A renewable urokinase silica gel adsorbent, a preparation method and application thereof

Abstract

This invention discloses a regenerable urokinase silica gel adsorbent, its preparation method, and its applications, belonging to the field of new materials. The regenerable urokinase silica gel adsorbent is prepared through the following steps: Step 1, preparing a composite template emulsion; Step 2, preparing a sol-gel system; Step 3, preparing a silica gel precursor; Step 4, preparing the regenerable urokinase silica gel adsorbent. This invention avoids the use of expensive chemical ligands and precipitation reagents, and the regeneration process only requires inexpensive buffer solutions without the need for high-concentration salts or denaturants. Therefore, the material and process costs of this invention are significantly reduced compared to existing technologies. Simultaneously, the simplified process greatly reduces water and electricity consumption and chemical waste discharge, providing a solid technical foundation for the large-scale, low-cost, and continuous production of urokinase.

Description

Technical Field

[0001] This invention relates to the field of new materials technology, and in particular to a renewable urokinase silica adsorbent, its preparation method, and its application. Background Technology

[0002] Urokinase is an important serine protease produced by kidney cells. It can directly activate plasminogen, thereby dissolving blood clots, and is a key thrombolytic drug for treating cardiovascular and cerebrovascular diseases such as acute myocardial infarction and cerebral thrombosis. Currently, the main source of commercially available urokinase is extraction and purification from large quantities of human urine. Therefore, developing efficient and low-cost urokinase purification technologies has significant economic and social value.

[0003] Urokinase is an important serine protease produced by kidney cells. It can directly activate plasminogen, thereby dissolving blood clots, and is a key thrombolytic drug for treating cardiovascular and cerebrovascular diseases such as acute myocardial infarction and cerebral thrombosis. Currently, the main source of commercially available urokinase is extraction and purification from large quantities of human urine. Therefore, developing efficient and low-cost urokinase purification technologies has significant economic and social value.

[0004] Extracting urokinase from urine is an extremely challenging process. Urine has a complex composition, containing not only urokinase but also large amounts of urea, salts, pigments, extraneous proteins (such as albumin and globulin), and suspended solids like cell debris. Traditional purification processes typically follow the classic "precipitation-chromatography" route, including: collecting urine, adjusting pH, adding a precipitant, adsorption or salting out, centrifuging to collect the precipitate, and redissolving the precipitate, followed by fine purification using ion exchange chromatography, affinity chromatography, or gel filtration chromatography. While these traditional processes have been used for many years, they suffer from lengthy processes, high production costs, and significant environmental pollution. Furthermore, the core purification material, the adsorption and separation material, currently relies heavily on chemically modified silica gel or agarose gels. For example, ion-exchange silica gels adsorb urokinase via electrostatic interactions by bonding quaternary ammonium salts and other functional groups. However, this chemical adsorption is strong and often requires high-ionic-strength solutions for elution, potentially damaging urokinase activity. More importantly, the high salt content and extraneous proteins in the original urine severely interfere with adsorption, making it impossible to directly process the original urine and necessitating complex pretreatment. Meanwhile, repeated regeneration with strong acids / bases can lead to hydrolysis and shedding of the bonded phase, resulting in a limited lifespan. While affinity chromatography packing materials offer high selectivity, their ligands (such as antibodies and peptides) are extremely expensive, have poor stability, and can only be reused a limited number of times, failing to meet the cost control requirements of large-scale industrial production. To overcome the shortcomings of chemisorption materials, those skilled in the art have attempted to use unmodified porous silica gel, hoping to simplify the process through physical adsorption (such as van der Waals forces). However, it is generally understood that in the near-neutral pH environment of urine, the surface of bare silica gel becomes negatively charged due to the dissociation of silanol groups, and urokinase, with its alkaline isoelectric point, is also negatively charged. The electrostatic repulsion between the two is considered to severely hinder effective physical adsorption. Therefore, a technical bias exists in the field, suggesting that unmodified bare silica gel is inherently unsuitable for adsorbing and enriching urokinase from urine. This bias has led research efforts almost entirely to complex chemical modification pathways, neglecting the possibility of overcoming this challenge through precise control of the physical properties of the bare silica gel itself.

[0005] Therefore, developing an adsorbent material that can efficiently and selectively adsorb urokinase directly from primary urine without complex pretreatment, and that can be fully regenerated and recycled multiple times through a mild and simple process, thereby significantly reducing production costs, has become a long-standing technical challenge that the field has been eager to solve but has yet to achieve a breakthrough in. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention proposes a regenerable urokinase silica adsorbent, its preparation method, and its application.

[0007] This invention provides a method for preparing a regenerative urokinase silica gel adsorbent, comprising the following steps:

[0008] Step 1: Dissolve and disperse hexadecyltrimethylammonium bromide and polystyrene microspheres together in a mixture of anhydrous ethanol and deionized water, and stir to form a homogeneous composite template emulsion;

[0009] Step 2: Add tetraethyl orthosilicate dropwise to the composite template emulsion and stir to obtain a sol-gel system;

[0010] Step 3: Add ammonia to the sol-gel system to gel. When the gel point is near, add an aqueous solution of calcium chloride and urea to the sol-gel system and stir to obtain a wet gel. Transfer the wet gel to a closed reaction vessel and allow it to age statically to obtain a gel. Wash the gel with a mixture of anhydrous ethanol and deionized water and dry it to obtain the silica gel precursor.

[0011] Step 4: Place the silica precursor obtained in Step 3 into a temperature-controlled muffle furnace and heat it twice. After the temperature of the temperature-controlled muffle furnace cools down to room temperature, remove it to obtain the regenerable urokinase silica adsorbent.

[0012] Further, the specific steps of step one are as follows: 5-10 parts by weight of hexadecyltrimethylammonium bromide and 10-20 parts by weight of polystyrene microspheres are dissolved and dispersed together in a mixture of 300-400 parts by weight of anhydrous ethanol and deionized water, and stirred in a water bath at 400-60°C at 400-800 rpm for 1.5-2.5 hours to form a homogeneous composite template emulsion.

[0013] Furthermore, in step one, the polystyrene microspheres have a particle size of 300-400 nm, and the volume ratio of anhydrous ethanol to deionized water in the mixture of anhydrous ethanol and deionized water is 1-2:0.5-1.

[0014] Furthermore, the specific steps of step two are as follows: 100-200 parts by mass of tetraethyl orthosilicate are slowly added dropwise to the composite template emulsion obtained in step one at a rate of 1-2 mL / min, and hydrolyzed by stirring at 400-800 rpm for 3-4 hours to obtain a sol-gel system.

[0015] Further, the specific steps of step three are as follows: 50-100 parts by weight of ammonia water are added to the sol-gel system in step two to prepare for gelation. After 25-35 minutes, when the gelation point is near, 20-25 parts by weight of an aqueous solution of calcium chloride and urea are rapidly added to the sol-gel system. The mixture is stirred at 200-400 rpm for 1-1.5 hours to obtain a wet gel. The wet gel is transferred to a sealed reactor and statically aged at 60-80°C for 20-24 hours to obtain a gel. After static aging, the gel is washed three times with a mixture of 100-120 parts by weight of anhydrous ethanol and deionized water, and then dried at 50-70°C for 10-12 hours to obtain the silica gel precursor.

[0016] Furthermore, in step three, the mass ratio of calcium chloride, urea, and deionized water in the aqueous solution of calcium chloride and urea is 1-1.5:2-3:15-20, and the volume ratio of anhydrous ethanol to deionized water in the mixture of anhydrous ethanol and deionized water is 6-7:2-3.

[0017] Furthermore, in step four, the specific steps of performing two heating cycles are as follows: the first heating cycle is performed, with a heating rate of 1-2℃ / min from room temperature to 220-280℃, and the temperature is maintained for 2-3 hours to remove physically adsorbed water and some residual solvent; then the second heating cycle is performed, with a heating rate of 2-3℃ / min to 430-470℃, and the temperature is maintained for 4-5 hours.

[0018] The present invention also provides a regenerative urokinase silica adsorbent prepared according to the method described.

[0019] The present invention also provides the application of the regenerable urokinase silica adsorbent in the extraction of urokinase.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] This invention overcomes the technical obstacle commonly believed in the art—the electrostatic repulsion between bare silica gel and urokinase, which prevents effective adsorption—by precisely controlling the physical parameters (Zeta potential, pore size, and surface hydroxyl density) of the regenerable urokinase silica gel adsorbent. It enables the efficient enrichment of urokinase directly in raw urine using the regenerable urokinase silica gel adsorbent without any pretreatment, eliminating the most cumbersome and time-consuming steps in traditional processes, significantly shortening the production cycle, and substantially reducing the loss of activity in the target product.

[0022] This invention creatively designs a composite structure with gradient channels and nano-CaCO3 particles. The large pores in the outer shell effectively prevent clogging, while the channels in the inner core enable the size sieving of impurity proteins and the specific adsorption of urokinase. Simultaneously, the introduced nano-CaCO3 particles can trigger physical elution and material regeneration simply by changing the pH of the buffer solution, solving the core problems of traditional chemical adsorbents such as easy contamination, difficult regeneration, and short lifespan.

[0023] This invention avoids the use of expensive chemical ligands (such as affinity ligands and ion exchange groups) and precipitation reagents, and the regeneration process only requires inexpensive buffer solutions without the need for high-concentration salts or denaturants. Therefore, the material and process costs of this invention are significantly lower than those of existing technologies. At the same time, the simplified process greatly reduces water and electricity consumption and chemical waste discharge, meeting the requirements of green manufacturing. This provides a solid technical foundation for the large-scale, low-cost, and continuous production of urokinase, and has enormous market application potential. Attached Figure Description

[0024] Figure 1 This is a flowchart illustrating the preparation process of the regenerable urokinase silica adsorbent in this invention.

[0025] Figure 2 This is an electron micrograph of the regenerable urokinase silica adsorbent in Example 1 of this invention.

[0026] Figure 1 In the middle: 1: composite template emulsion; 2: tetraethyl orthosilicate; 3: ammonia water; 4: aqueous solution of calcium chloride and urea; 5: closed reaction vessel; 6: silica gel precursor; 7: regenerable urokinase silica gel adsorbent; 71: outer shell layer of regenerable urokinase silica gel adsorbent; 72: inner core layer of regenerable urokinase silica gel adsorbent. Detailed Implementation

[0027] To better illustrate the preparation process of the regenerative urokinase silica adsorbent involved in this invention and its advantages over existing technologies, it will be based on... Figure 1 Further explanation will follow. Example

[0028] As shown in the attached figure, a method for preparing a regenerable urokinase silica gel adsorbent includes the following steps:

[0029] Step 1: Dissolve and disperse 5g of cetyltrimethylammonium bromide and 10g of polystyrene microspheres with a particle size of 300nm in a mixture of 200mL of anhydrous ethanol and 100mL of deionized water, and stir at 600rpm for 2 hours in a 40℃ water bath to form a homogeneous composite template emulsion.

[0030] Step 2: Add 100 mL of tetraethyl orthosilicate dropwise to the composite template emulsion obtained in Step 1 at a rate of 1 mL / min, and continue to stir at 600 rpm for 4 hours to hydrolyze the mixture and obtain a sol-gel system. At this time, the sol-gel system gradually changes from clear to milky white and the viscosity increases.

[0031] Step 3: Add 1.5g of calcium chloride and 3g of urea to 20ml of deionized water to prepare an aqueous solution of calcium chloride and urea. Add 50ml of ammonia to the sol-gel system from Step 2 to induce gelation. After about 30 minutes, when the gelation reaches near the gel point, quickly add the pre-prepared aqueous solution of calcium chloride and urea to the sol-gel system. Stir at 200rpm for 1 hour to obtain a wet gel. Then transfer the wet gel to a sealed reactor and statically age it at 80℃ for 24 hours to obtain a gel. After static aging, wash the gel three times with a mixture of 70ml of anhydrous ethanol and 30ml of deionized water to remove some unassembled surfactants and ions. Then dry it at 60℃ for 12 hours to obtain the silica gel precursor.

[0032] Step 4: Place the silica precursor obtained in Step 3 into a temperature-controlled muffle furnace. Perform the first heating at a rate of 1℃ / min from room temperature to 280℃, and hold for 2 hours. Then perform the second heating at a rate of 2℃ / min to 450℃, and hold for 4 hours. After the temperature-controlled muffle furnace cools to room temperature, remove the product to obtain the regenerable urokinase silica adsorbent. Example

[0033] Except for the 10g polystyrene microspheres with a particle size of 350nm added in step one, the other steps are the same as in Example 1. Example

[0034] Except for the 10g polystyrene microspheres added in step one with a particle size of 400nm, the other steps are the same as in Example 1. Example

[0035] Except for the addition of 3g of cetyltrimethylammonium bromide in step one, the remaining steps are the same as in Example 1. Example

[0036] Except for the addition of 7g of cetyltrimethylammonium bromide in step one, the remaining steps are the same as in Example 1. Example

[0037] Except for step four, in which the temperature is increased to 430°C at a rate of 2°C / min and held for 4 hours, the remaining steps are the same as in Example 1. Example

[0038] Except for step four, in which the temperature is increased to 470°C at a rate of 2°C / min and held at that temperature for 4 hours, the remaining steps are the same as in Example 1.

[0039] The principle of this invention:

[0040] In step one, the pore size is measured using a Micromeritics ASAP 2460 or equivalent fully automated surface area and pore size analyzer. The polystyrene microspheres, with a particle size of 300–400 nm, act as pore-forming agents, forming a macroscopic accumulation within the silica gel network. When they are eventually removed, the remaining spaces constitute the macropores (100–600 nm) in the outer shell of the regenerable urokinase silica gel adsorbent, providing high-speed transport channels for fluids.

[0041] In step one, hexadecyltrimethylammonium bromide acts as a surfactant, and its molecules self-assemble in solution to form micelles of approximately 3–4 nm. During the formation of the sol-gel system in step two, these micelles act as templates, guiding the condensation of silica generated in situ from the hydrolysis and condensation of tetraethyl orthosilicate during the sol-gel system formation process in step two, forming pores in the core layer of the regenerable urokinase silica adsorbent of 10–45 nm.

[0042] In step two, due to differences in diffusion and reaction rates, silicate ions generated from the hydrolysis of tetraethyl orthosilicate diffuse from the bulk solution to the sol-gel interface during the formation of the sol-gel system. Outside the sol-gel system, the reactant concentration is high, and the sol-gel process is rapid, forming loose macropores around the polystyrene microspheres. As the sol-gel system reacts inward, the concentration gradient of unhydrolyzed tetraethyl orthosilicate and the hydrolyzed silanol monomers decreases, and the micelle template formed by the self-assembly of hexadecyltrimethylammonium bromide plays a dominant role, thus forming micropores with gradually decreasing pore sizes. This reaction-diffusion process from the outside in naturally creates a pore size gradient.

[0043] In step three, adding calcium chloride and urea near the gel point takes advantage of the initial formation of the sol-gel system while still having sufficient porosity, allowing Ca to... 2+ Urea molecules are uniformly dispersed throughout the three-dimensional network framework of the sol-gel system. Urea will subsequently be released as slowly released CO3. 2- source.

[0044] In step four, during the heating process from 250 to 450°C, polystyrene and hexadecyltrimethylammonium bromide are completely decomposed and oxidized, and discharged in the form of CO2 and H2O. The space they originally occupied is permanently fixed, thus forming a continuous and stable gradient channel.

[0045] In step four, Ca is uniformly distributed in the silica precursor. 2+CO3 produced by the hydrolysis of urea 2- Through combination, in-situ crystallization generates nano-sized CaCO3 particles. The specific process is as follows:

[0046] (NH2)2CO + H2O → 2NH3 + CO2, CO2 + H2O → H2CO3 → CO3 2- + 2H + CO3 2- + Ca 2+ →CaCO3, these CaCO3 particles are physically embedded in the silica matrix, rather than chemically bonded.

[0047] The heat treatment temperature of 50°C was carefully selected. At this temperature, CaCO3 is very stable and will not decompose, ensuring its long-term presence in the regenerable urokinase silica adsorbent.

[0048] The heat treatment temperature has a decisive influence on the type and density of silanol groups (-SiOH) on the surface of the silica gel precursor. At 450℃, some adjacent silanol groups will condense to form siloxane bonds (-Si-O-Si-), which will reduce the density of surface silanol groups and tend to a stable and moderate state.

[0049] According to the surface coordination theory, the silanol group is the source of the surface charge of the regenerable urokinase silica gel adsorbent (-SiOH + H2O). -SiO - + H3O + The moderate reduction in hydroxyl density directly resulted in the regenerable urokinase silica adsorbent having its Zeta potential at pH 7.2 precisely tuned from above -30mV for ordinary silica to the "optimal adsorption window" of -18mV to -25mV required by this invention. This window ensures a moderate, reversible electrostatic attraction with urokinase molecules, rather than strong repulsion or irreversible adsorption.

[0050] Example of effect 1:

[0051] Pore ​​size was measured using a Micromeritics ASAP 2460 or equivalent fully automated surface area and pore size analyzer. Specific pore size values ​​under different experimental conditions in Examples 1-7 are shown in Table 1.

[0052] Table 1: Specific values ​​of pore size under different experimental conditions in Examples 1-7:

[0053]

[0054] Example 2:

[0055] 1. Specific steps for adsorbing urokinase in urine using regenerable urokinase silica gel adsorbent:

[0056] Step 1: Pack 10g of the regenerable urokinase silica gel adsorbent prepared in Example 1 into a suitable chromatography column with a diameter of 1.5cm and a height of 20cm using a wet packing method, ensuring uniform packing without air bubbles or gaps. Flow 5 column volumes of pH 7.4 phosphate-buffered saline (PBS, 10mM) through the adsorption column at a constant flow rate of 1.0mL / min until the pH and conductivity of the effluent match those of PBS, creating a stable chemical environment to ensure the consistency and reproducibility of subsequent adsorption behavior.

[0057] Step two: The pretreated raw urine is continuously pumped into the pre-equilibrated adsorption column at a flow rate of 1.5 mL / min. The flow-through liquid is monitored at 280 nm using a UV detector. When the absorbance reaches 80%–90% of the initial absorbance of the sample, adsorption saturation can be considered.

[0058] Step 3: After adsorption is complete, wash the adsorption column with 5 column volumes of pH 7.4 PBS buffer at a flow rate of 2.0 mL / min until the UV absorption signal of the flow-through solution drops to baseline.

[0059] Step 4: Elution with a mild acidic buffer. Elute using 5 column volumes of 0.1 M sodium acetate buffer at pH 5.5 at a flow rate of 1.0 mL / min. Immediately collect all elution peaks.

[0060] Step 5: Rinse the adsorption column in reverse at low speed with 5 column volumes of deionized water to thoroughly remove residual elution buffer and dissolved products. Rinse the adsorption column with 3 column volumes of 50 mM Tris-HCl buffer at pH 8.5. Finally, reequilibrate the adsorption column with 5 column volumes of PBS buffer at pH 7.4 for the next cycle.

[0061] 2. Recyclable silica gel adsorption performance test:

[0062] (1) Calculation of capacity retention rate:

[0063] By loading the silica gel adsorbent after the nth regeneration into a chromatography column, allowing a urokinase solution of known concentration to flow through it, measuring the urokinase concentration in the flow solution, and determining the initial dynamic adsorption capacity during the first use, the adsorption capacity for this cycle is calculated. The capacity retention rate is then calculated using the following formula:

[0064] Capacity retention rate η= ×100%;

[0065] in, The dynamic adsorption capacity (IU / g) of the regenerable urokinase silica adsorbent for urokinase after the nth adsorption-desorption cycle is given. is the initial dynamic adsorption capacity (IU / g) of silica gel adsorbent when used for the first time, and n is the number of adsorption-desorption cycles.

[0066] ;

[0067] in, The dynamic adsorption capacity (IU / g) of the regenerable urokinase silica adsorbent for urokinase after the nth adsorption-desorption cycle is given. The initial concentration of the sample loading solution (mg / g) The concentration of the flow-through solution (mg / g) This refers to the volume of the sample solution (ml). This represents the mass of the adsorbent (mg).

[0068] (2) Calculation of urokinase activity recovery rate after elution:

[0069] The total activity of biologically active urokinase in the eluent was determined by either the chromogenic substrate method (using specific substrates such as Chromozym U) or the fibrin plate lysis zone method. The urokinase activity recovery rate after elution was calculated by calculating the difference in urokinase activity in the solution before and after loading.

[0070] The calculation formula is:

[0071] ;

[0072] in, The total activity (IU) of urokinase eluted from the silica gel adsorbent. The total activity (IU) of urokinase adsorbed by the silica gel adsorbent.

[0073] ;

[0074] This represents the total activity of urokinase in the sample solution. The total activity of urokinase in the flow-through fluid. The total activity of urokinase in the washing solution.

[0075] The various indicators of Examples 1-7 were measured using the above formula and are shown in Table 2.

[0076] Table 2: Values ​​of various indicators in Examples 1-7:

[0077]

[0078] The steps in the method of this invention can be adjusted, combined, or deleted according to actual needs. In this invention, the descriptions of each embodiment have their own emphasis; parts not detailed or described in a certain embodiment can be referred to in the relevant descriptions of other embodiments. The technical features of this invention can be combined arbitrarily. To keep the description concise, not all possible combinations of the technical features in the embodiments are described; however, as long as these combinations of technical features do not contradict each other, they should be considered within the scope of this invention.

[0079] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A method for preparing a regenerable urokinase silica gel adsorbent, characterized in that, Includes the following steps: Step 1: Dissolve and disperse 5-10 parts by weight of hexadecyltrimethylammonium bromide and 10-20 parts by weight of polystyrene microspheres together in a mixture of 300-400 parts by weight of anhydrous ethanol and deionized water, and stir to form a uniform composite template emulsion. The particle size of the polystyrene microspheres is 300-400 nm. Step 2: Tetraethyl orthosilicate is added dropwise to the composite template emulsion and stirred to obtain a sol-gel system; Step 3: Ammonia water is added to the sol-gel system to gel. When the gel point is near, an aqueous solution of calcium chloride and urea is added to the sol-gel system and stirred to obtain a wet gel. The wet gel is transferred to a closed reaction vessel and statically aged to obtain a gel. The gel is washed with a mixture of anhydrous ethanol and deionized water and dried to obtain the silica gel precursor. Step four: Place the silica precursor obtained in step three into a temperature-controlled muffle furnace for the first heating, raising the temperature from room temperature to 220-280°C at a rate of 1-2°C / min, and holding it at this temperature for 2-3 hours to remove physically adsorbed water and some residual solvent; then perform a second heating, raising the temperature to 430-470°C at a rate of 2-3°C / min, and holding it at this temperature for 4-5 hours. After the temperature of the temperature-controlled muffle furnace cools to room temperature, remove the material to obtain the regenerable urokinase silica adsorbent.

2. The method for preparing the regenerable urokinase silica gel adsorbent as described in claim 1, characterized in that, The specific steps for stirring in step one are as follows: stirring at 400-800 rpm for 1.5-2.5 hours in a water bath at 40-60℃.

3. The method for preparing the regenerable urokinase silica gel adsorbent as described in claim 2, characterized in that, In step one, the volume ratio of anhydrous ethanol to deionized water in the mixture of anhydrous ethanol and deionized water is 1-2:0.5-1.

4. The method for preparing the regenerable urokinase silica gel adsorbent as described in claim 3, characterized in that, The specific steps of step two are as follows: 100-200 parts by mass of tetraethyl orthosilicate are slowly added dropwise to the composite template emulsion obtained in step one at a rate of 1-2 mL / min, and hydrolyzed by stirring at 400-800 rpm for 3-4 hours to obtain a sol-gel system.

5. The method for preparing the regenerable urokinase silica gel adsorbent as described in claim 4, characterized in that, The specific steps of step three are as follows: 50-100 parts by weight of ammonia water are added to the sol-gel system in step two to prepare for gelation. After 25-35 minutes, when the gelation point is near, 20-25 parts by weight of an aqueous solution of calcium chloride and urea are quickly added to the sol-gel system. The mixture is stirred at 200-400 rpm for 1-1.5 hours to obtain a wet gel. The wet gel is transferred to a sealed reactor and statically aged at 60-80°C for 20-24 hours to obtain a gel. After static aging, the gel is washed three times with a mixture of 100-120 parts by weight of anhydrous ethanol and deionized water, and then dried at 50-70°C for 10-12 hours to obtain the silica gel precursor.

6. The method for preparing the regenerable urokinase silica gel adsorbent as described in claim 5, characterized in that, In step three, the mass ratio of calcium chloride, urea and deionized water in the aqueous solution of calcium chloride and urea is 1-1.5:2-3:15-20, and the volume ratio of anhydrous ethanol and deionized water in the mixture is 6-7:2-3.

7. The regenerable urokinase silica adsorbent prepared by the preparation method according to any one of claims 1-6.

8. The application of the regenerable urokinase silica adsorbent as described in claim 7 in the extraction of urokinase.

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