Method for measuring bovine serum albumin by utilizing resonance Rayleigh scattering spectrum of gold and silver nano molecularly imprinted polymer

Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for measuring bovine serum albumin by using a gold and silver nano molecularly imprinted polymer resonance Rayleigh scattering spectrum. The method comprises the following steps: firstly, preparing a BSA standard solution system and a blank control solution system with known concentrations by utilizing Au-AgNP-coated MIP, BSA and the like, measuring the intensity value of a resonance scattering peak at 340nm, calculating the intensity value difference delta I of the resonance scattering peak between a standard solution and the blank control solution, and making a working curve for the concentration relationship of the BSA according to the delta I; then replacing the BSA standard solution with a sample solution to obtain a resonance scattering peak intensity value difference delta I sample between the sample solution and the blank control solution, and finally calculating the BSA content of the sample solution according to the working curve. Compared with the prior art, the method has the advantages that the RRS spectrum method in which Au-AgNP-coated MIP is specifically combined with BSA is adopted, the operation is simple, convenient and rapid, the sensitivity is high, and the accuracy is high.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and in particular relates to a method for measuring bovine serum albumin by utilizing resonance Rayleigh scattering spectra of gold-silver nanomolecular imprinted polymers. Background Art

[0002] BSA is a common abbreviation in biology and biochemistry. It stands for "bovine serum albumin". Bovine serum albumin is a protein found in bovine blood. It belongs to the serum albumin family and has good water solubility and stability. It can maintain its structure and function over a wide range of pH and temperature. The molecular weight of BSA is about 66.5 kDa. It is composed of 583 amino acid residues, including 17 disulfide bonds. These structural features make BSA highly stable and soluble in solution, and also make it widely used in biological research and medical fields. As an important biological reagent, BSA plays an irreplaceable role in biological and biochemical research and is widely used in drug development, clinical diagnosis, biotechnology industry and other fields. The applications of BSA mainly include: (1) Carrier protein: BSA can bind to a variety of substances with poor water solubility, such as fatty acids, hormones, drugs, etc., to help them transport and distribute in the blood. This property makes BSA have potential application value in drug delivery systems. (2) Stabilizer: In biochemical experiments, BSA is often used as a stabilizer to protect biological molecules (such as enzymes, antibodies and other proteins) from the negative effects of experimental conditions (such as changes in pH, ionic strength, temperature, etc.), thereby reducing protein aggregation and denaturation, and improving the accuracy and repeatability of the experiment. (3) Blocking agent: In cell culture, tissue sections and molecular biology experiments, BSA can be used to block non-specific binding sites on the surface of containers or carriers, which helps to reduce background noise and false positive signals in the experiment and improve the accuracy of experimental results. (4) Standard: In quantitative analysis, BSA is often used as a standard for protein concentration. By measuring the absorbance, fluorescence intensity or other physical properties of unknown samples and BSA standards, the concentration of proteins in unknown samples can be determined by comparison. With the continuous development of science and technology, the preparation process and application technology of BSA will continue to improve and improve, providing more reliable and efficient tools for research and application in the field of life sciences. At present, the main methods for detecting BSA at home and abroad include ELISA, Kjeldahl nitrogen determination, biuret method, Folin-phenol reagent method, ultraviolet absorption method, Coomassie brilliant blue method, fluorescence spectrophotometry, resonance light scattering method, electrochemical method and capillary electrophoresis. Among them, ELISA kit has the advantage of high detection specificity, but due to the use of spectrophotometry, it has the disadvantages of easy pollution and high background value, which often reduces the detection sensitivity. Kjeldahl nitrogen determination, biuret method, Folin-phenol reagent method and Coomassie brilliant blue method are all measured by spectrophotometry, which are the same as ultraviolet absorption method, fluorescence spectrophotometry, resonance light scattering method and electrochemical method, and all have the disadvantages of low detection specificity, sensitivity and accuracy.

[0003] Gold-silver nanomolecularly imprinted polymers are composite materials made by introducing gold-silver nanoparticles into a molecularly imprinted polymer matrix. Molecular imprinting technology is used to copolymerize template molecules (i.e., target analytes) with functional monomers and crosslinkers under specific conditions to form polymers with specific recognition sites. After removing the template molecules, cavities that match the shape, size, and chemical functional groups of the template molecules are left in the polymer. These cavities can specifically rebind with the template molecules. Gold-silver nanoparticles can significantly improve the detection sensitivity due to their unique surface plasmon resonance (SPR) effect. At present, a variety of methods for preparing gold-silver nanomolecularly imprinted polymers have been developed, mainly including the following: (1) In-situ reduction method: in-situ reduction of gold-silver salts in the molecularly imprinted polymer matrix to generate gold-silver nanoparticles and embed them into the polymer matrix. (2) Copolymerization method: co-polymerization of gold-silver nanoparticles with functional monomers and crosslinkers to form molecularly imprinted polymers containing gold-silver nanoparticles. (3) Post-modification method: first prepare molecularly imprinted polymers, and then modify gold-silver nanoparticles on the surface or inside of the polymer by physical or chemical methods. At the same time, gold and silver nano-molecular imprinting polymers have many excellent properties: (1) High selectivity: molecular imprinting polymers can specifically recognize target analytes and reduce nonspecific adsorption. (2) High sensitivity: the SPR effect of gold and silver nanoparticles can significantly enhance the detection signal and improve the detection sensitivity. (3) Good stability: gold and silver nanoparticles have good chemical stability and biocompatibility, which enables the composite material to maintain stable performance in complex environments. In addition, gold and silver nano-molecular imprinting polymers are widely used, and the main application areas include: (1) Environmental monitoring: used to detect pollutants in water, soil and air, such as heavy metal ions, organic pollutants, etc. (2) Biological detection: used to detect biomarkers, drug residues, etc. in biological samples, such as specific components in blood, urine and saliva. (3) Food safety: used to detect additives, pesticide residues and harmful microorganisms in food. (4) Drug analysis: used for drug screening, drug metabolism research and drug quality control.

[0004] Resonance Rayleigh Scattering (RRS) is an analytical method based on the resonance between light scattering and molecular light absorption. When light scattering is located at or close to the molecular absorption band, the scattering intensity may be greatly increased due to the scattering-absorption-rescattering process. This phenomenon is called resonance light scattering. It is a spectral analysis technology with good selectivity and high sensitivity. It has important application value in food, hygiene, safety, clinical and other fields. As an analytical method with development prospects, resonance Rayleigh scattering spectroscopy will play a more important role in bioanalysis, materials science, environmental monitoring and other fields. With the deepening of research and the continuous advancement of technology, the sensitivity and selectivity of this method will be further improved, and the scope of application will continue to expand. Although there are many reports on the detection technology of resonance Rayleigh scattering spectroscopy, there is no research on the determination of bovine serum albumin using resonance Rayleigh scattering spectroscopy.

[0005] In summary, if a new method for determining BSA can be developed using gold and silver nanomolecular imprinted polymers and resonance Rayleigh scattering spectroscopy, it will be expected to improve the sensitivity and accuracy of BSA detection. Summary of the invention

[0006] In order to overcome the shortcomings of the above-mentioned prior art, the present invention proposes a method for determining bovine serum albumin using resonance Rayleigh scattering spectroscopy of gold-silver nanomolecular imprinted polymer. The determination method adopts RRS spectroscopy of Au-AgNP@MIP specifically binding to BSA, which is simple to operate, rapid, highly sensitive and accurate.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The present invention provides a method for determining bovine serum albumin by using resonance Rayleigh scattering spectrum of gold-silver nanomolecular imprinted polymer, the method comprising the following steps:

[0009] S1, mixing a BSA standard solution of known concentration, a gold-silver nanomolecular imprinted polymer (Au-AgNP@MIP) and a HNO3 solution, and allowing the mixture to react for 10-30 minutes to prepare a BSA standard solution of known concentration;

[0010] S2, mix Au-AgNP@MIP and HNO3 solution, let it react for 10-30 minutes to prepare a blank control solution system;

[0011] S3. Determine the resonance scattering peak intensity value I of the BSA standard solution system at 340nm, and simultaneously determine the resonance scattering peak intensity value I0 of the blank control solution system, and calculate ΔI, ΔI=II 0；

[0012] S4. Make a working curve based on the relationship between ΔI and BSA concentration;

[0013] S5. Mix the sample solution, Au-AgNP@MIP and HNO3 solution, let it stand for 10-30 minutes, and then measure the resonance scattering peak intensity value I of the sample solution. 样品 , calculate ΔI 样品 , ΔI 样品 =I 样品 -I0;

[0014] S6. Calculate the BSA content of the sample solution according to the working curve of step S4;

[0015] The preparation method of Au-AgNP@MIP comprises the following steps:

[0016] S11, adding HAuCl4 aqueous solution to the cetyltrimethylammonium bromide solution to prepare a seed solution, and then adding an ice-cold NaBH4 solution under stirring, and obtaining an Au seed solution after aging;

[0017] S12, adding hexadecyltrimethylammonium chloride solution, ascorbic acid solution, AgNO3 solution and NaOH solution into water, and then adding the Au seed solution of step S1 under stirring, and obtaining Au-AgNP after water bath;

[0018] S13, adding Au-AgNP and β-cyclodextrin to phosphate buffer, stirring, then adding triethylene glycol dimethacrylate, ammonium persulfate and phosphate buffer, mixing and reacting, washing until no template protein is detected, and drying to obtain Au-AgNP@MIP.

[0019] Preferably, S1 is to mix 80 μL-800 μL of 10 μmol / L BSA standard solution, 10 μL-50 μL of 1 g / L Au-AgNP@MIP, and 50 μL-500 μL of 1 mol / L HNO3 solution to prepare a BSA standard solution of known concentration.

[0020] Preferably, the concentration of the cetyltrimethylammonium bromide solution in S11 is 100 mM, the concentration of the HAuCl4 aqueous solution is 25 mM, and the volume ratio of the cetyltrimethylammonium bromide solution to the HAuCl4 aqueous solution is 1-5:0.1-0.5.

[0021] Preferably, the concentration of the NaBH4 solution in S11 is 10 mM, and the volume ratio of the cetyltrimethylammonium bromide solution to the NaBH4 solution is 1-5:0.1-0.6.

[0022] Preferably, the aging in S11 is aging in a water bath at 25-35° C. for 2-4 hours.

[0023] Preferably, the concentration of the hexadecyltrimethylammonium chloride solution in S12 is 100mM, the concentration of the ascorbic acid solution is 100mM, the concentration of the AgNO3 solution is 10mM, the concentration of the NaOH solution is 1μM, and the volume ratio of the hexadecyltrimethylammonium chloride solution, ascorbic acid solution, AgNO3 solution, NaOH solution and water is 1-4:0.1-1:0.01-0.05:0.1-0.5:2-4.

[0024] Preferably, the volume ratio of the hexadecyltrimethylammonium chloride solution to the Au seed solution in S12 is 1-4:0.1-2.

[0025] Preferably, the step S12 is performed in a water bath at 25-40° C. for 7-15 hours.

[0026] Preferably, the usage ratio of Au-AgNP, β-cyclodextrin, triethylene glycol dimethacrylate and ammonium persulfate in S13 is 1-10 mL: 10-500 mg, 1-5 mmol: 5-50 mg.

[0027] Preferably, the reaction in S13 is carried out under stirring at 37-80° C. for 10-15 hours.

[0028] Preferably, S13 dissolves the template protein in phosphate buffer to make the concentration of the template protein be 1-100 mg / 20 mL; the amount of phosphate buffer added the second time is 1 / 2 of the amount added the first time.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The invention discloses a method for determining bovine serum albumin by using a gold-silver nanomolecular imprinting polymer resonance Rayleigh scattering spectrum. The method comprises the following steps: firstly, using a gold-silver nanomolecular imprinting polymer Au-AgNP@MIP and BSA to prepare a BSA standard solution system and a blank control solution system with known concentrations, and then determining a resonance scattering peak intensity value at 340 nm, calculating a resonance scattering peak intensity value difference ΔI between the standard solution and the blank control solution, and making a working curve based on the relationship between ΔI and the concentration of BSA; then, replacing the BSA standard solution with a sample solution, determining the resonance scattering peak intensity value of the sample solution, and calculating a resonance scattering peak intensity value difference ΔI between the sample solution and the blank control solution. 样品 , and finally, the BSA content of the sample solution was calculated according to the working curve. Compared with the existing BSA determination method, this determination method uses the RRS spectroscopy method of Au-AgNP@MIP specifically binding to BSA, which is simple to operate, rapid, highly sensitive and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1Characterization of Au-AgNP@MIP: (a) resonance Rayleigh scattering spectrum; (b) infrared spectrum of Au-AgNP@MIP and Au-AgNP@MIP combined with BSA.

[0032] Figure 2 This is the XPS full spectrum characterization diagram of MIP / Au-AgNP@MIP.

[0033] Figure 3 This is the resonance Rayleigh scattering spectrum of bovine serum albumin measured using gold and silver nanomolecular imprinting polymer (the corresponding concentrations from bottom to top are 0.04, 0.08, 0.16, 0.24, 0.32, and 0.4 μmol / L, respectively). DETAILED DESCRIPTION

[0034] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0036] Example 1

[0037] This embodiment provides a method for preparing a gold-silver nanomolecularly imprinted polymer (Au-AgNP@MIP), comprising the following steps:

[0038] (1) Preparation of Au seeds:

[0039] A seed solution was prepared by adding HAuCl4 aqueous solution (25 mM, 0.1 mL) to hexadecyltrimethylammonium bromide aqueous solution (100 mM, 5 mL). Freshly prepared NaBH4 (10 mM, 0.6 mL) was then quickly injected into the mixture under vigorous stirring and stirred for 2 min. The mixture was then placed in a 28 °C water bath for aging for 3 h to obtain an Au seed solution.

[0040] (2) Preparation of Au-AgNPs:

[0041] In a 10 mL conical flask, H2O (4 mL), hexadecyltrimethylammonium chloride aqueous solution (100 mM, 1 mL), ascorbic acid aqueous solution (100 mM, 750 μL), AgNO3 aqueous solution (10 mM, 30 μL) and NaOH aqueous solution (1 μM, 120 μL) were added. Then, the Au seed solution (1 mL) prepared in step (1) was quickly injected into the above mixture under vigorous stirring. After stirring for 5 min, the flask was transferred to a 28 ° C water bath for 12 h to obtain Au-AgNP.

[0042] (3) Preparation of Au-AgNP@MIP:

[0043] Add 20mL of phosphate buffer (0.02mol / L, pH=7.4) to a 50mL flask, then add 5mL of Au-AgNP and 100mg of β-cyclodextrin, and stir for 1h. Then add 1.8mmol of triethylene glycol dimethacrylate, 10mg of ammonium persulfate and 10mL of phosphate buffer (0.02mol / L, pH=7.4), ultrasonicate until the solution is dispersed and mixed, and stir at 60℃ for 12 hours. Then wash the obtained product several times with 0.5% sodium dodecyl sulfate until no template protein is detected, and finally freeze-dry in vacuum to obtain Au-AgNP@MIP.

[0044] Figure 1 The resonance Rayleigh scattering spectrum of (a) shows that the Au-AgNP@MIP in the solution system (before drying) has good dispersion, and the RRS signal gradually increases with the increase of Au-AgNP@MIP concentration.

[0045] (4) Preparation of Au-AgNP@MIP-BSA:

[0046] 10 mg BSA was added to a 50 mL flask and dissolved in 20 mL phosphate buffer (0.02 mol / L, pH = 7.4), and then 5 mL Au-AgNP and 100 mg β-cyclodextrin were added and stirred for 1 h. Then 1.8 mmol triethylene glycol dimethacrylate, 10 mg ammonium persulfate and 10 mL phosphate buffer (0.02 mol / L, pH = 7.4) were added, and the solution was dispersed and mixed by ultrasound, and then stirred at 60 ° C for 12 hours. The obtained product was then washed several times with 0.5% sodium dodecyl sulfate until no template protein was detected, and finally Au-AgNP@MIP-BSA was obtained after vacuum freeze drying.

[0047] Figure 1 The infrared spectrum of (b) shows that BSA successfully binds to Au-AgNP@MIP; Figure 2It shows that after BSA is combined with Au-AgNP@MIP, the proportion of N element increases significantly, which comes from the amino acid N source in BSA.

[0048] Example 2

[0049] This embodiment provides a method for determining bovine serum albumin using resonance Rayleigh scattering spectroscopy of gold-silver nanomolecular imprinted polymers, comprising the following steps:

[0050] (1) Prepare a BSA standard solution system with known concentrations: add 80 μL, 160 μL, 320 μL, 480 μL, 640 μL, and 800 μL of 1 μmol / L BSA standard solution, 50 μL of 1 g / L Au-AgNP@MIP, and 400 μL of 1 mol / L HNO3 solution to a graduated test tube, dilute to 2.0 mL with ultrapure water, shake well, and let react for 20 minutes;

[0051] (2) Preparing a blank control solution system: Prepare a standard solution without BSA using the method of step (1) to serve as a blank control solution system;

[0052] (3) Pour the BSA standard solution system and the blank control solution system prepared in steps (1) and (2) into a quartz cuvette respectively, set the instrument parameters on a Hitachi 7100 fluorescence spectrometer, scan and obtain the resonance scattering spectrum of the system under the conditions of a slit of 5 nm and a voltage of 350 V, measure the resonance scattering peak intensity value I of the BSA standard solution system at 340 nm, and simultaneously measure the resonance scattering peak intensity value I0 of the blank control solution system, and calculate ΔI, ΔI=I-I0;

[0053] (4) Make a working curve based on the relationship between ΔI and BSA concentration;

[0054] (5) preparing a sample solution (Mengniu milk sample) according to the method of step (1), wherein the added BSA standard solution is replaced by the sample solution, and the resonance scattering peak intensity value I of the sample solution is measured according to the method of step (3). 样品 , calculate ΔI 样品 , ΔI 样品 =I 样品 -I0;

[0055] (6) Calculate the BSA content of the sample solution based on the working curve of step (4). Figure 3 The spectrum of BSA detected by Au-AgNP@MIP-HNO3 system is shown. It can be seen that the detection limit of the method of the present invention is 0.04 μmol / L, while the detection limit measured by conventional flavonoid reaction principle and spectrophotometer is 0.15 μmol / L, indicating that the method of the present invention has high sensitivity.

[0056] (7) Validation of detection methods:

[0057] Take three portions of the milk sample in step (5), take 1g of the milk sample, mix it with 5mL of 5% methanol, and then centrifuge it at 5000rpm for 10min. The obtained supernatant is filtered through a 0.2μm filter membrane, diluted 100 times with PBS, and then added with a BSA standard solution with a final concentration of 200nmol / L of BSA. The recovery experiments are carried out, and the recovery rates are 97.7%, 98.8%, and 99.3%, respectively, and the relative standard deviations are 1.5%, 3.2%, and 1.8%, respectively. The above results show that the method of the present invention is accurate and reliable.

[0058] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A method for determining bovine serum albumin using resonance Rayleigh scattering spectroscopy of gold and silver nanomolecular imprinted polymers, characterized in that: The following steps are involved: S1. Mix the BSA standard solution with known concentration, Au-AgNP@MIP and HNO3 solution, and let it react for 10-30 minutes to prepare a BSA standard solution with known concentration; S2, mix Au-AgNP@MIP and HNO3 solution, let it react for 10-30 minutes to prepare a blank control solution system; S3. Determine the resonance scattering peak intensity value I of the BSA standard solution system at 340nm, and simultaneously determine the resonance scattering peak intensity value I0 of the blank control solution system, and calculate ΔI, ΔI=II 0； S4. Make a working curve based on the relationship between ΔI and BSA concentration; S5. Mix the sample solution, Au-AgNP@MIP and HNO3 solution, let it stand for 10-30 minutes, and then measure the resonance scattering peak intensity value I of the sample solution. 样品 , calculate ΔI 样品 , ΔI 样品 =I 样品 -I0; S6. Calculate the BSA content of the sample solution according to the working curve of step S4; The preparation method of Au-AgNP@MIP comprises the following steps: S11, adding HAuCl4 aqueous solution to the cetyltrimethylammonium bromide solution to prepare a seed solution, and then adding an ice-cold NaBH4 solution under stirring, and obtaining an Au seed solution after aging; S12, adding hexadecyltrimethylammonium chloride solution, ascorbic acid solution, AgNO3 solution and NaOH solution into water, and then adding the Au seed solution of step S1 under stirring, and obtaining Au-AgNP after water bath; S13, adding Au-AgNP and β-cyclodextrin to phosphate buffer, stirring, then adding triethylene glycol dimethacrylate, ammonium persulfate and phosphate buffer, mixing and reacting, washing until no template protein is detected, and drying to obtain Au-AgNP@MIP.

2. The method for determining bovine serum albumin using resonance Rayleigh scattering spectroscopy of gold and silver nanomolecular imprinted polymers according to claim 1, characterized in that: S1 is to mix 80 μL-800 μL of 10 μmol / L BSA standard solution, 10 μL-50 μL of 1 g / L Au-AgNP@MIP, and 50 μL-500 μL of 1 mol / L HNO3 solution to prepare a BSA standard solution of known concentration.

3. The method for determining bovine serum albumin using resonance Rayleigh scattering spectroscopy of gold and silver nanomolecular imprinted polymers according to claim 1, characterized in that: S11 The concentration of the cetyltrimethylammonium bromide solution is 100 mM, the concentration of the HAuCl4 aqueous solution is 25 mM, and the volume ratio of the cetyltrimethylammonium bromide solution to the HAuCl4 aqueous solution is 1-5:0.1-0.

5.

4. The method for determining bovine serum albumin using resonance Rayleigh scattering spectroscopy of gold and silver nanomolecular imprinted polymers according to claim 1, characterized in that: S11 The concentration of the NaBH4 solution is 10 mM, and the volume ratio of the cetyltrimethylammonium bromide solution to the NaBH4 solution is 1-5:0.1-0.

6.

5. The method for determining bovine serum albumin using resonance Rayleigh scattering spectroscopy of gold and silver nanomolecular imprinted polymers according to claim 1, characterized in that: The aging in S11 is aging in a water bath at 25-35° C. for 2-4 h.

6. The method for determining bovine serum albumin using resonance Rayleigh scattering spectroscopy of gold-silver nanomolecularly imprinted polymers according to claim 1, characterized in that: S12: The concentration of the hexadecyltrimethylammonium chloride solution is 100 mM, the concentration of the ascorbic acid solution is 100 mM, the concentration of the AgNO3 solution is 10 mM, the concentration of the NaOH solution is 1 μM, and the volume ratio of the hexadecyltrimethylammonium chloride solution, the ascorbic acid solution, the AgNO3 solution, the NaOH solution and water is 1-4: 0.1-1：0.01-0.05：0.1-0.5：2-4。 7. The method for determining bovine serum albumin using resonance Rayleigh scattering spectroscopy of gold and silver nanomolecular imprinted polymers according to claim 1, characterized in that: S12 The volume ratio of the hexadecyltrimethylammonium chloride solution to the Au seed solution is 1-4:0.1-2.

8. The method for determining bovine serum albumin using resonance Rayleigh scattering spectroscopy of gold and silver nanomolecular imprinted polymers according to claim 1, characterized in that: S12 is carried out in a water bath at 25-40° C. for 7-15 hours.

9. The method for determining bovine serum albumin using resonance Rayleigh scattering spectroscopy of gold-silver nanomolecularly imprinted polymers according to claim 1, characterized in that: The usage ratios of Au-AgNP, β-cyclodextrin, triethylene glycol dimethacrylate and ammonium persulfate in S13 are 1-10 mL: 10-500 mg and 1-5 mmol: 5-50 mg.

10. The method for determining bovine serum albumin using resonance Rayleigh scattering spectroscopy of gold-silver nanomolecularly imprinted polymers according to claim 1, characterized in that: The reaction in S13 is stirred at 37-80° C. for 10-15 hours.

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