Chitosan-modified gold nanoclusters, preparation method and application in erythrocyte immobilization

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a chitosan-modified gold nanoclusters, their preparation method, and their application in erythrocyte immobilization. The invention first provides a method for synthesizing CS-GSH-AuNCs clusters. This method utilizes a two-step low-temperature modification process: first, well-structured GSH-AuNCs core nanoparticles are synthesized; then, chitosan is modified through electrostatic interactions. The reaction conditions are low temperature and nitrogen protection, resulting in mild reaction conditions, high product reproducibility, and a more complete original GSH-AuNCs structure. Experiments show that the CS-GSH-AuNCs clusters synthesized in this invention exhibit good acid-base tolerance and temperature tolerance, demonstrate good erythrocyte immobilization efficacy, and exhibit high biosafety.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a chitosan-modified gold nanocluster, its preparation method, and its application in erythrocyte immobilization. Background Technology

[0002] Red blood cells, the most numerous cells in the human circulatory system, possess unique biological functions, such as oxygen transport, carbon dioxide exchange, and immune regulation. In the biomedical field, red blood cells are often used as drug carriers, biosensor recognition elements, or in vitro diagnostic models. Achieving efficient and stable immobilization of red blood cells is a core prerequisite for expanding their applications.

[0003] Existing erythrocyte immobilization technologies mainly include physical adsorption, cross-linking agent methods, and carrier encapsulation methods, but all have some technical bottlenecks. For example, the ordinary physical adsorption method relies on the weak interaction between the carrier and the surface of erythrocytes, resulting in low immobilization efficiency and poor stability, and is easily affected by environmental pH and temperature, leading to cell detachment. The cross-linking agent method cross-links erythrocytes with the carrier through chemical bonds, which can improve stability, but the cytotoxicity of the cross-linking agent can damage the erythrocyte membrane structure, leading to loss of its biological activity. Although the carrier encapsulation method can encapsulate erythrocytes, it suffers from problems such as high mass transfer resistance and poor compatibility between the carrier and the cell, which limits the normal functioning of erythrocytes.

[0004] Gold nanoclusters (AuNCs) are a novel type of nanomaterial characterized by small particle size (1-5 nm) and good biocompatibility. However, unmodified gold nanoclusters (AuNCs) are prone to aggregation, making them unsuitable for long-term immobilization. Glutathione (GSH) is a natural small molecule containing thiol groups that can stabilize AuNCs through Au-S bonds and also exhibits good biocompatibility. However, GSH-AuNCs have a weakly negative surface charge, resulting in weak adsorption to most human erythrocytes (which carry a negative surface charge), and they are easily dispersed and lost in aqueous environments. Chitosan (CS) is a natural cationic polysaccharide with good film-forming properties, biocompatibility, and cell adsorption capacity (binding to negatively charged cells through electrostatic interactions). However, single chitosan materials have poor mechanical properties and lack specific binding sites for erythrocytes, leading to poor immobilization effects.

[0005] Patent CN116213706A, entitled "Chitosan-encapsulated gold nanoclusters and their preparation method and application," discloses a one-step reaction of a mixture of glutathione and chitosan with chloroauric acid solution in a water bath to generate gel-encapsulated gold nanoclusters. This material possesses film-forming properties, abundant binding sites, and is easily modified with biomolecules. The patent discloses the application of this gel-encapsulated gold nanoclusters in label-free electrochemiluminescence. However, the gold nanoclusters synthesized in this one-step high-temperature method are gel-like. The gel easily swells at low concentrations and easily aggregates and precipitates at high concentrations, resulting in unstable immobilization effects when used for erythrocyte fixation. Furthermore, the gel network and clusters are synthesized simultaneously in this method, making it difficult to control the proportion of surface groups (e.g., -SH, -NH2) on the clusters, leading to significant batch-to-batch variations in the product. The high-temperature water bath also causes partial degradation of chitosan, reducing cell adsorption capacity. Overall, this method fails to meet the requirements for high-quality erythrocyte immobilization materials.

[0006] In summary, there is an urgent need to develop an immobilization material that combines high immobilization efficiency, good biocompatibility, and stable preservation of erythrocyte activity to meet the field's demand for better immobilization of human erythrocytes and to supplement the shortcomings of existing technologies. Summary of the Invention

[0007] The purpose of this invention is to provide a chitosan-modified gold nanoclusters, a preparation method, and its application in erythrocyte immobilization, thereby partially solving or alleviating the above-mentioned deficiencies in the prior art. The specific technical solution adopted by this invention is as follows.

[0008] This invention provides a two-step method for synthesizing chitosan-modified gold nanoclusters.

[0009] A method for preparing chitosan-modified gold nanoclusters includes the following steps:

[0010] S01: Glutathione is added to chloroauric acid solution and stirred to react, wherein the molar ratio of glutathione to chloroauric acid is in the range of 1-3:1;

[0011] S02: Under nitrogen protection, a reducing agent is slowly added dropwise, wherein the molar ratio of the reducing agent to the chloroauric acid is in the range of 2-5:1. The reaction is carried out in the dark under conditions slightly above room temperature to obtain a crude reaction solution of GSH-AuNCs.

[0012] S03: The crude GSH-AuNCs reaction solution is purified by dialysis;

[0013] S04: Prepare a chitosan solution with a concentration of 0.5-2 mg / mL;

[0014] S05: The purified GSH-AuNCs solution and the chitosan solution are mixed at a volume ratio of 1:1-3, and stirred at room temperature to react fully to obtain a mixture containing CS-GSH-AuNCs. The mixture is centrifuged, and the precipitate is collected to obtain the chitosan-modified gold nanoclusters.

[0015] Furthermore, the molar ratio of glutathione to chloroauric acid includes 1:1, 2:1, or 3:1.

[0016] Preferably, the molar ratio of glutathione to chloroauric acid is 2:1.

[0017] Furthermore, the molar ratio of the reducing agent to the chloroauric acid includes 2:1, 3:1, 4:1 or 5:1; the reducing agent includes sodium borohydride or ascorbic acid.

[0018] Preferably, the molar ratio of the reducing agent to the chloroauric acid is 3:1.

[0019] Furthermore, SO2 reacts in the dark at 30-40℃.

[0020] Furthermore, the volume ratio of the purified GSH-AuNCs solution to the chitosan solution includes 1:1, 1:2, or 1:3.

[0021] Preferably, the volume ratio of the purified GSH-AuNCs solution to the chitosan solution is 1:2.

[0022] Furthermore, SO5 was stirred and reacted at 25-30 °C for 2-4 h.

[0023] Furthermore, the molecular weight of the chitosan ranges from 50 to 200 kDa.

[0024] Furthermore, the concentration of chloroauric acid is 0.1-1.0 mM.

[0025] In another aspect, the present invention provides chitosan-modified gold nanoclusters (CS-GSH-AuNCs) prepared by the above method.

[0026] Furthermore, in the chitosan-modified gold nanoclusters, chitosan and GSH-AuNCs are bound together by electrostatic adsorption.

[0027] Furthermore, the particle size range of the GSH-AuNCs core in the chitosan-modified gold nanoclusters is 2-10 nm, preferably 2-5 nm.

[0028] In another aspect, the present invention also provides the application of the chitosan-modified gold nanoclusters (CS-GSH-AuNCs) synthesized above.

[0029] The application of the chitosan-modified gold nanoclusters mentioned above in the preparation of erythrocyte immobilization reagents.

[0030] In another aspect, the present invention provides a method for preparing an immobilized human erythrocyte line, comprising the following steps:

[0031] S01: Prepare the above-mentioned CS-GSH-AuNCs powder;

[0032] SO2: Preparation concentration is 10 8 -10 9 Human erythrocyte suspension per mL;

[0033] S03: Mix the CS-GSH-AuNCs powder with human erythrocyte suspension at a mass-to-volume ratio of 1:5-10, incubate with shaking at 20-30 ℃ for 30-60 min, centrifuge and wash to obtain the immobilized human erythrocyte line.

[0034] Furthermore, the oscillation rate in SO3 is 100-150 rpm, the centrifugation speed is 4000 rpm, and the centrifugation time is 1-10 min.

[0035] Furthermore, the human erythrocyte lineage is mammalian human erythrocytes; the mammalian human erythrocytes include pancreatic islet erythrocytes or tumor erythrocytes.

[0036] Beneficial technical effects:

[0037] This invention first provides a method for synthesizing CS-GSH-AuNCs clusters. The method utilizes a two-step low-temperature modification approach: first, well-structured GSH-AuNCs core nanoparticles are synthesized; then, chitosan is modified via electrostatic interaction to form CS-GSH-AuNCs clusters. This two-step synthesis method allows for precise control of the surface group ratio and charge density. The reaction conditions of this invention are slightly above room temperature (30-40℃) under nitrogen protection, resulting in mild reaction conditions, high product reproducibility, and a more complete original GSH-AuNCs structure. Regarding dispersibility, electrostatic modification imparts a stable charge to the particle surface, reducing the likelihood of precipitation. In this invention, GSH-AuNCs first form uniform nuclei at small sizes, and then combine with chitosan via electrostatic interaction. This provides greater controllability of the surface modification layer, allowing for precise control of particle surface charge and size by adjusting the amount of chitosan.

[0038] Experiments revealed that the CS-GSH-AuNCs clusters synthesized in this invention exhibit good acid-base and temperature tolerance, demonstrate excellent immobilization effect on erythrocytes, and possess high biosafety. Erythrocytes immobilized by these CS-GSH-AuNCs clusters showed high activity under both pH and temperature stress experiments. This is because the small particle size (2-5 nm) of the GSH-AuNCs core and its chitosan-coated surface prevents erythrocyte membrane perforation or immune recognition, thus reducing apoptosis signal activation. The amino groups (-NH2) on the chitosan molecular chain can be protonated to regulate local pH, resisting the influence of external pH fluctuations on erythrocyte membrane potential and preventing membrane protein denaturation. The synergistic effect of GSH and gold nanoclusters can alleviate high-temperature-induced oxidative damage. High temperatures promote the production of reactive oxygen species (ROS) in cells, and excessive ROS can damage biomolecules such as DNA and proteins. GSH, being an endogenous antioxidant, can directly scavenge ROS. In addition, gold nanoclusters themselves have certain enzyme-like activities, which can assist in the catalytic decomposition of ROS, further reducing the damage of oxidative stress to erythrocytes and thus protecting cell activity. Therefore, they not only have a good effect on immobilizing erythrocytes, but also show that the environmental tolerance of erythrocytes immobilized by this material is significantly improved.

[0039] In summary, the CS-GSH-AuNCs clusters provided by this invention bind to erythrocytes through "electrostatic attraction + specific sugar interaction," which is a mild, non-covalent binding. The cluster material forms a uniform thin coating on the surface of erythrocytes, achieving stable fixation without hindering the exchange of substances between erythrocytes and the external environment, thus ensuring the normal metabolic needs of erythrocytes. This invention has broad application potential as a erythrocyte fixation reagent. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0041] Figure 1 Transmission electron microscopy images of GSH-AuNCs and CS-GSH-AuNCs nanoparticles prepared in one embodiment of the present invention (a is GSH-AuNCs with monodisperse morphology, b is CS-GSH-AuNCs with aggregated morphology).

[0042] Figure 2This is the result of co-culturing different concentrations of CS-GSH-AuNCs solution with erythrocytes on cell viability in one embodiment of the present invention;

[0043] Figure 3 The zeta potential of CS-GSH-AuNCs nanoparticles prepared in one embodiment of the present invention;

[0044] Figure 4 This is the fluorescence intensity result of different molar ratios of GSH and HAuCl4 in one embodiment of the present invention;

[0045] Figure 5 This is the fluorescence intensity result of different molar ratios of NaBH4 and HauCl4 in one embodiment of the present invention;

[0046] Figure 6 This is the fluorescence intensity result of GSH-AuNCs to CS at different volume ratios in one embodiment of the present invention;

[0047] Figure 7 This refers to the fluorescence intensity retention rate of CS-GSH-AuNCs under different pH conditions in one embodiment of the present invention.

[0048] Figure 8 The fluorescence intensity retention rate of CS-GSH-AuNCs under different temperature conditions is shown in one embodiment of the present invention. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0050] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0051] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0052] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0053] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0054] Example 1

[0055] I. Preparation method of CS-GSH-AuNCs

[0056] 1. Synthesis of GSH-AuNCs

[0057] (1) Chloroauric acid (HAuCl4) Dissolve HAuCl4 in ultrapure water to prepare a 0.1-1.0 mM HAuCl4 solution.

[0058] (2) Add glutathione (GSH) to the above solution, control the molar ratio of GSH to HAuCl4 in the range of 1-3:1, and stir magnetically for 10-20 min until the solution is clear.

[0059] (3) Under nitrogen protection, slowly add reducing agent (sodium borohydride or ascorbic acid), control the molar ratio of reducing agent to HAuCl4 in the range of 2-5:1, react at 30-40℃ in the dark for 1-3 h, and obtain light yellow crude GSH-AuNCs reaction solution.

[0060] (4) Place the crude reaction solution in a dialysis bag (molecular weight cutoff 3500 Da) and dialyze with ultrapure water for 24-48 h (replace the water every 6 h) to remove unreacted GSH and salts, and obtain purified GSH-AuNCs solution.

[0061] 2. CS Preprocessing

[0062] (1) Dissolve chitosan (molecular weight range 50-200 kDa) in 1.0% (v / v) glacial acetic acid solution to prepare a CS solution with a concentration of 0.5-2.0 mg / mL.

[0063] (2) Adjust the pH of the CS solution to 5.5-6.5 with 0.1 M NaOH solution, stir magnetically for 30 min, filter through a 0.22 μm filter membrane to remove insoluble impurities, and obtain a clear CS solution.

[0064] 3. Preparation of CS-GSH-AuNCs

[0065] (1) The purified GSH-AuNCs solution and CS solution were mixed at a volume ratio of 1:1 to 1:3 and magnetically stirred at 25-30℃ for 2-4 h to obtain a mixture containing CS-GSH-AuNCs. The amino group (-NH2) of CS and the carboxyl group (-COOH) of GSH form a strong electrostatic interaction, and CS achieves surface modification of GSH-AuNCs.

[0066] (2) Centrifuge the mixture (8000-10000 rpm, 10-15 min) and collect the precipitate; wash the precipitate with ultrapure water 3-5 times to remove unbound free CS.

[0067] (3) The precipitate was redispersed in ultrapure water and sonicated for 5-10 min to obtain a CS-GSH-AuNCs dispersion with a concentration of 0.5-1.5 mg / mL. It was stored at 4℃ in the dark for later use.

[0068] II. Application of CS-GSH-AuNCs in Human Erythrocyte Immobilization

[0069] 1. Material Pretreatment

[0070] The CS-GSH-AuNCs dispersion was freeze-dried to form a powder for later use.

[0071] 2. Red blood cell pretreatment

[0072] Collect fresh anticoagulated whole blood, separate red blood cells by density gradient centrifugation, wash 2-3 times with PBS buffer, and adjust the red blood cell concentration to 1×10⁻⁶. 6 -1×10 7 The red blood cell suspension was obtained by dividing the cells by 1 / mL.

[0073] 3. Erythrocyte immobilization

[0074] CS-GSH-AuNCs were dispersed in PBS buffer to prepare a carrier suspension with a concentration of 0.1-1 mg / mL. The carrier suspension was mixed with red blood cell suspension at a volume ratio of 1-5:1 and incubated at 25-37℃ with gentle shaking (shaking rate 100-150 rpm) for 30-60 min. Unbound red blood cells were removed by centrifugation to obtain immobilized red blood cell complexes.

[0075] Example 2

[0076] An example of a specific preparation method for CS-GSH-AuNCs is provided.

[0077] 1. Synthesis of GSH-AuNCs: Take 100 mL of 0.5 mM HAuCl4 solution, add GSH (molar ratio of GSH to HAuCl4 is 2:1), and stir for 15 min; under nitrogen protection, add 2 mM NaBH4 solution (molar ratio of NaBH4 to HAuCl4 is 3:1), and react at 37℃ in the dark for 2 h; dialyze for 36 h to obtain crude GSH-AuNCs reaction solution, dialyze and purify to obtain GSH-AuNCs solution.

[0078] 2. CS pretreatment: Dissolve CS with a molecular weight of 100 kDa in 1% glacial acetic acid to prepare a 1.0 mg / mL solution; adjust the pH to 6.0, filter and set aside.

[0079] 3. CS modification: GSH-AuNCs solution and CS solution were mixed at a volume ratio of 1:2 and stirred at 28℃ for 3 h; centrifuged at 10000 rpm for 12 min, washed 3 times, and ultrasonically dispersed to obtain a CS-GSH-AuNCs dispersion of 1.0 mg / mL.

[0080] 4. Co-culture with red blood cells:

[0081] (1) Prepare CS-GSH-AuNCs solutions of different concentrations: 0, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL.

[0082] (2) Preparation of red blood cell suspension: Take 5 mL of fresh anticoagulated whole blood from a healthy person and dilute it with an equal volume of PBS buffer; slowly add the diluted blood to the surface of lymphocyte separation medium (density 1.077 g / mL), centrifuge at 2000 r / min for 20 min, and aspirate the red blood cell pellet below the middle layer. Then, wash the red blood cell pellet three times with PBS buffer, centrifuging at 1500 r / min for 10 min after each wash to remove plasma proteins and white blood cells; finally, resuspend the red blood cells in PBS buffer and adjust the red blood cell concentration to 5 × 10⁻⁶. 6 The red blood cell suspension was obtained by measuring cells / mL and set aside for later use.

[0083] (3) Mix CS-GSH-AuNCs solutions of different concentrations with red blood cell suspension, incubate gently with shaking at 37°C for 60 min, and count the red blood cells.

[0084] 4. Characterization results:

[0085] (1) Figure 1Transmission electron microscopy (TEM) showed that the particle size of GSH-AuNCs nanoparticles was 2-5 nm. Compared with monodisperse GSH-AuNCs, CS-GSH-AuNCs exhibited an aggregated morphology, indicating that CS successfully bound to GSH-AuNCs.

[0086] (2) Figure 2 Human erythrocyte toxicity assays showed that even at high concentrations of CS-GSH-AuNCs solution, human erythrocytes still exhibited good activity, indicating that CS-GSH-AuNCs can maintain high erythrocyte activity.

[0087] (3) In this invention, the modification of GSH-AuNCs by chitosan is by electrostatic adsorption: the carboxyl group (-COOH) of GSH dissociates into -COO in aqueous solution. - This causes the surface of GSH-AuNCs to carry a negative charge; while the amino group (-NH2) of CS will protonate to -NH3 under neutral / weakly acidic conditions. + The two components, carrying a positive charge, bind through electrostatic interactions, enabling chitosan to modify GSH-AuNCs. This binding mechanism can be demonstrated using zeta potential characterization. Figure 3 As shown, the potential of GSH-AuNCs was -20.6 mV, and the potential changed to +10.7 mV after modification with chitosan. The significant change in potential proves the electrostatic adsorption effect.

[0088] Example 3

[0089] Verification of CS-GSH-AuNCs fixed erythrocytes prepared in Example 2.

[0090] 1. CS-GSH-AuNCs fixation of human erythrocytes

[0091] CS-GSH-AuNCs powder was dispersed in PBS buffer to prepare a carrier suspension with a concentration of 0.5 mg / mL. 3 mL of the carrier suspension was mixed with 1 mL of erythrocyte suspension and placed in a 37°C constant temperature shaking incubator. The mixture was gently shaken at 50 r / min for 45 min to complete cell immobilization, serving as the experimental group.

[0092] Two control groups, Control Group 1 and Control Group 2, were set up. Control Group 1 consisted of pure chitosan solution (2 mg / mL), and Control Group 2 consisted of PBS. Both solutions were mixed with red blood cell suspension and incubated in a 37°C constant temperature shaking incubator at 50 rpm for 45 min to complete cell immobilization.

[0093] The fixation rate of both the experimental and control groups was determined by centrifugation sedimentation (fixation rate = number of fixed human erythrocytes / total number of human erythrocytes × 100%). The results are shown in Table 1. Compared with control groups 1 and 2, the fixation rate of the experimental group was as high as 90%, which was 30.2% higher than that of pure chitosan fixation, indicating a better fixation effect.

[0094] Table 1

[0095]

[0096] 2. Preservation of erythrocyte viability under stress conditions

[0097] (1) Temperature stress: The fixed red blood cells of the above experimental group, control group 1 and control group 2 were placed in a constant temperature incubator at 42℃ for 12 h.

[0098] (2) pH stress: The fixed red blood cells of the above experimental group, control group 1 and control group 2 were placed in PBS buffer at pH 5.0 and pH 9.0 respectively and incubated at 37°C for 24 h.

[0099] The retention rate of erythrocytes in each group was detected by flow cytometry combined with Annexin V-FITC / PI double staining. Three replicates were set up for each group, and the average value was taken. The results are shown in Table 2. Compared with control groups 1 and 2, the erythrocyte viability retention rate in the experimental groups was above 70%.

[0100] Table 2

[0101]

[0102] Example 4

[0103] The key parameters and steps of the preparation method in Example 1 were verified.

[0104] 1. Synthesis of GSH-AuNCs

[0105] (1) Chloroauric acid (HAuCl4) Dissolve HAuCl4 in ultrapure water to prepare a 0.1-1.0 mM HAuCl4 solution.

[0106] (2) Add glutathione (GSH) to the above solution, control the molar ratio of GSH to HAuCl4 to be 1-3:1, and stir magnetically for 10-20 min until the solution is clear.

[0107] (3) Under nitrogen protection, slowly add reducing agent (sodium borohydride or ascorbic acid), control the molar ratio of reducing agent to HAuCl4 to be 2-5:1, and react at 30-40℃ in the dark for 1-3 h to obtain light yellow crude GSH-AuNCs reaction solution.

[0108] (4) Place the crude reaction solution in a dialysis bag (molecular weight cutoff 3500 Da) and dialyze with ultrapure water for 24-48 h (replace the water every 6 h) to remove unreacted GSH and salts, and obtain purified GSH-AuNCs solution.

[0109] In the synthesis of GSH-AuNCs, GSH is the ligand of AuNCs. The molar ratio of GSH to HAuCl4 affects the size and fluorescence quantum yield of AuNCs (too low a ratio will lead to cluster aggregation, while too high a ratio will result in ligand redundancy).

[0110] The molar ratio of reducing agent to HAuCl4 affects the degree of Au reduction (insufficient reduction will leave Au residue). 3+ Excessive amounts may disrupt the coordination structure of GSH.

[0111] Nitrogen protection combined with light avoidance and low temperature reaction can prevent GSH oxidation and maintain the stability of Au-S bonds. If the conditions are out of control, AuNCs will undergo fluorescence quenching and structural collapse.

[0112] Figure 4 The results for different molar ratios of GSH and HAuCl4 are shown. Higher fluorescence intensity indicates better performance of the synthesized GSH-AuNCs. Figure 5 The results show the results of different molar ratios of reducing agent NaBH4 and HAuCl4. Higher fluorescence intensity indicates better performance of the synthesized GSH-AuNCs.

[0113] 2. CS Preprocessing

[0114] (1) Dissolve chitosan (molecular weight range 50-200 kDa) in 1.0% (v / v) glacial acetic acid solution to prepare a CS solution with a concentration of 0.5-2.0 mg / mL.

[0115] (2) Adjust the pH of the CS solution to 5.5-6.5 with 0.1 M NaOH solution, stir magnetically for 30 min, filter through a 0.22 μm filter membrane to remove insoluble impurities, and obtain a clear CS solution.

[0116] 3. Preparation of CS-GSH-AuNCs

[0117] (1) The purified GSH-AuNCs solution and CS solution were mixed at a volume ratio of 1:1 to 1:3 and magnetically stirred at 25-30℃ for 2-4 h to obtain a mixture containing CS-GSH-AuNCs. The surface modification of GSH-AuNCs by CS is achieved by the strong electrostatic interaction between the amino group (-NH2) of CS and the carboxyl group (-COOH) of GSH.

[0118] (2) Centrifuge the mixture (8000-10000 rpm, 10-15 min) and collect the precipitate; wash the precipitate with ultrapure water 3-5 times to remove unbound free CS.

[0119] (3) The precipitate was redispersed in ultrapure water and sonicated for 5-10 min to obtain a CS-GSH-AuNCs dispersion with a concentration of 0.5-1.5 mg / mL. It was stored at 4℃ in the dark for later use.

[0120] In the preparation of CS-GSH-AuNCs, the volume ratio of GSH-AuNCs solution to CS solution affects the amount of chitosan modification (insufficient modification leads to unstable surface charge of the material, while excessive modification will encapsulate AuNCs, resulting in weakened fluorescence).

[0121] Stirring time affects the uniformity of electrostatic adsorption (too short a time will result in an uneven modified layer, while too long a time may lead to AuNCs agglomeration).

[0122] Centrifugation speed / ultrasonic dispersion affects the final dispersibility of the material (insufficient centrifugation leaves behind free CS, while excessive ultrasonication will destroy the AuNCs structure).

[0123] Figure 6 The results of different volumes of GSH-AuNCs solution and CS solution are shown. Higher fluorescence intensity indicates better performance of the synthesized CS-GSH-AuNCs.

[0124] Example 5

[0125] Other properties of the CS-GSH-AuNCs prepared by the preferred method in Example 2 were verified.

[0126] Figure 7 The results show the fluorescence intensity retention rate of CS-GSH-AuNCs under different pH conditions. The results show that the fluorescence intensity retention rate of CS-GSH-AuNCs does not change much in the pH 4 to pH 8 environment, and is basically maintained at about 80%, which proves that CS-GSH-AuNCs have good acid and alkali tolerance.

[0127] Figure 8 The fluorescence intensity retention rate of CS-GSH-AuNCs under different temperature conditions is shown. The results show that the fluorescence intensity retention rate of CS-GSH-AuNCs does not change much in an environment of 25℃ ~ 60℃, and basically remains at about 60%, which proves that CS-GSH-AuNCs also have good temperature tolerance.

[0128] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0129] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A method for preparing chitosan-modified gold nanoclusters, characterized in that, Includes the following steps: S01: Glutathione is added to chloroauric acid solution and stirred to react, wherein the molar ratio of glutathione to chloroauric acid is in the range of 1-3:1; S02: Under nitrogen protection, a reducing agent is slowly added dropwise, wherein the molar ratio of the reducing agent to the chloroauric acid is in the range of 2-5:

1. The reaction is carried out in the dark under conditions slightly above room temperature to obtain a crude reaction solution of GSH-AuNCs. S03: The crude GSH-AuNCs reaction solution is purified by dialysis; S04: Prepare a chitosan solution with a concentration of 0.5-2 mg / mL; S05: The purified GSH-AuNCs solution and the chitosan solution are mixed at a volume ratio of 1:1-3, and stirred at room temperature to react fully to obtain a mixture containing CS-GSH-AuNCs. The mixture is centrifuged, and the precipitate is collected to obtain the chitosan-modified gold nanoclusters.

2. The preparation method according to claim 1, characterized in that, The molar ratio of glutathione to chloroauric acid includes 1:1, 2:1, or 3:

1.

3. The preparation method according to claim 1, characterized in that, The molar ratio of the reducing agent to the chloroauric acid includes 2:1, 3:1, 4:1 or 5:1; the reducing agent includes sodium borohydride or ascorbic acid.

4. The preparation method according to claim 1, characterized in that, SO2 reacts in the dark at 30-40℃.

5. The preparation method according to claim 1, characterized in that, The volume ratio of the purified GSH-AuNCs solution to the chitosan solution includes 1:1, 1:2, or 1:

3.

6. The preparation method according to claim 1, characterized in that, The molecular weight range of the chitosan is 50-200 kDa.

7. Chitosan-modified gold nanoclusters prepared by the preparation method according to any one of claims 1-6.

8. The chitosan-modified gold nanoclusters as described in claim 7, characterized in that, In the chitosan-modified gold nanoclusters, chitosan and GSH-AuNCs are bound together by electrostatic adsorption.

9. The chitosan-modified gold nanoclusters as described in claim 7, characterized in that, The particle size range of the GSH-AuNCs core in the chitosan-modified gold nanoclusters is 2-10 nm.

10. The application of the chitosan-modified gold nanoclusters prepared by the preparation method according to any one of claims 1-6 or the chitosan-modified gold nanoclusters according to claim 7 in the preparation of erythrocyte immobilization reagents.

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