CN108085364B - Polypeptide functionalized magnetic ball for detecting protease and colorimetric detection method - Google Patents
Polypeptide functionalized magnetic ball for detecting protease and colorimetric detection method Download PDFInfo
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Abstract
The polypeptide functionalized magnetic ball for detecting the protease comprises a magnetic ball, a layer of gold is wrapped outside the magnetic ball, and the polypeptide is fixed on the gold, wherein the sequence characteristic of the polypeptide is as follows: can be cleaved by a corresponding protease, and the third amino acid of the cleavage site in the direction of the carbon terminal is histidine, and the 5 amino acids of the carbon terminal is PPPPPPC. The colorimetric detection method for detecting the protease, which adopts the polypeptide functionalized magnetic ball, comprises the following steps: a: preparation of polypeptide functionalized magnetic spheres: adding the polypeptide into a gold-coated magnetic ball, performing magnetic separation, washing with secondary water, and adding a mercaptohexanol solution to obtain a polypeptide functionalized magnetic ball; b: and (4) detecting the protease. The method has the advantages of simplicity, sensitivity, intuition, high flux, no need of special instruments and the like, and the detection of different types of protease can be realized by the successful development of the technology.
Description
Technical Field
The invention relates to protease detection, in particular to a polypeptide functionalized magnetic ball for protease detection and a detection method, and belongs to the field of chemistry.
Background
Proteases are capable of hydrolyzing peptide bonds in proteins or polypeptides. It has decisive effects in digestion and absorption, wound healing, functional immunity, etc. Some serious diseases occur in close relation to the change of the content and activity of protease, such as cancer, apoptosis, Alzheimer disease, AIDS, etc. Therefore, protease detection is of great importance in the diagnosis and therapeutic research of these serious diseases. The existing methods for detecting the protease mainly comprise high performance liquid chromatography, gel chromatography, mass spectrometry, fluorescence spectroscopy, electrochemistry and the like, but the high performance liquid chromatography, the gel chromatography and the mass spectrometry need special instruments, and have low sensitivity and high analysis cost. Fluorescence spectroscopy and electrochemical methods often require labeling of both ends of the enzyme substrate (usually a polypeptide), which is time-consuming and labor-consuming; in addition, the efficiency of enzyme-catalyzed cleavage may be affected when the two ends of the polypeptide are labeled. The important role of protein cleavage reaction in the research of life activities and disease prevention and treatment, and the establishment of a method which is low in cost, simple and rapid and is suitable for monitoring the content or activity change of protease in general laboratories or clinics is one of the hot spots of the current research.
The visual detection analysis method is a method of determining the concentration of a substance to be detected in a solution by observing the color of the solution to be detected (or the color developed after adding a reagent) with the eye (or a visual colorimeter), comparing the color depth of the solution, or performing measurement with an electro-colorimeter. The method has the advantages of low analysis cost, high detection speed, no need of special instruments and the like, and is suitable for high-throughput analysis. Gold nanoparticles have high molar absorption, and in recent years, colorimetric analysis based on color change of gold nanoparticles has shown wide application prospects in detection of metal ions, proteins, nucleic acids and biological small molecules, drug screening, environmental monitoring and the like. The polypeptide with a specific sequence can cause gold nanoparticles to agglomerate and change color before (or after) being cut by protease, and colorimetric analysis of protease can be realized based on the principle. However, such detection methods often require careful design of the polypeptide sequence, for example, by introducing specific amino acids (e.g., cysteine, positively charged amino acids, etc.) into the polypeptide sequence. However, cysteine is easily oxidized and easily reacts with a disulfide bond in a protease to affect the activity of the protease. In addition, some components (such as nucleic acid, protein, sulfhydryl compound, inorganic salt and the like) in the biological matrix sample are easily adsorbed to the surface of the gold nanoparticles, so that the stability of the gold nanoparticles is influenced, and the biological matrix sample has large interference on the experiment of polypeptide-induced color change of the gold nanoparticles; the color and UV absorption of some biological matrix samples (such as blood) themselves also severely affect the visual detection of proteases. Therefore, it is necessary to develop a simple and sensitive colorimetric assay method with strong anti-interference capability and high throughput for visual assay and detection of protease.
Disclosure of Invention
The invention aims to overcome the problems in the conventional protease detection and provides a polypeptide functionalized magnetic ball for protease detection and a colorimetric detection method.
In order to realize the purpose of the invention, the following technical scheme is adopted: the polypeptide functionalized magnetic ball for detecting the protease comprises a magnetic ball, a layer of gold is wrapped outside the magnetic ball, and the polypeptide is fixed on the gold, wherein the sequence characteristic of the polypeptide is as follows: can be cleaved by a corresponding protease, and the third amino acid of the cleavage site in the direction of the carbon terminal is histidine, and the 5 amino acids of the carbon terminal is PPPPPPC.
The colorimetric detection method for detecting the protease, which adopts the polypeptide functionalized magnetic ball, comprises the following steps:
a: preparation of polypeptide functionalized magnetic spheres: adding a polypeptide into a gold-coated magnetic sphere, wherein the sequence characteristics of the polypeptide are as follows: the magnetic ball can be cut by corresponding protease, the third amino acid of the cutting site towards the carbon end is histidine, the 5 amino acids at the carbon end are PPPPC, the mixture is shaken for 4 to 6 hours, the magnetic separation is carried out, mercaptohexanol solution is added after the mixture is washed by secondary water, the magnetic separation is carried out after the mixture is continuously shaken for a period of time, and the mixture is washed by ethanol, thus obtaining the polypeptide functionalized magnetic ball;
b: and (3) detection of protease: adding acetic acid or phosphoric acid buffer solution of protease to be detected into polypeptide functionalized magnetic beads, reacting for 20-60 minutes, separating by using a magnet, washing with secondary water, and discarding supernatant; then adding a phosphoric acid buffer solution containing copper sulfate, mixing and oscillating, adding a phosphoric acid buffer solution containing ascorbic acid, and oscillating for reaction; magnetic separation, taking out supernatant, adding phosphoric acid buffer solution containing hexadecyl trimethyl ammonium chloride, slowly dropping chloroauric acid water solution, and observing the color change of the solution with naked eyes or measuring the absorption value of the solution at 530 nm with a spectrophotometer.
Further, the gold-coated magnetic ball is prepared by the following method:
(1) preparation of a gold colloid solution with positive charges: adding a mercaptoethylamine aqueous solution into a chloroauric acid solution by taking sodium borohydride as a reducing agent and mercaptoethylamine as a stabilizing agent, oscillating for 20 minutes, slowly dropwise adding a newly prepared sodium borohydride aqueous solution, continuing oscillating for about 1-3 hours, gradually turning the solution from turbid yellow brown to transparent wine red, stopping oscillating, and obtaining a gold colloid solution with positive charges, and storing the gold colloid solution at 4 ℃ for later use;
(2) preparing a gold colloid-magnetic ball compound: adding the gold colloid solution prepared in the step (1) into the uniformly mixed magnetic ball solution, keeping out of the sun, carrying out mild oscillation reaction, separating by using a magnet, removing supernatant, adding the gold colloid solution again, and repeating the operation until the added gold colloid solution does not fade any more;
(3) preparation of gold-coated magnetic spheres: and (3) adding a mixed solution of polyvinyl alcohol, hydrogen peroxide and chloroauric acid into the gold colloid-magnetic sphere compound obtained in the step (2), keeping away from light, gently shaking for 12 hours, carrying out magnetic separation, and washing for 3 times by using secondary water to obtain the gold-coated magnetic sphere.
The invention has the following advantages and effects: (1) the polypeptide is fixed on the surface of the magnetic ball, and the solution after protease cutting is separated by adopting a magnet, so that the interference of other components in the biological matrix sample can be avoided; (2) the colorimetric analysis method for detecting the protease is established by reducing the chloroauric acid by the ascorbic acid to form a red AuNPs solution in situ, has the advantages of simplicity, sensitivity, intuition, high flux, no need of special instruments and the like, and the detection of different types of protease can be realized by successfully developing the technology.
Drawings
FIG. 1 is a graph of the UV absorbance for various concentrations of β -secretase.
FIG. 2 is the selectivity of the assay.
FIG. 3 is a linear plot of the assay against caspase-3 detection.
Detailed Description
In order to more fully explain the implementation of the present invention, examples of the implementation of the present invention are provided. These examples are merely illustrative of the process and do not limit the scope of the present invention, and the present invention is described by the following examples, but not limited to the following examples, and any modified embodiments are included in the technical scope of the present invention.
The sequence of the polypeptide of the invention is characterized by being capable of being cleaved by a corresponding protease, the corresponding protease is the protease to be detected, and the third amino acid of the cleavage site in the direction of the carbon end is histidine, the 5 amino acids at the carbon end are PPPPPPC. the following examples take β -secretase and caspase-3 as examples to be specifically illustrated, further explanation is given to the figure, FIG. 1 is the relationship between the ultraviolet absorption value of the solution at 530 nM and the β -secretase concentration, β -secretase concentration is 0,1, 5, 10, 25, 50, 75, 100, 125 nM. FIG. 2 is the influence of different components in the biological matrix on the ultraviolet absorption value, corresponding in order from 1 to 11 are blank experiments, 50 ng/mL bovine serum albumin, 50 ng/mL thrombin, 50 ng/mL trypsin, 10 μ M amyloid peptide, 10 μ M dopamine, 50 μ M uric acid, 10 μ M glutathione, 50 μ M ascorbic acid, 10% caspase, 10% 2 ng/mL, 539 2, 5-75 mg-150 nM-150 mg-150 nM-5-mm-5 nM-5-mm-5-mm-K-.
Example 1: preparation of polypeptide functionalized magnetic spheres:
(1) preparation of a gold colloid solution with positive charges: the preparation method of the gold colloid adopts sodium borohydride as a reducing agent and mercaptoethylamine as a protective agent, and comprises the following specific operations: adding 73 mu L of 213 mM mercaptoethylamine aqueous solution into 7.3 mL of 1.42 mM chloroauric acid aqueous solution, shaking for 20 minutes at the rotating speed of 3000 r/min on a shaker, and completely mixing to obtain a solution which is brown; regulating the rotating speed to 500 r/min to ensure that the solution does not splash out of the centrifugal tube, slowly dropwise adding 5 mu L of 10 mM newly prepared sodium borohydride aqueous solution, covering a centrifugal tube cover after dropwise adding, quickly regulating the rotating speed to 3000 r/min, shaking for about 1-3 hours, gradually turning the solution from turbid yellow brown to transparent wine red, stopping shaking to obtain a gold colloid solution with positive charges, and storing at 4 ℃ for later use;
(2) preparing a gold colloid-magnetic ball compound: putting 200 mu L of vibrated and uniformly mixed magnetic sphere solution into a centrifuge tube, adding 3mL of the gold colloid solution with positive charge prepared in the step (1), keeping out of the sun and carrying out mild vibration reaction, putting the gold colloid solution at the bottom of the centrifuge tube by using a magnet, removing supernatant, adding the gold colloid solution again, repeating the operation for 3-5 times until the added gold colloid solution does not fade any more, and finally separating by using the magnet and removing supernatant;
(3) preparation of gold-coated magnetic spheres: and (3) adding 2-4 mL of mixed solution containing 0.01 wt% of polyvinyl alcohol, 4.4 mM of hydrogen peroxide and 2.1 mM of chloroauric acid into the gold colloid-magnetic ball compound obtained in the step (2), keeping away from light, gently shaking for more than 12 hours, carrying out magnetic separation, removing supernatant, and washing with secondary water for three times to obtain the gold-coated magnetic balls (Au-MMBs).
(4) Preparation of polypeptide functionalized magnetic spheres: adding 0.5 mL of 1 mM polypeptide Ac-EVNLDAHFWADRPPPPC (or Ac-GDEVDSGHGPPPPC) into 50 mg of Au-MMBs obtained in the step (3), mixing and shaking for 4-6 hours, performing magnetic separation, and washing with secondary water for three times; and adding 1 mL of 1 mM mercaptohexanol solution, continuing shaking for 15 minutes, performing magnetic separation, washing with ethanol for three times, and removing supernatant to obtain the polypeptide Ac-EVNLDAHFWADRPPPPC (or Ac-GDEVDSGHGPPPPC) functionalized magnetic ball.
EXAMPLE 2 detection of β -secretase
Adding 0.5 mL of acetic acid buffer solution (2 mM, pH 4.5) containing β -secretase with different concentrations to 4 mg of polypeptide Ac-EVNLDAHFWADRPPPPC functionalized magnetic beads, reacting for about 20 minutes, separating with a magnet, washing 3 times with secondary water, discarding the supernatant, adding 0.15 mL of phosphate buffer solution (10 mM, pH 7.2) containing 3.2 μ M copper sulfate, mixing and shaking for 1 minute, adding 0.15 mL of phosphate buffer solution containing 400 μ M ascorbic acid, shaking for 30 minutes, magnetically separating, taking out 0.25 mL of supernatant, adding 0.2 mL of phosphate buffer solution containing 0.5 mM cetyltrimethylammonium chloride, adding 50 μ L of 2 mM chloroauric acid aqueous solution slowly, observing the color change of the solution with the naked eye, or measuring the secreted absorption value of the solution at 530 nM with a spectrophotometer, FIG. 1 is a UV absorption value for β -secretase with different concentrations, from which is shown in the figure, the graph, the larger the concentration of β -secretase is, the absorption value of the secreted enzyme in the AU solution at 530 nM is detected by a linear change in the absorbance range of absorbance of the produced by a high light spectrum analyzer, which is detected by measuring the absorbance of the enzyme produced by a photometer, which is observed by a linear spectrum of the absorbance of the enzyme produced at 530 nM, which is observed by a linear range of the absorbance of the enzyme produced at 530 nM, which is found by a linear range of the absorbance of the enzyme, which is found by a linear range of.
Example 3: selectivity is
Referring to the procedure of example 2, the β -secretase enzyme in step (2) was changed to the substance to be tested, and the conditions of the other steps were not changed, and the experimental results are shown in FIG. 2. As can be seen from the figure, only β -secretase enzyme can cause the solution to turn red, and other substances (including serum) do not cause the solution to turn red, and the change of the ultraviolet absorption value caused by these substances is negligible, indicating that the method has good selectivity for the detection of β -secretase.
Example 4: detection of caspase-3
The results show that the color of the solution is redder the greater the caspase-3 concentration is, and the change in the color of the solution is visually observed when the caspase-3 concentration is greater than 1 ng/mL, FIG. 3 is the UV absorbance for various concentrations of caspase-3, and it can be seen that the absorbance increases with increasing caspase-3 concentration, the linear range is 0.5-50 ng/mL, the detection limit is 0.2 ng/mL, when a control experiment is performed using the magnetic spheres functionalized with the polypeptide of example 2, no significant change in the red and absorbance of the solution is observed, because the polypeptide of example 2 cannot be cleaved by the caspase-3, and thus the magnetic spheres are converted into magnetic spheres for detecting the polypeptide fragments corresponding to the AC-GDEVDSGHGPPPPC, and the polypeptide fragments can be analyzed by a method of magnetic bead GDEVDSGHGPPPPC method if the polypeptide fragments are used for detecting the polypeptide fragments.
The mechanism discussion of the invention is as follows: the ascorbic acid can reduce chloroauric acid into nanogold (AuNPs) to form a red solution, but copper ions can catalyze the oxidation of the ascorbic acid, so that the AuNPs are not favorably formed; however, when the polypeptide on the surface of the magnetic bead is cleaved by the protease, the polypeptide fragment DAHFWADRPPPPC (or SGHGPPPPC) remaining on the surface of the magnetic bead will complex copper ions, and the formed copper ion complex cannot catalyze the oxidation of ascorbic acid, so that the ascorbic acid in the solution can reduce chloroauric acid to form a red AuNPs solution. This change can be monitored visually or by a spectrophotometer. The specific technical scheme is as follows: adding protease solutions with different concentrations into polypeptide functionalized magnetic beads, reacting for 20-60 minutes, performing magnetic separation, washing for 3 times with secondary water, and removing supernatant; then adding a copper sulfate solution, mixing and oscillating for 1 minute, adding an ascorbic acid solution, and oscillating for reaction for 30 minutes; magnetic separation, taking supernatant liquid, adding the supernatant liquid into a solution containing hexadecyl trimethyl ammonium chloride, and then slowly dripping chloroauric acid solution. Finally, the solution was observed visually for color change, or the absorbance of the solution at 530 nm was measured with a spectrophotometer.
After the embodiments of the present invention have been described in detail, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention, and it is intended that all simple modifications, equivalent changes and modifications made to the above embodiments based on the technical spirit of the present invention shall fall within the technical scope of the present invention, and the present invention shall not be limited to the embodiments illustrated in the description.
Sequence listing
<110> Anyang college of teachers and schools
<120> polypeptide functionalized magnetic ball for protease detection and colorimetric detection method
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<170>SIPOSequenceListing 1.0
<210>1
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<212>PRT
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
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Glu Val Asn Leu Asp Ala His Phe Trp Ala Asp Arg Pro Pro Pro Pro
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Cys
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<211>14
<212>PRT
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
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Gly Asp Glu Val Asp Ser Gly His Gly Pro Pro Pro Pro Cys
1 5 10
Claims (1)
1. The method for detecting the protease by using the polypeptide functionalized magnetic spheres is characterized by comprising the following steps: comprises the following steps
(1) Preparation of gold-coated magnetic spheres: the substeps are as follows:
(1.1) preparation of a positively charged gold colloid solution: adding a mercaptoethylamine aqueous solution into a chloroauric acid solution by taking sodium borohydride as a reducing agent and mercaptoethylamine as a stabilizing agent, oscillating for 20 minutes, slowly dropwise adding a newly prepared sodium borohydride aqueous solution, continuing oscillating for 1-3 hours, gradually changing the solution from turbid yellow brown to transparent wine red, stopping oscillating, and obtaining a gold colloid solution with positive charges, and storing the gold colloid solution at 4 ℃ for later use;
(1.2) preparing a gold colloid-magnetic sphere compound: adding the gold colloid solution prepared in the step (1.1) into the uniformly mixed magnetic ball solution, keeping out of the sun and carrying out mild oscillation reaction, separating by using a magnet, removing supernatant, adding the gold colloid solution again, and repeating the operation until the added gold colloid solution does not fade any more;
(1.3) preparation of gold-coated magnetic spheres: adding a mixed solution of polyvinyl alcohol, hydrogen peroxide and chloroauric acid into the gold colloid-magnetic ball compound obtained in the step (1.2), keeping out of the sun, gently shaking for 12 hours, carrying out magnetic separation, and washing for 3 times by using secondary water to obtain a gold-coated magnetic ball;
(2) preparation of polypeptide functionalized magnetic spheres: adding a polypeptide into a gold-coated magnetic sphere, wherein the sequence characteristics of the polypeptide are as follows: the target protease can be used for cutting, the third amino acid from the cutting site to the carbon end is histidine, the 5 amino acids from the carbon end are PPPPPPC, the cut carbon end is reserved on the surface of the magnetic sphere, the mixture is shaken for 4 to 6 hours, and the magnetic separation is carried out for three times by using secondary water; adding 1 mM mercaptohexanol solution, continuing to vibrate for 15 minutes, performing magnetic separation, and washing for 3 times by using ethanol to obtain a polypeptide functionalized magnetic ball;
(3) colorimetric detection: adding acetic acid or phosphoric acid buffer solution of protease to be detected into the polypeptide functionalized magnetic ball, reacting for 20-60 minutes, separating by using a magnet, washing by using secondary water, and discarding supernatant; then adding a phosphoric acid buffer solution of copper sulfate, mixing and oscillating, adding a phosphoric acid buffer solution of ascorbic acid, and oscillating for reaction; magnetic separation, taking out supernatant, adding phosphoric acid buffer solution containing hexadecyl trimethyl ammonium chloride, slowly dropping chloroauric acid water solution, and observing the color change of the solution with naked eyes or measuring the absorption value of the solution at 530 nm with a spectrophotometer.
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| Title |
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| A signal-on electrochemical strategy for protease detection based on the formation of ATCUN-Cu(II);Ning Xia;《Sensors and Actuators B: Chemical》;20160326;第232卷;557–563 * |
| Fluorescent gold clusters as nanosensors for copper ions in live cells;Durgadas C V;《Analyst》;20111231;第136卷(第5期);933-940 * |
| Ning Xia.A signal-on electrochemical strategy for protease detection based on the formation of ATCUN-Cu(II).《Sensors and Actuators B: Chemical》.2016,第232卷557–563. * |
| 金纳米粒的绿色合成及其在铜离子检测中的应用;何紫君;《化学与生物工程》;20151202(第11期);40-43 * |
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