A polypeptide-based molecularly imprinted polymer / quantum composite, preparation method and application
By preparing a peptide-based molecularly imprinted polymer/quantum composite material, the problem of low efficiency in the separation and analysis of amino acids and peptides was solved, achieving efficient and rapid separation and recognition of peptides and amino acids.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- QILU UNIVERSITY OF TECHNOLOGY (SHANDONG ACADEMY OF SCIENCES)
- Filing Date
- 2023-10-11
- Publication Date
- 2026-07-21
AI Technical Summary
In existing technologies, the separation and analysis methods for amino acids and peptides are inefficient, time-consuming, and have weak intermolecular forces between inorganic polymers and organic molecules, making them difficult to disperse in a carrier and resulting in poor separation performance.
Using peptides as template molecules, molecularly imprinted polymer/quantum composite materials based on peptides are prepared. By combining inorganic quantum dots with organic polymers, the separation and recognition capabilities are improved through fluorescence recognition performance, thereby shortening the separation time.
It achieves efficient separation and recognition of a variety of amino acids and peptides, with a detection limit below 3.0 nmol/L, and the separation time is shortened to half an hour, reducing costs and simplifying the separation process.
Smart Images
Figure CN117362739B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecularly imprinted polymer separation materials, specifically relating to a polypeptide-based molecularly imprinted polymer / quantum composite material, its preparation method, and its application. Background Technology
[0002] Amino acids are commonly used in drug synthesis, and D-amino acids or L-amino acids are usually chosen as raw materials to form a single enantiomer of the drug. However, since most non-natural amino acids exist in racemic form, it is necessary to develop a method for the efficient separation and analysis of D- and L-amino acids.
[0003] A polypeptide is a peptide composed of two or more amino acids linked together by peptide bonds. If it consists of only two amino acids, it is called a dipeptide, and so on. Polypeptides possess biological activity and have a wide range of applications; therefore, methods for the separation and analysis of polypeptides are of great significance for their application.
[0004] Molecularly imprinted polymer materials are increasingly widely used in the field of separation and purification. Molecularly imprinted polymers are polymers synthesized using molecular imprinting technology that exhibit specific recognition and selective adsorption of specific target molecules (template molecules) and their structural analogs.
[0005] Inorganic quantum dots, based on their fluorescence recognition properties, can effectively improve the ability to recognize and separate target molecules when combined with molecularly imprinted polymers to form quantum composite materials.
[0006] In existing technologies, the template molecules in molecularly imprinted polymers are generally the same as the target molecules. Existing techniques for detecting a single target molecule using a single template molecule include: Chinese literature (Liu Luying, et al. Specific adsorption and detection of riboflavin using a molecularly imprinted carbon quantum dot fluorescent sensor, *Journal of Analytical Science*, 2002, Vol. 38, No. 4, pp. 503-507) describes the specific adsorption and detection of riboflavin using a molecularly imprinted carbon quantum dot fluorescent sensor; and Chinese literature (Zhao Fei, et al. Preparation of quantum dot-imprinted polymers and their application in the determination of L-arginine, *Modern Chemical Industry Research*, 2023, No. 11, pp. 86-88) utilizes quantum dot / imprinted polymers prepared with L-arginine as a template to determine L-arginine. Existing technologies that use multiple template molecules to detect multiple corresponding target molecules, such as Chinese patent CN113209948A (Preparation method and application of a multi-template molecularly imprinted composite material) and CN112007621A (Preparation and application method of a multi-template molecularly imprinted magnetic composite material for tetracycline antibiotics), require all separated molecules to be used as template molecules in order to achieve simultaneous recognition of multiple molecules.
[0007] In addition, existing technologies all use inorganic polymers as carriers. The intermolecular forces between organic molecules such as amino acids and peptides and inorganic polymers are small, making them difficult to disperse in polymer carriers, resulting in poor separation effect and long separation time. It takes more than 2 hours to reach absorption equilibrium. Summary of the Invention
[0008] To address the problems of existing technologies, this invention provides a method for preparing a molecularly imprinted organic polymer / quantum composite material using a single polypeptide as a template. This method is applied to the separation and recognition of various amino acids and their combined polypeptides, exhibiting high separation and recognition performance. This achieves the goal of effectively separating and recognizing multiple target molecules with a single material, while significantly shortening the separation time.
[0009] To achieve the above objectives, the present invention adopts the following technical solution:
[0010] This invention provides a method for preparing a polypeptide-based molecularly imprinted polymer / quantum composite material, comprising the following steps:
[0011] (1) The inorganic quantum dot material was uniformly dispersed in anhydrous ethanol to obtain an inorganic quantum dot material dispersion;
[0012] (2) Take the inorganic quantum dot material dispersion from step (1), add a tripeptide composed of L-alanine, L-leucine and L-valine as a template molecule, continue to add acrylamide, diacetone acrylamide and initiator, then add ammonia and deionized water and stir to mix evenly, react at 60-80℃ in the dark, and collect the polymerization product by filtration.
[0013] (3) The polymerization product from step (2) is eluted and post-treated to remove the template molecules and obtain the polypeptide-based molecularly imprinted polymer / quantum composite material.
[0014] For ease of use, the composite material is ground and crushed into particles with an average particle size of 10-30 micrometers.
[0015] Preferably, the inorganic quantum dot material mentioned in step (1) is selected from carbon quantum dots or graphene quantum dots, with a particle size of 4-8 nm.
[0016] Preferably, the uniform dispersion in step (1) is achieved by ultrasonic dispersion for 30-40 minutes.
[0017] The tripeptide mentioned in step (2) is composed of L-alanine, L-leucine and L-valine, that is, the tripeptide is any one of alanine-leucine-valine tripeptide, alanine-valine-leucine tripeptide, valine-leucine-alanine tripeptide, valine-alanine-leucine tripeptide, leucine-alanine-valine tripeptide or leucine-valine-alanine tripeptide.
[0018] Preferably, the initiator in step (2) is potassium persulfate or ammonium persulfate.
[0019] Preferably, in steps (1) to (2), the amounts of each component are as follows:
[0020] 1-2 parts of inorganic quantum dot material;
[0021] 2-3 portions of tripeptide template molecule;
[0022] 10-20 parts acrylamide;
[0023] 5-10 parts of diacetone acrylamide
[0024] Initiator 0.1-0.2 parts;
[0025] The weight-to-volume ratio of inorganic quantum dot material to anhydrous ethanol is (1-2):(10-20) g / ml;
[0026] The volume ratio of anhydrous ethanol, ammonia, and deionized water is (10-20):(20-30):(40-80);
[0027] The ammonia content of the water is 28%.
[0028] Preferably, the reaction time in step (2) is 2 hours.
[0029] Preferably, the elution and post-treatment steps in step (3) are as follows: the polymer product is loaded into a chromatography column, eluted with 5% ammonia solution until template molecules are not detected in the eluent, then washed three times with 50 mL to 60 mL of deionized water, filtered and dried, and finally vacuum dried at 60 °C for 12 h.
[0030] The present invention also provides a polypeptide-based molecularly imprinted polymer / quantum composite material prepared by the above method.
[0031] This invention also provides applications of the above-mentioned polypeptide-based molecularly imprinted polymer / quantum composite materials for the quantitative detection of polypeptides or amino acids, wherein the amino acids are selected from L-alanine, L-valine, or L-leucine; the polypeptides are dipeptides or tripeptides obtained by dehydration condensation of different amino acids. The dipeptides are selected from propionylvaline dipeptide, valine-propionyl dipeptide, propionyl-leucine dipeptide, leucine-propionyl dipeptide, valine-leucine dipeptide, or leucine-valine dipeptide; the tripeptides are selected from propionylvaline-leucine tripeptide, propionyl-leucine-valine tripeptide, valine-leucine-propionyl tripeptide, valine-propionyl-leucine tripeptide, leucine-propionylvaline tripeptide, or leucine-valine-propionyl tripeptide.
[0032] Preferably, the detection method in the application includes the following steps:
[0033] (1) The fluorescence of the polypeptide-based molecularly imprinted polymer / quantum composite material was tested using a fluorescence spectrophotometer to obtain the maximum fluorescence emission wavelength of the composite material;
[0034] (2) Perform analysis and testing at the maximum fluorescence emission wavelength obtained in step (1):
[0035] 1) Take 0.1g of the polypeptide-based molecularly imprinted polymer / quantum composite material and add it to a 5mL centrifuge tube. Disperse it in 2mL of distilled water to obtain a composite material dispersion. Take 9 portions of the composite material dispersion and add 5mL of a standard solution of an amino acid or polypeptide target molecule with concentrations of 0.2μmol / L, 0.5μmol / L, 1μmol / L, 2μmol / L, 3μmol / L, 4μmol / L, 5μmol / L, 6μmol / L, 8μmol / L, and 10μmol / L respectively to obtain a set of mixed solutions. Shake at 180rpm for 0.5h and measure the fluorescence value of the set of mixed solutions. Plot the obtained fluorescence value on the ordinate and the standard solution concentration of the target molecule on the abscissa to obtain the working curve of the target molecule. Take the upper and lower limits of the linear portion of the working curve as the working interval for quantitative detection of the target molecule.
[0036] 2) Take the composite material dispersion described in step 1), add the test solution of the target molecule, shake at 180 rpm for 0.5 h to obtain the test mixed solution, measure the fluorescence value of the test mixed solution, and calculate the concentration of the test molecule in the test mixed solution according to the working curve;
[0037] 3) The detection limit of the amino acid molecule to be tested is determined by using the signal-to-noise ratio method, S / N = 3, where S is the signal intensity and N is the noise intensity.
[0038] The detection limit is the concentration of the analyte when the ratio of the fluorescence emission peak signal to the baseline noise is 3.
[0039] Compared with the prior art, the present invention has the following beneficial effects:
[0040] (1) The present invention prepares molecularly imprinted polymer / quantum composite material by using tripeptide as template molecule. The composite material can realize the detection of various amino acid or polypeptide target molecules, thereby improving the detection efficiency.
[0041] (2) The tripeptide described in this invention is composed of L-alanine, L-valine and L-leucine. The resulting molecularly imprinted polymer / quantum composite material has good detection effect on the three amino acids and dipeptides or tripeptides composed of the amino acids. The detection limit is below 3.0 nmol / L. Moreover, the concentration of the target molecule can be quantitatively detected within a certain range.
[0042] (3) The molecularly imprinted polymer described in this invention can recognize 3 amino acids, 6 dipeptides and 6 tripeptides using only one polypeptide as a template, which greatly reduces costs, simplifies the separation process, and shortens the separation time from more than 2 hours to half an hour. Attached Figure Description
[0043] Figure 1 The fluorescence spectrum is shown in step (1) of Example 1;
[0044] Figure 2 The graph is the working curve obtained in step (2) of Example 1. Detailed Implementation
[0045] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.
[0046] Example 1
[0047] A method for preparing a polypeptide-based molecularly imprinted polymer / quantum composite material includes the following steps:
[0048] (1) Weigh 1 gram of carbon quantum dot material, add it to 10 ml of anhydrous ethanol and ultrasonically disperse it for 30-40 minutes to obtain an inorganic quantum dot material dispersion, and store it at 4°C in the dark.
[0049] (2) Take the inorganic quantum dot material dispersion from step (1), add 2.2 g of propyl-valine-leucine tripeptide as a template molecule, and continue to add 15 g of acrylamide, 7 g of diacetone acrylamide and 0.12 g of potassium persulfate. Then add 20 ml of ammonia water (mass fraction of 28%) and 50 ml of deionized water, stir and disperse evenly, and react at 65°C in the dark for 2 h. Collect the polymerization product by filtration.
[0050] (3) The polymerization product from step (2) was loaded into a chromatography column and eluted with 5% ammonia solution until no template molecules were detected in the eluent. Then it was washed three times with 50 ml of deionized water, filtered and dried, and finally vacuum dried at 60 °C for 12 h to obtain a polypeptide-imprinted polymer / quantum composite material. The material was then ground into particles with an average particle size of 10-30 micrometers.
[0051] The above-mentioned polypeptide-based molecularly imprinted polymer / quantum composite material is used to detect amino acids, dipeptides, or tripeptides, including the following steps:
[0052] (1) The fluorescence of the molecularly imprinted polymer / quantum composite material prepared above was tested using a fluorescence spectrophotometer, and the results are as follows: Figure 1 As shown in the figure, its maximum fluorescence emission wavelength was found to be 360 nm. (2)
[0054] 1) Take 0.1g of the above-mentioned polypeptide-based molecularly imprinted polymer / quantum composite material and add it to a 5mL centrifuge tube. Disperse it in 2mL of distilled water to obtain a composite material dispersion. Take 9 portions of the composite material dispersion and add 5mL of L-alanine standard solution with concentrations of 0.2μmol / L, 0.5μmol / L, 1μmol / L, 2μmol / L, 3μmol / L, 4μmol / L, 5μmol / L, 6μmol / L, 8μmol / L, and 10μmol / L respectively to obtain a set of mixed solutions. Shake at 180rpm for 0.5h and measure the fluorescence value of the set of mixed solutions at a wavelength of 360nm. Plot the obtained fluorescence value on the ordinate and the concentration of L-alanine standard solution on the abscissa to obtain the working curve of L-alanine, as shown in the figure. Figure 2 The upper and lower limits of the linear portion of the working curve are taken as the working range for quantitative detection of L-alanine.
[0055] 2) Take the composite material dispersion described in step 1), add the L-alanine solution to be tested, and shake under the same conditions as in step 1). Then measure the fluorescence value and calculate the concentration of the L-alanine molecules to be tested based on the working curve.
[0056] 3) The detection limit of L-alanine to be tested was determined by using the signal-to-noise ratio method (S / N = 3, where S is the signal intensity and N is the noise intensity), which is the concentration of the analyte when the ratio of the fluorescence emission peak signal to the baseline noise is 3.
[0057] Using the methods described in steps 1)-3) above, the detection limits for each of the other target amino acids, dipeptides, and tripeptides were determined.
[0058] The detection limits and upper and lower limits of concentration for detecting the separated amino acids, dipeptides and tripeptides using the composite material of this embodiment are shown in Table 1.
[0059] Table 1. Detection results of target molecules by composite materials
[0060]
[0061]
[0062] Example 2
[0063] A method for preparing a polypeptide-based molecularly imprinted polymer / quantum composite material includes the following steps:
[0064] (1) Weigh 1.2 g of graphene quantum dot material, add 15 ml of anhydrous ethanol and ultrasonically disperse for 30-40 minutes to obtain inorganic quantum dot material dispersion, and store at 4°C in the dark.
[0065] (2) Take the inorganic quantum dot material dispersion from step (1), add 2.2 g of propionyl-leucine-valley tripeptide as a template molecule, and continue to add 20 g of acrylamide, 10 g of diacetone acrylamide and 0.15 g of ammonium persulfate. Then add 22 ml of ammonia water (mass fraction 28%) and 60 ml of deionized water, stir and mix evenly, and polymerize at 80°C for 2 hours. Collect the polymerization product by centrifugation.
[0066] (3) The polymerization product from step (2) is loaded into a chromatography column and eluted with 5% ammonia solution until template molecules are no longer detectable in the eluent. Then, it is washed three times with 60 ml of deionized water, filtered and dried, and finally vacuum dried at 60 °C for 12 h to obtain a polypeptide-imprinted polymer / quantum composite material. The material is then ground into particles with an average particle size of 10-30 micrometers.
[0067] The above-mentioned peptide-based molecularly imprinted polymer / quantum composite material is used to detect amino acids, dipeptides, and tripeptides, including the following steps:
[0068] (1) The molecularly imprinted polymer / quantum composite material prepared above was subjected to fluorescence test using a fluorescence spectrophotometer, and its maximum fluorescence emission wavelength was found to be 535 nm. (2)
[0070] 1) Take 0.1g of molecularly imprinted polymer / quantum composite material and add it to a 5mL centrifuge tube. Disperse it in 2mL of distilled water to obtain a composite material dispersion. Take 9 portions of the composite material dispersion and add 5mL of L-alanine standard solution with concentrations of 0.2μmol / L, 0.5μmol / L, 1μmol / L, 2μmol / L, 3μmol / L, 4μmol / L, 5μmol / L, 6μmol / L, 8μmol / L, and 10μmol / L respectively to obtain a set of mixed solutions. Shake at 180rpm for 0.5h and measure the fluorescence value of the set of mixed solutions at a wavelength of 535nm. Plot the obtained fluorescence value on the ordinate and the concentration of L-alanine standard solution on the abscissa to obtain the working curve of L-alanine. Take the upper and lower limits of the linear part of the working curve as the working interval for quantitative detection of L-alanine.
[0071] 2) Take the composite material dispersion described in step 1), add the L-alanine solution to be tested, and shake under the same conditions as in step 1). Then measure the fluorescence value and calculate the concentration of the L-alanine molecules to be tested based on the working curve.
[0072] 3) The detection limit of L-alanine to be tested was determined by using the signal-to-noise ratio method (S / N = 3, where S is the signal intensity and N is the noise intensity), which is the concentration of the analyte when the ratio of the fluorescence emission peak signal to the baseline noise is 3.
[0073] Using the methods described in steps 1)-3) above, the detection limits of each other target amino acid molecule and the corresponding dipeptide and tripeptide were determined.
[0074] The detection limits and upper and lower limits of concentration for detecting the separated amino acids, dipeptides and tripeptides using the composite material of this embodiment are shown in Table 2.
[0075] Table 2. Detection results of target molecules by composite materials
[0076]
[0077]
[0078] Example 3
[0079] A method for preparing a polypeptide-based molecularly imprinted polymer / quantum composite material includes the following steps:
[0080] (1) Weigh 1 gram of graphene quantum dot material, add it to 10 ml of anhydrous ethanol and ultrasonically disperse it for 40 minutes to obtain an inorganic quantum dot material dispersion, and store it at 4°C in the dark.
[0081] (2) Take the inorganic quantum dot material dispersion from step (1), add 2.1 g of va-leucine-propane tripeptide as a template molecule, and continue to add 12 g of acrylamide, 8 g of diacetone acrylamide and 0.12 g of potassium persulfate. Then add 25 ml of ammonia water (mass fraction 28%) and 70 ml of deionized water, sonicate for 20 min, and polymerize at 60 °C for 2 h. Collect the polymerization product by filtration.
[0082] (3) The polymerization product from step (2) was loaded into a chromatography column and eluted with 5% ammonia solution until no template molecules were detected in the eluent. Then it was washed three times with 50 ml of deionized water, filtered and dried, and finally vacuum dried at 60 °C for 12 h to obtain a polypeptide-imprinted polymer / quantum composite material. The material was then ground into particles with an average particle size of 10-30 micrometers.
[0083] The above-mentioned peptide-based molecularly imprinted polymer / quantum composite material is used to detect target molecules such as amino acids, dipeptides, and tripeptides, including the following steps:
[0084] (1) The molecularly imprinted polymer / quantum composite material prepared above was subjected to fluorescence test using a fluorescence spectrophotometer, and its maximum fluorescence emission wavelength was found to be 540 nm. (2)
[0086] 1) Take 0.2g of molecularly imprinted polymer / quantum composite material and add it to a 5mL centrifuge tube. Disperse it in 2mL of distilled water to obtain a composite material dispersion. Take 9 portions of the composite material dispersion and add 5mL of L-alanine standard solution with concentrations of 0.2μmol / L, 0.5μmol / L, 1μmol / L, 2μmol / L, 3μmol / L, 4μmol / L, 5μmol / L, 6μmol / L, 8μmol / L, and 10μmol / L respectively to obtain a set of mixed solutions. Shake at 180rpm for 0.5h and measure the fluorescence value of the set of mixed solutions at a wavelength of 540nm. Plot the obtained fluorescence value on the ordinate and the concentration of L-alanine standard solution on the abscissa to obtain the working curve of L-alanine. Take the upper and lower limits of the linear part of the working curve as the working interval for quantitative detection of L-alanine.
[0087] 2) Take the composite material dispersion described in step 1), add the L-alanine solution to be tested, and shake under the same conditions as in step 1). Then measure the fluorescence value and calculate the concentration of the L-alanine molecules to be tested based on the working curve.
[0088] 3) The detection limit of L-alanine to be tested was determined by using the signal-to-noise ratio method (S / N = 3, where S is the signal intensity and N is the noise intensity), which is the concentration of the analyte when the ratio of the fluorescence emission peak signal to the baseline noise is 3.
[0089] Using the methods described in steps 1)-3) above, the detection limits of each other target molecule were determined.
[0090] The detection limits and upper and lower limits of concentration for common separated target molecules using the composite material of this embodiment are shown in Table 3.
[0091] Table 3. Detection results of target molecules by composite materials
[0092]
[0093]
[0094] Example 4
[0095] A method for preparing a polypeptide-based molecularly imprinted polymer / quantum composite material includes the following steps:
[0096] (1) Weigh 1 gram of carbon quantum dot material, add it to 10 ml of anhydrous ethanol and ultrasonically disperse it for 30 minutes to obtain an inorganic quantum dot material dispersion, and store it at 4°C in the dark.
[0097] (2) Take the inorganic quantum dot material dispersion from step (1), add 2 g of va-propane-leucine tripeptide as a template molecule, and continue to add 12 g of acrylamide, 6 g of diacetone acrylamide and 0.18 g of potassium persulfate. Then add 20 ml of ammonia water (mass fraction 28%) and 60 ml of deionized water. After stirring and mixing evenly, polymerize at 60 °C for 2 h. Collect the polymerization product by filtration.
[0098] (3) The polymerization product from step (2) was loaded into a chromatography column and eluted with 5% ammonia solution until no template molecules were detected in the eluent. Then it was washed three times with 50 ml of deionized water, filtered and dried, and finally vacuum dried at 60 °C for 12 h to obtain a polypeptide-imprinted polymer / quantum composite material. The material was then ground into particles with an average particle size of 10-30 micrometers.
[0099] The above-mentioned peptide-based molecularly imprinted polymer / quantum composite material is used to detect target molecules such as amino acids, including the following steps:
[0100] (1) The molecularly imprinted polymer / quantum composite material prepared above was subjected to fluorescence test using a fluorescence spectrophotometer, and its maximum fluorescence emission wavelength was found to be 365 nm. (2)
[0102] 1) Take 0.1g of molecularly imprinted polymer / quantum composite material and add it to a 5mL centrifuge tube. Disperse it in 2mL of distilled water to obtain a composite material dispersion. Take 9 portions of the composite material dispersion and add 5mL of L-alanine standard solution with concentrations of 0.2μmol / L, 0.5μmol / L, 1μmol / L, 2μmol / L, 3μmol / L, 4μmol / L, 5μmol / L, 6μmol / L, 8μmol / L, and 10μmol / L respectively to obtain a set of mixed solutions. Shake at 180rpm for 0.5h and measure the fluorescence value of the set of mixed solutions at a wavelength of 365nm. Plot the obtained fluorescence value on the ordinate and the concentration of L-alanine standard solution on the abscissa to obtain the working curve of L-alanine. Take the upper and lower limits of the linear part of the working curve as the working interval for quantitative detection of L-alanine.
[0103] 2) Take the composite material dispersion described in step 1), add the L-alanine solution to be tested, and shake under the same conditions as in step 1). Then measure the fluorescence value and calculate the concentration of the L-alanine molecules to be tested based on the working curve.
[0104] 3) The detection limit of L-alanine to be tested was determined by using the signal-to-noise ratio method (S / N = 3, where S is the signal intensity and N is the noise intensity), which is the concentration of the analyte when the ratio of the fluorescence emission peak signal to the baseline noise is 3.
[0105] Using the methods described in steps 1)-3) above, the detection limits of each other target molecule were determined.
[0106] The detection limits and upper and lower limits of concentration for detecting common separated target molecules using the composite material of this embodiment are shown in Table 5.
[0107] Table 4. Detection results of target molecules by composite materials
[0108] tested molecules Detection linear range (μmol / L) Limit of detection (nmol / L) L-alanine 0.25-8.67 2.87 L-valine 0.18-9.20 2.16 L-Leucine 0.17-8.97 2.21 Propylvaline dipeptide 0.27-9.14 2.87 Val-malondipeptide 0.24-9.28 1.96 Propionyl leucide 0.25-9.12 1.89 Leucine-Propionide 0.31-8.96 2.97 Leucine-valine dipeptide 0.24-8.87 2.07 Val-Leucide 0.31-8.99 2.71 Propyl-valine-leucine tripeptide 0.22-9.02 2.77 Propionyl-leucine-valine tripeptide 0.30-8.87 2.81 Val-Propionyl-Leucide Tripeptide 0.15-9.57 1.24 Val-Leucine Tripeptide 0.31-8.75 2.90 Leucine-pro-valve tripeptide 0.37-9.04 2.87 Leucine-valine-protereptide 0.31-9.22 2.73
[0109] Example 5
[0110] A method for preparing a polypeptide-based molecularly imprinted polymer / quantum composite material includes the following steps:
[0111] (1) Weigh 1 gram of carbon quantum dot material, add it to 10 ml of anhydrous ethanol and ultrasonically disperse it for 30 minutes to obtain an inorganic quantum dot material dispersion, and store it at 4°C in the dark.
[0112] (2) Take the inorganic quantum dot material dispersion from step (1), add 2.1 g of leuc-propane-valley tripeptide as a template molecule, and continue to add 16 g of acrylamide, 10 g of diacetone acrylamide and 0.2 g of ammonium persulfate. Then add 20 ml of ammonia water (mass fraction 28%) and 60 ml of deionized water, stir and mix evenly, react at 70°C for 2 h, and collect the polymerization product by filtration.
[0113] (3) The polymerization product from step (2) was loaded into a chromatography column and eluted with 5% ammonia solution until no template molecules were detected in the eluent. Then it was washed three times with 50 ml of deionized water, filtered and dried, and finally vacuum dried at 60 °C for 12 h to obtain a polypeptide-imprinted polymer / quantum composite material. The material was then ground into particles with an average particle size of 10-30 micrometers.
[0114] The above-mentioned peptide-based molecularly imprinted polymer / quantum composite material is used to detect target molecules such as amino acids, including the following steps:
[0115] (1) The molecularly imprinted polymer / quantum composite material prepared above was subjected to fluorescence test using a fluorescence spectrophotometer, and its maximum fluorescence emission wavelength was found to be 362 nm. (2)
[0117] 1) Take 0.1g of molecularly imprinted polymer / quantum composite material and add it to a 5mL centrifuge tube. Disperse it in 2mL of distilled water to obtain a composite material dispersion. Take 9 portions of the composite material dispersion and add 5mL of L-alanine standard solution with concentrations of 0.2μmol / L, 0.5μmol / L, 1μmol / L, 2μmol / L, 3μmol / L, 4μmol / L, 5μmol / L, 6μmol / L, 8μmol / L, and 10μmol / L respectively to obtain a set of mixed solutions. Shake at 180rpm for 0.5h and measure the fluorescence value of the set of mixed solutions at a wavelength of 362nm. Plot the obtained fluorescence value on the ordinate and the concentration of L-alanine standard solution on the abscissa to obtain the working curve of L-alanine. Take the upper and lower limits of the linear part of the working curve as the working interval for quantitative detection of L-alanine.
[0118] 2) Take the composite material dispersion described in step 1), add the L-alanine solution to be tested, and shake under the same conditions as in step 1). Then measure the fluorescence value and calculate the concentration of the L-alanine molecules to be tested based on the working curve.
[0119] 3) The detection limit of L-alanine to be tested was determined by using the signal-to-noise ratio method (S / N = 3, where S is the signal intensity and N is the noise intensity), which is the concentration of the analyte when the ratio of the fluorescence emission peak signal to the baseline noise is 3.
[0120] Using the methods described in steps 1)-3) above, the detection limits of each other target molecule were determined.
[0121] The detection limits and upper and lower limits of concentration for detecting common separated target molecules using the composite material of this embodiment are shown in Table 5.
[0122] Table 5. Detection results of target molecules by composite materials
[0123] tested molecules Detection linear range (μmol / L) Limit of detection (nmol / L) L-alanine 0.32-8.69 2.91 L-valine 0.28-9.07 2.55 L-Leucine 0.22-8.95 2.31 Propylvaline dipeptide 0.21-9.17 2.17 Val-malondipeptide 0.25-9.11 2.86 Propionyl leucide 0.25-9.01 2.89 Leucine-Propionide 0.11-9.13 2.10 Leucine-valine dipeptide 0.29-8.88 2.87 Val-Leucide 0.31-8.91 2.85 Propyl-valine-leucine tripeptide 0.28-8.99 2.87 Propionyl-leucine-valine tripeptide 0.32-8.90 2.88 Val-Propionyl-Leucide Tripeptide 0.28-9.44 2.74 Val-Leucine Tripeptide 0.31-8.98 2.89 Leucine-pro-valve tripeptide 0.17-9.54 1.87 Leucine-valine-protereptide 0.31-9.34 2.85
[0124] Example 6
[0125] A method for preparing a polypeptide-based molecularly imprinted polymer / quantum composite material includes the following steps:
[0126] (1) Weigh 1 gram of carbon quantum dot material, add it to 10 ml of anhydrous ethanol and ultrasonically disperse it for 30 minutes to obtain an inorganic quantum dot material dispersion, and store it at 4°C in the dark.
[0127] (2) Take the inorganic quantum dot material dispersion from step (1), add 2 g of leuc-val-propane as a template molecule, and continue to add 12 g of acrylamide, 7 g of diacetone acrylamide and 0.15 g of potassium persulfate. Then add 20 ml of ammonia water (mass fraction 28%) and 60 ml of deionized water, stir and mix evenly, and react at 70°C for 2 h. Collect the polymerization product by filtration.
[0128] (3) The polymerization product from step (2) was loaded into a chromatography column and eluted with 5% ammonia solution until no template molecules were detected in the eluent. Then it was washed three times with 50 ml of deionized water, filtered and dried, and finally vacuum dried at 60 °C for 12 h to obtain a polypeptide-imprinted polymer / quantum composite material. The material was then ground into particles with an average particle size of 10-30 micrometers.
[0129] The above-mentioned peptide-based molecularly imprinted polymer / quantum composite material is used to detect target molecules such as amino acids, including the following steps:
[0130] (1) The molecularly imprinted polymer / quantum composite material prepared above was subjected to fluorescence test using a fluorescence spectrophotometer, and its maximum fluorescence emission wavelength was found to be 358 nm. (2)
[0132] 1) Take 0.1g of molecularly imprinted polymer / quantum composite material and add it to a 5mL centrifuge tube. Disperse it in 2mL of distilled water to obtain a composite material dispersion. Take 9 portions of the composite material dispersion and add 5mL of L-alanine standard solution with concentrations of 0.2μmol / L, 0.5μmol / L, 1μmol / L, 2μmol / L, 3μmol / L, 4μmol / L, 5μmol / L, 6μmol / L, 8μmol / L, and 10μmol / L respectively to obtain a set of mixed solutions. Shake at 180rpm for 0.5h and measure the fluorescence value of the set of mixed solutions at a wavelength of 358nm. Plot the obtained fluorescence value on the ordinate and the concentration of L-alanine standard solution on the abscissa to obtain the working curve of L-alanine. Take the upper and lower limits of the linear part of the working curve as the working interval for quantitative detection of L-alanine.
[0133] 2) Take the composite material dispersion described in step 1), add the L-alanine solution to be tested, and shake under the same conditions as in step 1). Then measure the fluorescence value and calculate the concentration of the L-alanine molecules to be tested based on the working curve.
[0134] 3) The detection limit of L-alanine to be tested was determined by using the signal-to-noise ratio method (S / N = 3, where S is the signal intensity and N is the noise intensity), which is the concentration of the analyte when the ratio of the fluorescence emission peak signal to the baseline noise is 3.
[0135] Using the methods described in steps 1)-3) above, the detection limits of each other target molecule were determined.
[0136] The detection limits and upper and lower limits of concentration for detecting common separated target molecules using the composite material of this embodiment are shown in Table 6.
[0137] Table 6. Detection results of target molecules by composite materials
[0138]
[0139]
[0140] As can be clearly seen from the data listed in the tables of the various embodiments, all molecularly imprinted polymer / quantum composite materials have a wide linear measurement range for target molecules, can quantitatively detect target molecules over a wide concentration range, and have detection limits below 3.0 nmol / L, and the analysis and separation time can be shortened to 0.5 hours.
[0141] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. A method for preparing a polypeptide-based molecularly imprinted polymer / quantum composite material, characterized in that, The steps include: (1) uniformly dispersing inorganic quantum dot materials in anhydrous ethanol to obtain an inorganic quantum dot material dispersion; (2) Take the inorganic quantum dot material dispersion from step (1), add a tripeptide composed of L-alanine, L-leucine and L-valine as a template molecule, continue to add acrylamide, diacetone acrylamide and initiator, then add ammonia and deionized water and stir to mix evenly, react at 60-80℃, and collect the polymerization product by filtration. (3) The polymerization product from step (2) is eluted and post-treated to remove template molecules, and then ground into particles with an average particle size of 10-30 micrometers to obtain the polypeptide-based molecularly imprinted polymer / quantum composite material; The inorganic quantum dot material mentioned in step (1) is selected from carbon quantum dots or graphene quantum dots, with a particle size of 4-8 nm; In steps (1) and (2), the amounts of each component are as follows: 1-2 parts of inorganic quantum dot material; 2-3 parts of template molecule; 10-20 parts acrylamide; 5-10 parts of diacetone acrylamide; Initiator 0.1-0.2 parts; The weight-to-volume ratio of inorganic quantum dot material to anhydrous ethanol is (1-2):(10-20) g / ml; The volume ratio of anhydrous ethanol, ammonia, and deionized water is (10-20):(20-30):(40-80); The ammonia content of the water is 28%.
2. The method for preparing a polypeptide-based molecularly imprinted polymer / quantum composite material according to claim 1, characterized in that, The uniform dispersion in step (1) is achieved by ultrasonic dispersion for 30-40 minutes.
3. The method for preparing a polypeptide-based molecularly imprinted polymer / quantum composite material according to claim 1, characterized in that, The initiator mentioned in step (2) is potassium persulfate or ammonium persulfate.
4. The method for preparing a polypeptide-based molecularly imprinted polymer / quantum composite material according to claim 1, characterized in that, The reaction time described in step (2) is 2 hours.
5. The method for preparing a polypeptide-based molecularly imprinted polymer / quantum composite material according to claim 1, characterized in that, The elution and post-treatment steps in step (3) are as follows: the polymer product is loaded into a chromatography column, eluted with 5% ammonia solution until no template molecules are detected in the eluent, then washed three times with 50 mL to 60 mL of deionized water, filtered and dried, and finally vacuum dried at 60 °C for 12 h.
6. The polypeptide-based molecularly imprinted polymer / quantum composite material prepared by the preparation method according to any one of claims 1 to 5.
7. The application of the polypeptide-based molecularly imprinted polymer / quantum composite material as described in claim 6, for the quantitative detection of polypeptides or amino acids, wherein the amino acid is selected from L-alanine, L-valine, or L-leucine; the polypeptide is a dipeptide or a tripeptide; the dipeptide is selected from propylvaline dipeptide, valine-propylvaline dipeptide, propyl-leucine dipeptide, leucine-propylvaline dipeptide, valine-leucine dipeptide, or leucine-valine dipeptide; the tripeptide is selected from propylvaline-leucine tripeptide, propyl-leucine-valine tripeptide, valine-leucine-propylvaline tripeptide, valine-propyl-leucine tripeptide, leucine-propylvaline tripeptide, or leucine-valine-propylvaline tripeptide.
8. The application of the polypeptide-based molecularly imprinted polymer / quantum composite material according to claim 7, characterized in that, The detection method in the application includes the following steps: (1) The fluorescence of the polypeptide-based molecularly imprinted polymer / quantum composite material was tested using a fluorescence spectrophotometer to obtain the maximum fluorescence emission wavelength of the composite material; (2) Perform analysis and testing at the maximum fluorescence emission wavelength obtained in step (1): 1) Take 0.1g of the polypeptide-based molecularly imprinted polymer / quantum composite material and add it to a 5mL centrifuge tube. Disperse it in 2mL of distilled water to obtain a composite material dispersion. Take 9 portions of the composite material dispersion and add 5mL of a standard solution of an amino acid or polypeptide target molecule with concentrations of 0.2μmol / L, 0.5μmol / L, 1μmol / L, 2μmol / L, 3μmol / L, 4μmol / L, 5μmol / L, 6μmol / L, 8μmol / L, and 10μmol / L respectively to obtain a set of mixed solutions. Shake at 180rpm for 0.5h and measure the fluorescence value of the set of mixed solutions. Plot the obtained fluorescence value on the ordinate and the standard solution concentration of the target molecule on the abscissa to obtain the working curve of the target molecule. Take the upper and lower limits of the linear portion of the working curve as the working interval for quantitative detection of the target molecule. 2) Take the composite material dispersion described in step 1), add the test solution of the target molecule, shake at 180 rpm for 0.5 h to obtain the test mixed solution, measure the fluorescence value of the test mixed solution, and calculate the concentration of the test molecule in the test mixed solution according to the working curve; 3) The detection limit of the analyte is determined by using the signal-to-noise ratio method, S / N = 3, where S is the signal intensity and N is the noise intensity.