Functionalized thermoresponsive surfactants for selective removal of hrp from crude froth flotation effluent and methods of making and using the same

Abstract

This invention belongs to the field of functionalized materials technology, and discloses a functionalized thermosensitive surfactant for selective foam flotation of HRP in crude extract, its preparation method, and its application. This invention uses hydrophilic thermosensitive monomer acrylamide (AAm), hydrophobic monomer acrylonitrile (AN), and epoxy group-containing monomer glycidyl methacrylate (GMA) as polymerizing monomers, and performs RAFT polymerization to obtain a thermosensitive surfactant P(AAm-co-AN-co-GMA). Surface tension is controlled by adjusting the length of the hydrophobic segments and temperature. Its performance and the effect of switchable foam stability control are tested. Based on the excellent foam-stabilizing properties of the thermosensitive surfactant and the highly specific binding ability of the aptamer to the target enzyme, the aptamer is modified onto the thermosensitive surfactant to prepare the selectively functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt. Using HRP in the crude extract obtained from fresh horseradish as the research target, the high affinity binding of P(AAm-co-AN-co-GMA)-Apt to HRP and selective foam flotation are achieved.

Description

Technical Field

[0001] This invention belongs to the field of functionalized materials technology, specifically relating to a functionalized thermosensitive surfactant for horseradish peroxidase (HRP) in selective foam flotation crude extract, its preparation method, and its application. Background Technology

[0002] Enzyme biocatalysts, characterized by high catalytic efficiency, strong substrate specificity, and mild reaction conditions, are widely used in food, pharmaceuticals, and energy. Enzymes can be obtained from extracts of animals, plants, and microorganisms. Compared to traditional enzyme separation techniques such as salting out, ultrafiltration, chromatography, and electrophoresis, foam flotation offers advantages such as high separation efficiency, simple equipment, ease of operation, low cost, and minimal environmental pollution. Furthermore, foam separation is particularly suitable for separating target enzymes in complex systems with low concentrations. It utilizes air bubbles as carriers, forming a foam layer by injecting bubbles into the solution, and then separating the foam layer from the bulk liquid phase. During flotation, the air bubbles provide a large surface area, allowing solutes to adsorb at the gas-liquid interface. If a solute can selectively adsorb at the gas-liquid interface, its concentration in the foam layer will be much higher than its concentration in the bulk liquid phase. Therefore, by controlling the adsorption characteristics of the solute at the gas-liquid interface, selective separation of the solute can be achieved. However, due to the low concentration and complex composition of the target enzyme in the enzyme extract, foam flotation technology suffers from insufficient selectivity and low efficiency in the separation of target enzymes.

[0003] Aptamers are short oligonucleotide sequences or peptides obtained through in vitro screening. They possess high affinity and specificity, enabling them to bind efficiently and rapidly to specific targets. Compared to traditional antibodies, aptamers offer advantages such as small molecular weight, structural stability, and ease of synthesis and modification. Once an aptamer acts on a target enzyme, it adaptively folds into a conformation complementary to the target enzyme's shape, thus preserving the enzyme's native conformation and high enzymatic activity. Multiple interactions between the aptamer and target enzyme ensure highly specific binding.

[0004] Based on the different aggregation states of temperature-sensitive surfactants at the gas-liquid interface at different temperatures, switchable controllable foam stability is achieved, resulting in high foam stability at high temperatures and low foam stability at low temperatures. An aptamer with specific binding ability is introduced onto the temperature-sensitive surfactant for selective foam flotation of HRP in complex crude extracts. Leveraging the multiple interactions between the aptamer and HRP and the advantages of foam flotation technology, the problems of single driving force and poor selectivity for target enzyme separation in low-concentration, complex crude extract systems are solved. Summary of the Invention

[0005] This invention uses hydrophilic thermosensitive monomers acrylamide (AAm), hydrophobic monomers acrylonitrile (AN), and glycidyl methacrylate (GMA), a monomer with epoxy groups, as polymerizable monomers to perform RAFT polymerization to obtain a thermosensitive surfactant P(AAm-co-AN-co-GMA). Surface tension is controlled by adjusting the length of the hydrophobic segments and temperature. Its performance and the effect of switchable control over foam stability are tested. Based on the excellent foam-stabilizing properties of the thermosensitive surfactant and the highly specific binding ability of the aptamer to the target enzyme, the aptamer is modified onto the thermosensitive surfactant to prepare a selectively functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt. Using HRP in the crude extract obtained from fresh horseradish as the research target, high affinity binding of P(AAm-co-AN-co-GMA)-Apt to HRP and selective foam flotation are achieved.

[0006] A method for preparing selectively functionalized thermosensitive surfactants includes the following steps:

[0007] (1) Preparation of RAFT chain transfer agent (BTPA)

[0008] 3-Mercaptopropionic acid and carbon disulfide were slowly added dropwise to an aqueous solution of potassium hydroxide under stirring. The resulting mixture was reacted in an oil bath. Then, benzyl bromide was slowly added dropwise, and the oil bath was heated under reflux overnight. After the reaction was completed, the mixture was cooled to room temperature, chloroform was added, and hydrochloric acid was added dropwise until the solution separated into phases. The upper aqueous phase was removed, and the lower organic phase was washed repeatedly with excess deionized water until the upper aqueous phase was clear. After the organic phase was concentrated under reduced pressure, an appropriate amount of dichloroform was added, and the mixture was placed in a refrigerator for recrystallization. The precipitated yellow crude product was filtered and washed with water. Next, the crude product was dissolved in chloroform, concentrated under reduced pressure again, recrystallized with dichloroform, and frozen to precipitate. The product was washed with excess deionized water. The purification operation needs to be repeated 2 to 3 times. Finally, the obtained product was placed in a vacuum drying oven and dried at room temperature to obtain a yellow solid, which is the chain transfer agent BTPA.

[0009] In step (1), the ratio of potassium hydroxide aqueous solution, 3-mercaptopropionic acid, carbon disulfide and benzyl bromide is 35-40 mL: 32.4 mmol: 4-6 mL: 36.2 mmol, wherein the concentration of potassium hydroxide aqueous solution is 2.07 M;

[0010] In step (1), the reaction temperature in the oil bath is 20-30℃ and the reaction time is 4-6 hours;

[0011] In step (1), the temperature of the heating reflux is 85-95℃ and the time is 12-14h.

[0012] (2) Preparation of thermosensitive surfactant P(AAm-co-AN-co-GMA)

[0013] The chain transfer agent BTPA prepared in step (1), glycidyl methacrylate (GMA), acrylamide (AAm), acrylonitrile (AN) and azobisisobutyronitrile (AIBN) in different proportions were dissolved in N,N'-dimethylformamide (DMF), and the mixed solution was transferred to a round-bottom flask. The mixed solution was purged with nitrogen and bubbled to remove oxygen. The reaction was sealed under a nitrogen atmosphere. After the reaction was completed, the round-bottom flask was placed in an ice-water bath to quench the reaction. The resulting mixture was dropped into methanol to precipitate, and centrifuged to obtain a solid product. The solid product was dried under vacuum and dissolved in deionized water. Unreacted monomers and small molecules were further removed by dialysis. The collected solution was freeze-dried to obtain surfactant P (AAm-co-AN-co-GMA).

[0014] In step (2), the ratio of BTPA, glycidyl methacrylate, acrylamide, acrylonitrile, azobisisobutyronitrile and N,N'-dimethylformamide is 1.2 mmol: 2 mmol: 30 mmol: 7-9 mmol: 121 μmol: 20-25 mL;

[0015] In step (2), the nitrogen purging time is 30-60 min; the temperature of the sealed reaction is 70-80℃, and the sealing reaction time is 3-5 h; the ice water quenching time is 10-20 min; the volume of methanol used for the precipitate is 100-150 mL; the dialysis bag used in this invention has a molecular weight cutoff of 10000D, and the dialysis time is 48-60 h.

[0016] (3) aptamer functionalization of thermosensitive surfactant P(AAm-co-AN-co-GMA)

[0017] The thermosensitive surfactant P(AAm-co-AN-co-GMA) solution obtained in step (2) was mixed evenly with the horseradish peroxidase aptamer (Apt) solution. After the mixture was reacted at a constant temperature, glycine was added to the reaction solution to continue the reaction. After the reaction was completed, the crude product was obtained by low-temperature centrifugation. The crude product was dissolved in water again, and the dissolution-precipitation cycle was repeated 2 to 4 times to remove residual glycine and Apt. The product obtained was the selectively functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt.

[0018] In step (3), the ratio of P(AAm-co-AN-co-GMA) solution, horseradish peroxidase aptamer solution, and glycine is 400 μL: 200 μL: 1.2 mg; wherein the concentration of P(AAm-co-AN-co-GMA) solution is 1 mg / mL, the concentration of horseradish peroxidase aptamer solution is 10 μM, and the solvent is deionized water;

[0019] In step (3), the constant temperature reaction is 30-40℃ and the time is 22-24h; glycine is added and the reaction continues for 5-8h.

[0020] In step (3), the aptamer sequence of horseradish peroxidase is: 5'-NH2-GTC CGC AAG TTG TCG CGCGAT AAG CTT ATG GCT GGT TGA-3'.

[0021] Based on the thermosensitive surfactant P(AAm-co-AN-co-GMA), this invention introduces a highly selective horseradish peroxidase aptamer to successfully prepare a selectively functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt material, which is then used for selective foam flotation of HRP in crude extract.

[0022] This invention leverages the excellent foam-stabilizing properties of thermosensitive surfactants and the highly specific binding ability of aptamers to target enzymes. By modifying thermosensitive surfactants with aptamers, a selectively functionalized thermosensitive surfactant, P(AAm-co-AN-co-GMA)-Apt, is prepared. Using HRP in the crude extract as the target, this invention achieves high affinity binding of P(AAm-co-AN-co-GMA)-Apt to HRP and selective foam flotation.

[0023] The beneficial effects of this invention are as follows:

[0024] Based on the highly specific binding ability of aptamers to target enzymes, a temperature-sensitive surfactant with selective recognition function for horseradish peroxidase (HRP) was constructed to achieve selective adsorption of HRP at the gas-liquid interface. The surface activity of the temperature-sensitive surfactant can be controlled by changing the temperature, thereby achieving selective foam flotation, efficient separation and purification, and rapid recovery of HRP in crude HRP extract. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the synthesis process of the thermosensitive surfactant P(AAm-co-AN-co-GMA).

[0026] Figure 2(a) FT-IR spectra of thermosensitive surfactants P1, P2, and P3; (b) 1 H NMR spectrum and (c) GPC test pattern.

[0027] Figure 3 (a) Changes in transmittance of solutions P1, P2, and P3 at 30 mg / mL; (b) T values ​​of solutions P1, P2, and P3 at different concentrations. cp .

[0028] Figure 4 (a) Graphs showing the surface tension of solutions of thermosensitive surfactants P1, P2 and P3 as a function of concentration, and (b) Graphs showing the contact angles of thermosensitive surfactants P1, P2 and P3.

[0029] Figure 5 This is a schematic diagram of the synthesis of P(AAM-co-AN-co-GMA)-Apt.

[0030] Figure 6 The FT-IR spectra of (a) Apt, P(AAm-co-AN-co-GMA) and P(AAm-co-AN-co-GMA)-Apt are shown, and the thermogravimetric analysis plots of P(AAm-co-AN-co-GMA) and P(AAm-co-AN-co-GMA)-Apt are shown.

[0031] Figure 7 The reaction heat release and (b) molar enthalpy change of (a) HRP solution titration of P(AAm-co-AN-co-GMA)-Apt solution, and (c) the specific binding of P(AAm-co-AN-co-GMA)-Apt to HRP and the nonspecific binding of P(AAm-co-AN-co-GMA)-Apt to trypsin, bromelain, lipase, soy protein and bovine serum albumin.

[0032] Figure 8 (a) Surface tension curves of P(AAm-co-AN-co-GMA) solution and P(AAm-co-AN-co-GMA)-Apt solution as a function of concentration; (b) t-co-AN-co-GMA)-Apt solution of different concentrations. 1 / 2 And FS.

[0033] Figure 9(a) Infrared spectra of free HRP and P(AAm-co-AN-co-GMA)-Apt; (b) Thermogravimetric analysis of P(AAm-co-AN-co-GMA)-Apt and P(AAm-co-AN-co-GMA)-Apt-HRP complex; (c) Gel electrophoresis analysis of HRP in crude extract. M: Standard protein molecular weight; Lane 1: Commercial HRP; Lane 2: Crude HRP extract; Lane 3: Defoaming solution; Lane 4: Defoaming solution for further low-temperature sedimentation; (d) CD spectra of free HRP and HRP obtained by foam flotation.

[0034] Figure 10 The effects of different P(AAm-co-AN-co-GMA)-Apt concentrations (a), HRP concentrations (b), gas flow rates (d), temperatures (e), and foam separation times (f) on the HRP foam flotation performance are shown in (c) Langmuir and Freundlich models.

[0035] Figure 11 (a) Schematic diagram of further processing of foam liquid based on the thermal response performance of P(AAm-co-AN-co-GMA)-Apt, (b) Purification factor under different experimental conditions.

[0036] Figure 12 The reusability of HRP in the crude extract of selective foam flotation of P(AAm-co-AN-co-GMA)-Apt is (a) and the recovery rate of reusable P(AAm-co-AN-co-GMA)-Apt is (b). Detailed Implementation

[0037] The present invention will be further illustrated below with specific examples and the technical solutions of the present invention will be described in detail with reference to the accompanying drawings. Obviously, the examples given are not all embodiments of the present invention. Any obvious improvements, substitutions or changes that can be made by those skilled in the art without departing from the essence of the present invention are within the protection scope of the present invention.

[0038] Example 1:

[0039] (1) Preparation of RAFT chain transfer agent (BTPA), the process is as follows: Figure 1 As shown in (a)

[0040] 3-Mercaptopropionic acid (2.8 mL, 32.4 mmol) and carbon disulfide (CS2, 5 mL) were slowly added dropwise to a 2.07 M, 36 mL aqueous solution of potassium hydroxide under stirring. The resulting mixture was reacted in an oil bath at 25 °C for 6 h. Then, benzyl bromide (6.2 g, 36.2 mmol) was slowly added dropwise, and the oil bath was heated to 90 °C and refluxed overnight for 12 h. After the reaction was complete, the mixture was cooled to room temperature and transferred to a 500 mL beaker. 80 mL of chloroform was added, and hydrochloric acid was added dropwise until the solution separated into phases. The upper aqueous phase was removed. The lower organic phase was then washed repeatedly with excess deionized water until the upper aqueous phase became clear. After the organic phase was concentrated under reduced pressure, an appropriate amount of dichloroform was added dropwise, and the mixture was placed in a refrigerator for recrystallization. The precipitated yellow crude product was filtered and washed with water. Next, the crude product was dissolved in chloroform, concentrated again under reduced pressure, recrystallized from dichloromethane, and frozen to precipitate. The product was then washed with excess deionized water. This purification process needed to be repeated 2-3 times. Finally, the obtained product was placed in a vacuum drying oven and dried at room temperature for 48 hours to obtain a yellow solid, which was the chain transfer agent BTPA.

[0041] (2) The preparation process of the thermosensitive surfactant P(AAm-co-AN-co-GMA) is as follows: Figure 1 As shown in (b).

[0042] Chain transfer agent BTPA (0.0318 g, 1.2 mmol), glycidyl methacrylate (GMA, 0.284 g, 2 mmol), acrylamide (AAm, 2.14 g, 30 mmol), and different proportions of acrylonitrile (AN, 0.371 g, 7 mmol; 0.425 g, 8 mmol; 0.478 g, 9 mmol) and azobisisobutyronitrile (AIBN, 20 mg, 121 μmol) were dissolved in N,N-dimethylformamide (DMF, 22 mL), and the mixture was transferred to a round-bottom flask. The mixture was purged with nitrogen for 30 min to remove oxygen. The round-bottom flask was then placed in an oil bath at 70 °C, and the mixture was stirred and sealed under a nitrogen atmosphere for 3 h. After the reaction was complete, the round-bottom flask was quenched in an ice-water bath for 10 min. The resulting mixture was added dropwise to methanol (100 mL) to precipitate, and the solid product was obtained by centrifugation. The solid product was dried under vacuum and dissolved in deionized water. After further dialyzing with a dialysis bag with a molecular weight cutoff of 10,000 D for 48 hours, the solution was collected and freeze-dried to obtain surfactant P (AAm-co-AN-co-GMA).

[0043] In step (2), the amount of AAm and GMA monomers is fixed, and the amount of AN is adjusted to 7, 8 and 9 mmol to prepare three temperature-sensitive surfactants P (AAm-co-AN-co-GMA) with different AN hydrophobic segment lengths, which are denoted as P1, P2 and P3 respectively.

[0044] like Figure 2 As shown in Figure (a) of the FT-IR spectrum, P1, P2, and P3 were found to be at 3350 cm⁻¹. -1 and 3199cm -1 Absorption peaks of the NH stretching vibration of AAm were observed at all locations, with the peak at 1664 cm⁻¹. -1 A C=O stretching vibration absorption band appears at 2242 cm⁻¹. -1 The characteristic absorption band of the C≡N stretching vibration of AN appears, and at 913 cm⁻¹ -1 and 838cm -1 A characteristic absorption band attributable to the stretching vibration of the epoxy groups on GMA was observed. Furthermore, with increasing AN feed amount, the characteristic C≡N absorption peak of AN (2242 cm⁻¹) was observed. -1 The strength gradually increases, proving that the AN hydrophobic segments of the thermosensitive surfactants gradually increase from P1 to P3. Figure 2 (b) shows the 1H NMR spectra of P1, P2, and P3, revealing that the trends of the three spectral lines are basically consistent. Finally, GPC analysis was performed to determine the molecular weights and distributions of P1, P2, and P3. Figure 2 As shown in (c), the GPC elution lines of P1, P2 and P3 are all single-peaked. The molecular weights of P1, P2 and P3 are 11136 Da, 12768 Da and 14132 Da, respectively. The molecular weight distribution (PDI) is relatively narrow, indicating that the molecular weight of the temperature-sensitive surfactant is monodisperse.

[0045] Depend on Figure 3 As shown in (a), the thermosensitive surfactants P1, P2, and P3 exhibit excellent thermosensitivity; the surfactant solution can be clarified by increasing the temperature and become turbid again by decreasing the temperature, thus enabling the recycling of the thermosensitive surfactants. At a concentration of 30 mg / mL, the cloud points of thermosensitive surfactants P1, P2, and P3 are 20.8, 23.6, and 26.2 °C, respectively. This indicates that from P1 to P3, the cloud point gradually increases with the growth of the hydrophobic segment AN. Further measurements were taken of the cloud points of P1, P2, and P3 solutions at different concentrations. Figure 3 As shown in (b), the T of the temperature-sensitive surfactant cp The concentration changes in a U-shaped trend. The UCST of surfactants P1, P2 and P3 are 27.1, 29.6 and 33.2℃, respectively, indicating that the cloud point of temperature-sensitive surfactants can be controlled by adjusting the length of the hydrophobic chain segment.

[0046] Depend on Figure 4As can be seen from (b), from P1 to P3, with the increase of the hydrophobic segment of AN, the contact angle of the temperature-sensitive surfactant increases from 59.58° to 95.19°, and the hydrophobicity gradually increases. Therefore, the ability to reduce surface tension should also increase positively, that is, the value of γmin gradually decreases. Figure 4 (a) The study investigated the relationship between surface tension and concentration of solutions of thermosensitive surfactants P1, P2, and P3 with different AN hydrophobic segment lengths at 30°C. Figure 4 As the concentration increases, the surface tension of solutions P1, P2, and P3 all show a trend of first decreasing and then remaining basically unchanged. The γmin values ​​of aqueous solutions of P1, P2, and P3 are 60.66, 52.46, and 63.62 mN / m, respectively, with solution P2 having a lower surface tension.

[0047] (3) aptamer functionalization of thermosensitive surfactant P(AAm-co-AN-co-GMA), the process is as follows: Figure 5 As shown.

[0048] A solution of the thermosensitive surfactant P(AAm-co-AN-co-GMA) (1 mg / mL, 400 μL) was mixed thoroughly with a horseradish peroxidase aptamer solution (Apt, 10 μM, 200 μL), and the mixture was reacted at 35 °C for 24 h. After the reaction was complete, 1.2 mg of glycine was added to the reaction solution, and the reaction was continued for another 6 h. After the reaction was completed, the crude product was obtained by low-temperature centrifugation. The crude product was then dissolved in water again, and the dissolution-precipitation cycle was repeated 2-4 times to remove residual glycine and Apt. The product obtained was the selectively functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt.

[0049] The FT-IR spectrum of P(AAm-co-AN-co-GMA)-Apt is as follows: Figure 6 As shown in (a), 3412cm -1 and 1691cm -1 The characteristic absorption peaks are attributed to the stretching vibrations of NH and C=O, while the 1638 cm⁻¹ peak... -1 The absorption peak at that point is the absorption peak of the NH deformation vibration. Comparing the FT-IR spectrum of P(AAm-co-AN-co-GMA), it can be seen that when Apt is grafted onto the thermosensitive surfactant P(AAm-co-AN-co-GMA), the FT-IR spectrum of P(AAm-co-AN-co-GMA)-Apt is located at 3412 cm⁻¹. -1 and 1691cm -1 The absorption peaks of the stretching vibrations of the amino group (NH) and the carbonyl group (C=O) are enhanced, and the peak at 1638 cm⁻¹ is also enhanced. -1The absorption peak of the NH deformation vibration on the amide bond was also correspondingly enhanced, indicating that when Apt was modified to P(AAm-co-AN-co-GMA)-Apt, the intensity of the overlapping absorption peaks was further strengthened. Based on the above results, it can be concluded that Apt was successfully incorporated into the thermosensitive surface active material, resulting in the P(AAm-co-AN-co-GMA)-Apt material.

[0050] The composition of P(AAm-co-AN-co-GMA) and P(AAm-co-AN-co-GMA)-Apt materials was further characterized by thermogravimetric analysis. Figure 6 As shown in (b), in the first stage (25-220℃), the thermal decomposition of both materials is mainly due to water loss, which is caused by the evaporation of moisture in the low-temperature range. Then, in the second stage (220-480℃), the materials begin to decompose further, with P(AAm-co-AN-co-GMA) and P(AAm-co-AN-co-GMA)-Apt experiencing weight losses of 57.87% and 60.95%, respectively, in this stage. By comparing the percentage weight loss of the two materials, it can be seen that the weight loss of Apt is approximately 3.08%, further confirming the successful preparation of the P(AAm-co-AN-co-GMA)-Apt material.

[0051] Example 2:

[0052] The affinity and selectivity of selectively functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt for HRP.

[0053] The binding chemostometry between P(AAm-co-AN-co-GMA)-Apt and HRP was quantitatively studied using ITC to verify the affinity of the aptamer on P(AAm-co-AN-co-GMA)-Apt for HRP. Figure 7 As shown in (a) and (b), the titration of P(AAm-co-AN-co-GMA)-Apt solution with HRP solution exhibits a significant heat change, demonstrating a strong binding interaction between the two. The test data were fitted using an Independent model to obtain parameters such as the dissociation constant Kd, stoichiometry n, enthalpy change ΔH, and entropy change ΔS. The large negative enthalpy change (ΔH = -22.66 kJ / mol) and positive entropy change (ΔS = 34.66 J / mol·K) indicate a conformational change in the aptamer on P(AAm-co-AN-co-GMA)-Apt, selectively binding HRP through hydrophobic interactions and hydrogen bonds. The Gibbs free energy change (ΔG) and dissociation constant (Kd) are -32.99 kJ / mol and 1.66 × 10⁻⁶ kJ / mol, respectively. -6Based on the above results, a possible binding mechanism is proposed. First, when the affinity aptamer on P(AAm-co-AN-co-GMA)-Apt comes into contact with HRP, it adaptively folds into a specific conformation. Second, the aptamer specifically binds to HRP through hydrogen bonding and hydrophobic interactions.

[0054] The specificity of P(AAm-co-AN-co-GMA)-Apt for HRP was further evaluated, and non-specific experiments were conducted using commonly used proteins (trypsin, bromelain, lipase, soy protein, and bovine serum albumin). Aptamers, as a novel type of ligand, possess the ability to specifically bind to target molecules. Compared to traditional physical adsorption and covalent binding, aptamers can achieve more efficient and specific adsorption of target substances. Figure 7 As shown in (c), 96% of HRP binds to P(AAm-co-AN-co-GMA)-Apt to form the P(AAm-co-AN-co-GMA)-Apt-HRP complex, while the binding rates of the other five proteins to P(AAm-co-AN-co-GMA)-Apt are all less than 10%. Therefore, P(AAm-co-AN-co-GMA)-Apt can specifically bind to HRP.

[0055] Example 3:

[0056] The bubble-stabilizing properties of selectively functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt

[0057] Figure 8 (a) shows the surface tension tests of the thermosensitive surfactant P(AAm-co-AN-co-GMA) and the selectively functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt. After selective functionalization of the thermosensitive surfactant, the γmin value decreased from 52.46 mN / m to 50.22 mN / m, demonstrating that P(AAm-co-AN-co-GMA)-Apt has better surface activity. The inflection point of the γ-lg C curve indicates that the cmc of P(AAm-co-AN-co-GMA)-Apt is 11.6 mg / mL, while that of P(AAm-co-AN-co-GMA) is 18.3 mg / mL. The foam-stabilizing performance of P(AAm-co-AN-co-GMA)-Apt was analyzed by half-life and foam stability index. Figure 8 From (b), we know that the t of P(AAm-co-AN-co-GMA)-Apt 1 / 2The functionalization, foaming properties, and applications of the temperature-sensitive surfactant P(AAm-co-AN-co-GMA) were compared with those of P(AAm-co-AN-co-GMA) at 75 min and 56.02% respectively. 1 / 2 (68 min) and FS (52.63%), it was found that the selective functionalization of temperature-sensitive surfactants enhances foam stability, namely P(AAm-co-AN-co-GMA)-Apt has excellent foam stability.

[0058] Example 4:

[0059] Selective functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt foam flotation of HRP in fresh horseradish

[0060] (1) Preparation of crude extract of horseradish peroxidase (HRP) from fresh horseradish

[0061] Rinse fresh horseradish thoroughly with deionized water, chop it, and weigh 100g of fresh horseradish. Add it to 4°C pre-cooled PBS buffer solution (pH = 7.5, 50mM, 100mL). Homogenize five times using a homogenizer, each time for 1 min, with a 2-min interval. After homogenization, filter the mixture. Incubate the filtrate in a 4°C ice-water bath and sonicate three times, each time for 15 min, with a 10-min interval. After sonication, centrifuge the extract at 4°C for 10 min to remove the precipitate. Centrifuge the supernatant again at a controlled temperature for 10 min. The final solution is the crude HRP extract.

[0062] (2) P(AAM-co-AN-co-GMA)-Apt selective foam flotation of HRP in horseradish crude extract

[0063] 50 mL of P(AAm-co-AN-co-GMA)-Apt and 50 mL of HRP crude extract of different concentrations were mixed thoroughly. The mixture was then incubated in a 30°C water bath for 30 min, and transferred to a glass flotation column at a constant temperature of 30°C. Nitrogen gas was introduced into the glass column through a gas distributor at a controlled flow rate. As a result, the P(AAm-co-AN-co-GMA)-Apt-HRP complex was adsorbed at the gas-liquid interface and, as the bubbles rose, further entered the foam phase from the aqueous phase. Bubbles carrying the P(AAm-co-AN-co-GMA)-Apt-HRP complex flowed out from the top of the glass column and were stored in a foam collector.

[0064] (3) Evaluation of the specificity and affinity of P(AAM-co-AN-co-GMA)-Apt for HRP

[0065] To demonstrate that P(AAm-co-AN-co-GMA)-Apt successfully and selectively adsorbed HRP from the crude extract, a P(AAm-co-AN-co-GMA)-Apt-HRP complex was obtained. The composition of the obtained complex was characterized using infrared spectroscopy and thermogravimetric analysis. First, based on... Figure 9 As shown in (a) FT-IR plot, the FT-IR spectrum of free HRP at 1530 cm⁻¹... -1 A characteristic absorption peak appeared at 1530 cm⁻¹, corresponding to the combined absorption peak of the bending vibration of the NH bond and the stretching vibration of the CN bond in the peptide backbone of HRP. The FT-IR spectra of P(AAm-co-AN-co-GMA)-Apt and the P(AAm-co-AN-co-GMA)-Apt-HRP complex were almost identical, the only difference being that the FT-IR spectrum of the complex also showed a characteristic absorption peak at 1530 cm⁻¹, which corresponds to the combined absorption peak of the bending vibration of the NH bond and the stretching vibration of the CN bond in the peptide backbone of HRP. This phenomenon indicates that HRP is selectively adsorbed at the gas-liquid interface, thus forming the P(AAm-co-AN-co-GMA)-Apt-HRP complex material.

[0066] Secondly, thermogravimetric analysis was further performed on the P(AAm-co-AN-co-GMA)-Apt-HRP complex, such as... Figure 9 As shown in (b), the thermal decomposition in the first stage (25-220℃) is mainly due to the loss of water from the material. In the second stage (220-450℃), the weight loss of P(AAm-co-AN-co-GMA)-Apt is 61.97%, while the weight loss of the P(AAm-co-AN-co-GMA)-Apt-HRP composite is 64.56%. Comparing the percentage weight loss of the two thermal decompositions, the weight loss percentage of HRP is approximately 2.59%, which further proves that HRP is selectively bound to the P(AAm-co-AN-co-GMA)-Apt material through foam flotation.

[0067] The effect of HRP in the foam flotation crude extract was analyzed using gel electrophoresis SDS-PAGE, such as... Figure 9As shown in (c), the crude HRP extract contained many contaminating proteins (lane 2). After specific binding of HRP in the crude extract by P(AAm-co-AN-co-GMA)-Apt followed by foam flotation, the number of contaminating proteins in the defoaming solution decreased (lane 3). Further purification using the thermal responsiveness of P(AAm-co-AN-co-GMA)-Apt resulted in only an HRP band of approximately 44.3 kDa on the protein lane (lane 4), consistent with the bands of commercially available HRP (lane 1). This indicates that P(AAm-co-AN-co-GMA)-Apt selectively bound HRP in the crude extract through foam separation.

[0068] Furthermore, circular dichroism (CD) spectra of free HRP and HRP obtained from selective foam flotation of P(AAm-co-AN-co-GMA)-Apt were recorded from 190 nm to 240 nm to demonstrate that the conformation of HRP remained essentially unchanged during adsorption at the gas-liquid interface and cooling sedimentation. Figure 9 As shown in (d), as a common feature of HRP, the negative peaks at 208 nm and 222 nm correspond to the α-helical structure, while the positive peak at approximately 196 nm corresponds to the β-sheet structure. By analyzing the spectral structure of HRP, it was found that HRP obtained from foam flotation also exhibited a similar spectral structure, and the trends of the two spectral lines were basically the same. This indicates that the adaptive folding of the aptamer on P(AAm-co-AN-co-GMA)-Apt forms a shape complementary to HRP, which can better maintain the original conformation of HRP during gas-liquid interface adsorption and cooling sedimentation.

[0069] Example 5:

[0070] Optimization of experimental conditions for selective foam flotation of HRP in horseradish crude extract using P(AAM-co-AN-co-GMA)-Apt

[0071] The effects of selectively functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt on HRP in crude extract by foam flotation under different experimental conditions were comprehensively explored from four aspects: protein enrichment ratio (Ep), protein recovery rate (Rp), enzyme activity enrichment ratio (Ee), and enzyme activity recovery rate (Re), so as to obtain the optimal experimental conditions.

[0072] The effect of different P(AAm-co-AN-co-GMA)-Apt concentrations on selective adsorption performance is as follows: Figure 10As shown in (a), when the concentration of P(AAm-co-AN-co-GMA)-Apt is 2-8 mg / mL, the recoveries Rp and Re gradually increase to 87.07% and 93.05%, respectively, while the enrichment ratios Ep and Ee decrease slightly to 1.41 and 1.78. When the concentration is greater than 8 mg / mL, the recoveries Rp and Re begin to decrease, while the enrichment ratios Ep and Ee do not change much.

[0073] The effect of different initial HRP concentrations in the crude extract on foam flotation is as follows: Figure 10 As shown in (b), within the initial HRP concentration range of 0.2-0.8 mg / mL, the recoveries Rp and Re significantly increased with increasing HRP concentration in the crude extract, while the enrichment ratios Ep and Ee did not change significantly. When the HRP concentration was 0.8 mg / mL, the recoveries Rp and Re reached their maximum values ​​of 87.18% and 96.29%, respectively, and the enrichment ratios Ep and Ee were 1.65 and 1.85, respectively.

[0074] Further analysis of the adsorption behavior of the P(AAm-co-AN-co-GMA)-Apt-HRP complex at the gas-liquid interface was conducted using Langmuir and Freundlich adsorption isotherm models. 2 =0.9384) compared to the Freundlich model (R 2 =0.8905) is more suitable for describing the adsorption behavior of the P(AAm-co-AN-co-GMA)-Apt-HRP complex at the gas-liquid interface. Figure 10 P(AAm-co-AN-co-GMA)-Apt-HRP is adsorbed as a monolayer at the gas-liquid interface.

[0075] The effect of different gas flow rates on the HRP foam flotation effect is as follows: Figure 10 As shown in (d), within the gas flow rate range of 50-100 mL / min, the recoveries Rp and Re significantly increased with increasing N2 flow rate, while the enrichment ratios Ep and Ee gradually decreased with increasing flow rate. At a gas flow rate of 100 mL / min, Rp and Re increased to 87.12% and 93.52%, respectively, while Ep and Ee decreased to 1.64 and 1.76. When the gas flow rate was greater than 100 mL / min, the rate of increase in recoveries slowed down, while the enrichment ratios still decreased.

[0076] The effect of different temperatures on the selective adsorption of HRP is as follows: Figure 10As shown in (e), as the temperature increases from 25°C to 30°C, the enrichment ratios Rp and Re increase to 87.15% and 94.52%, respectively, while Ep and Ee increase to 1.68 and 1.78, respectively. Further increases in temperature have little effect on the recovery rates (Rp, Re) and enrichment ratios (Ep, Ee).

[0077] The effect of different adsorption times on HRP foam flotation effect is as follows: Figure 10 As shown in (f), within the range of 5–20 min, the recoveries Rp and Re significantly increased with increasing flotation time, while the enrichment ratios Ep and Ee changed less. At 20 min, the recoveries Rp and Re reached their maximum values ​​of 90.20% and 98.70%, respectively, and the enrichment ratios Ep and Ee were 1.80 and 2.15, respectively.

[0078] Based on the analysis of the HRP flotation effect under different experimental conditions, the optimal flotation experimental conditions were obtained: the concentration of P(AAm-co-AN-co-GMA)-Apt was 8 mg / mL, the initial concentration of HRP was 0.8 mg / mL, the gas flow rate was 100 mL / min, the flotation temperature was 30℃, and the flotation time was 20 min. Under these optimal experimental conditions, the recoveries Rp and Re were 90.20% and 98.70%, respectively, and the enrichment ratios Ep and Ee were 1.80 and 2.15, respectively.

[0079] Example 6:

[0080] Further purification of selective foam flotation HRP foam liquid based on the thermal response properties of P(AAm-co-AN-co-GMA)-Apt

[0081] Temperature-sensitive surfactants exhibit better bubble stability above the Tcp, while bubble stability is poor below the Tcp, and low temperatures accelerate bubble collapse. Therefore, based on the thermal response properties of P(AAm-co-AN-co-GMA)-Apt, the foam liquid obtained after HRP foam flotation can be further processed. First, the temperature is lowered to 10℃ to desorb the P(AAm-co-AN-co-GMA)-Apt-HRP complex adsorbed at the gas-liquid interface from the gas-liquid interface, and then low-temperature centrifugation is performed to allow the P(AAm-co-AN-co-GMA)-Apt-HRP complex to fully aggregate and settle. Figure 11 (a)). After removing the supernatant, the temperature was increased to allow the aggregated complex to dissolve again. Further treatment using the thermal response properties of P(AAm-co-AN-co-GMA)-Apt increased the purification factor of HRP in the resulting solution to 9.75 (a). Figure 11 (b)

[0082] Example 7:

[0083] Cyclic performance of P(AAm-co-AN-co-GMA)-Apt

[0084] Previous studies have shown that the interactions between proteins and aptamers mainly involve hydrogen bonds and van der Waals forces, which can be disrupted by acidic solutions and washing with high ionic strength. In this study, HRP was released from the P(AAm-co-AN-co-GMA)-Apt-HRP complex by adding 0.1 M HAc solution. Further cooling, centrifugation, and drying yielded reusable P(AAm-co-AN-co-GMA)-Apt. This P(AAm-co-AN-co-GMA)-Apt was then used again for foam flotation of HRP in the crude extract. After six cycles, P(AAm-co-AN-co-GMA)-Apt still effectively promoted selective foam flotation of HRP, with Ep and Rp values ​​of 1.65% and 83.6%, respectively. Figure 12 (a)). Meanwhile, approximately 81.28% of P(AAm-co-AN-co-GMA)-Apt( Figure 12 (b) Therefore, by recovering P(AAm-co-AN-co-GMA)-Apt, the froth flotation of HRP in crude liquid can be effectively and selectively promoted.

[0085] The embodiments described herein are preferred embodiments of the present invention. All other embodiments obtained by those skilled in the art without making any obvious improvements, substitutions or modifications are within the scope of protection of the present invention.

Claims

1. A process for the preparation of functionalized thermoresponsive surfactants selective for HRP in a roughing stage froth, characterized by, Includes the following steps: (1) Preparation of RAFT chain transfer agent BTPA: 3-Mercaptopropionic acid and carbon disulfide were slowly added dropwise to an aqueous solution of potassium hydroxide under stirring. The resulting mixture was reacted in an oil bath. Benzyl bromide was then slowly added dropwise, and the oil bath was heated under reflux overnight. After the reaction was complete, the mixture was cooled to room temperature, chloroform was added, and hydrochloric acid was added dropwise until the solution separated into phases. The upper aqueous phase was removed, and the lower organic phase was washed repeatedly with excess deionized water until the upper aqueous phase became clear. The organic phase was concentrated under reduced pressure, and an appropriate amount of dichloroform was added. The mixture was then placed in a refrigerator for recrystallization. The precipitated yellow crude product was filtered and washed with water. Next, the crude product was dissolved in chloroform, concentrated again under reduced pressure, recrystallized with dichloroform, and frozen to precipitate. The product was washed with excess deionized water. The purification process needed to be repeated several times. Finally, the obtained product was placed in a vacuum drying oven and dried at room temperature to obtain a yellow solid, which is the chain transfer agent BTPA. (2) Preparation of thermosensitive surfactant P(AAm-co-AN-co-GMA): The chain transfer agent BTPA, glycidyl methacrylate GMA, acrylamide AAM, acrylonitrile AN and azobisisobutyronitrile AIBN prepared in step (1) were dissolved in N,N'-dimethylformamide DMF, and the mixed solution was transferred to a round-bottom flask. The mixed solution was purged with nitrogen and bubbled to remove oxygen. The reaction was sealed under a nitrogen atmosphere. After the reaction was completed, the round-bottom flask was placed in an ice-water bath to quench the reaction. The resulting mixture was dropped into methanol to precipitate, and centrifuged to obtain a solid product. The solid product was dried under vacuum and dissolved in deionized water. Unreacted monomers and small molecules were further removed by dialysis. The collected solution was freeze-dried to obtain surfactant P (AAm-co-AN-co-GMA). (3) aptamer functionalization of thermosensitive surfactant P(AAm-co-AN-co-GMA): The thermosensitive surfactant P(AAm-co-AN-co-GMA) obtained in step (2) was prepared into a solution and mixed evenly with the horseradish peroxidase aptamer Apt solution. After the mixture was reacted at a constant temperature, glycine was added to the reaction solution to continue the reaction. After the reaction was completed, the crude product was obtained by low-temperature centrifugation. The crude product was dissolved in water again, and the dissolution-precipitation cycle was repeated several times to remove the residual glycine and Apt. The product obtained is the selectively functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt.

2. The production method according to claim 1, wherein In step (1), the ratio of potassium hydroxide aqueous solution, 3-mercaptopropionic acid, carbon disulfide and benzyl bromide is 35~40 mL: 32.4 mmol: 4~6 mL: 36.2 mmol, wherein the concentration of potassium hydroxide aqueous solution is 2.07 M.

3. The production method according to claim 1, wherein In step (1), the reaction temperature in the oil bath is 20~30℃ and the time is 4~6 h; the reflux temperature is 85~95℃ and the time is 12~14 h.

4. The preparation method according to claim 1, characterized in that, In step (2), the ratio of BTPA, glycidyl methacrylate, acrylamide, acrylonitrile, azobisisobutyronitrile and N,N'-dimethylformamide is 1.2 mmol: 2 mmol: 30 mmol: 7~9 mmol: 121 μmol: 20~25 mL.

5. The preparation method according to claim 1, characterized in that, In step (2), the nitrogen purging time is 30-60 min; the temperature of the sealed reaction is 70-80℃, and the time of the sealed reaction is 3-5 h; the ice water quenching time is 10-20 min; the volume of methanol used for the precipitate is 100-150 mL; the molecular weight cutoff of the dialysis bag is 10000 D, and the dialysis time is 48-60 h.

6. The preparation method according to claim 1, characterized in that, In step (3), the ratio of P(AAm-co-AN-co-GMA) solution, horseradish peroxidase aptamer solution and glycine is 400 μL: 200 μL: 1.2 mg; The concentration of P(AAm-co-AN-co-GMA) solution was 1 mg / mL, the concentration of horseradish peroxidase aptamer solution was 10 μM, and the solvent was deionized water.

7. The preparation method according to claim 1, characterized in that, In step (3), the constant temperature reaction is 30~40℃ and the time is 22~24 h; glycine is added and the reaction continues for 5~8 h.

8. The preparation method according to claim 1, characterized in that, In step (3), the aptamer sequence of horseradish peroxidase is: 5'-NH2-GTC CGC AAG TTG TCG CGC GAT AAG CTT ATG GCT GGT TGA-3'.

9. The application of the functionalized thermosensitive surfactant P(AAm-co-AN-co-GMA)-Apt of HRP in the selective foam flotation crude extract prepared by the method according to any one of claims 1 to 8, characterized in that, Selective foam flotation for HRP in crude extract.

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