 is 1:(4-6):(35-45). The mass ratio of RNasin powder, HEPES buffer, dithiothreitol and ribose vanadate complex in step A3 is 1:(900-1000):(0.04-0.06):(0.15-0.25).

6. The lymphocyte preservation solution according to claim 1, characterized in that, The preparation steps of the hemoglobin-MnO2 nanoparticles are as follows: Step B1: Add potassium permanganate solution to 0.3 mol / L HCl solution, stir for 8-12 min, heat to 110-130℃, react for 5-7 h, cool to 25-30℃ to obtain MnO2 nanosheet suspension; Step B2: Under a nitrogen atmosphere, add lyophilized hemoglobin to deoxy PBS solution, heat to 4-6℃, stir for 3-5 min, add trehalose and ethylenediaminetetraacetic acid, let stand for 50-70 min, and obtain pretreatment solution; Step B3: Under an anaerobic environment, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added to the pretreatment solution, heated to 2-4℃, activated for 20-40 min, MnO2 nanosheet suspension is added, ultrasonic treatment is performed for 3-5 min, and the reaction is stirred for 10-14 h to obtain hemoglobin-MnO2 nanoparticles. The mass ratio of potassium permanganate solution to HCl solution is 1:(2.8-3.2). The mass ratio of hemoglobin, deoxyPBS solution, trehalose, and EDTA is 1:(45-50):(0.08-0.09):(0.01-0.02). The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, pretreatment solution and MnO2 nanosheet suspension is (8-12):(12-14):(90-100):

1.

7. The lymphocyte preservation solution according to claim 1, characterized in that, The preparation steps of the modified lactate oxidase immobilized microspheres are as follows: Step C1: Under a nitrogen atmosphere, N-isopropylacrylamide, acrylic acid, and crosslinking agent N,N'-methylenebisacrylamide are added to deionized water and stirred for 10-15 min. Then, ammonium persulfate and tetramethylethylenediamine are added as initiators, the temperature is raised to 30-40℃, and the reaction is carried out for 1.5-2.5 h. After filtration, the particle size is 150-250 nm, and hydrogel microspheres are obtained. Step C2: Add 2-(N-morpholino)ethanesulfonic acid to ultrapure water, adjust the pH to 4.5-5.5, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir for 8-12 min, add hydrogel microspheres, heat to 2-4℃, and shake for 0.8-1.2 h to obtain activated microspheres; Step C3: Add lactate oxidase and luciferase to the activated microspheres, heat to 2-4℃, shake for 10-14h to complete the reaction, add serum albumin, heat to 20-30℃, react for 20-40min, then add PBS solution containing 5%wt trehalose-gelatin, cool, and soak for 1-3h to obtain modified lactate oxidase immobilized microspheres; The mass ratio of N-isopropylacrylamide, acrylic acid, crosslinking agent N,N'-methylenebisacrylamide, ammonium persulfate, and tetramethylethylenediamine is (8-9):1:(0.1-0.2):(0.01-0.03):(0.015-0.016). The mass ratio of 2-(N-morpholino)ethanesulfonic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide to hydrogel microspheres is 1:(0.2-0.4):(0.3-0.4):(9-10). The mass ratio of lactate oxidase, luciferase, serum albumin, activated microspheres to PBS solution containing 5% wt trehalose-gelatin is (1.8-2.2):1:(0.4-0.6):(9-10):(18-20).

8. The lymphocyte preservation solution according to claim 1, characterized in that, The modified hydrogel microspheres are prepared as follows: Step D1: Under a nitrogen atmosphere, N-isopropylacrylamide, acrylic acid, sodium dodecyl sulfate and crosslinking agent N,N'-methylenebisacrylamide are added to deionized water, heated to 20-30℃, stirred for 20-40 min, ammonium persulfate and tetramethylethylenediamine are added, the temperature is raised to 60-80℃, and the reaction is carried out for 5-7 h. After the reaction is completed, the temperature is lowered to 20-30℃, centrifuged for 10-20 min, and freeze-dried to obtain porous microspheres. Step D2: Add porous microspheres to 2-morpholine ethanesulfonic acid buffer, cool to -10 to 0°C, sonicate for 2 to 4 min, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, heat to 20 to 30°C, and shake for 20 to 40 min to obtain an activated microsphere suspension; Step D3: Add phosphatidylserine affinity peptide to the activated microsphere suspension, adjust the pH to 6.8-7.2, heat to 30-40℃, shake for 3-5 hours. After the reaction is complete, add ethanolamine, cool to 20-25℃, shake for 20-40 minutes, wash, and obtain modified sensitive hydrogel microspheres. The mass ratio of N-isopropylacrylamide, acrylic acid, sodium dodecyl sulfate, crosslinking agent N,N'-methylenebisacrylamide, ammonium persulfate, and tetramethylethylenediamine is (12-16):1:(0.08-0.12):(0.25-0.35):(0.15-0.25):(0.015-0.025). The mass ratio of the porous microspheres, 2-morpholine ethanesulfonic acid buffer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:(8.5-9.0):(0.025-0.035):(0.015-0.025). The mass ratio of the phosphatidylserine affinity peptide, the activated microsphere suspension, and ethanolamine is 1:(170-190):(18-20).

9. A method for preparing a lymphocyte preservation solution according to any one of claims 1-8, characterized in that, The preparation steps are as follows: Step S1: Add trehalose to Tris-HCl buffer, heat to 2-4℃, stir for 20-40 min, add poloxamer 407 and polyethylene glycol-polylactic acid copolymer, heat to 30-50℃, stir for 15-25 min to obtain a mixed solution; Step S2: Under a nitrogen atmosphere, add quabustatin A and nuclease inhibitor to the mixed solution, stir for 10-20 min, add hemoglobin-MnO2 nanoparticles, sonicate for 3-7 min, then add modified lactate oxidase immobilized microspheres and modified sensitive hydrogel microspheres, stir for 1-3 min, filter through a 0.22 μm filter membrane to obtain lymphocyte preservation solution.

10. The application of a lymphocyte preservation solution according to any one of claims 1-9 in sample pretreatment, characterized in that, It is used for immunotherapy monitoring, rare cell analysis, and sampling in remote areas.

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