CN113155788B - C-reactive protein detection reagent and method based on aptamer and quantum dot quenching effect - Google Patents
C-reactive protein detection reagent and method based on aptamer and quantum dot quenching effect Download PDFInfo
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Abstract
The reagent is composed of a biological probe formed by combining C-reactive protein aptamers at two ends, which are respectively modified by sulfydryl and amino, carboxylated quantum dots and gold nanoparticles, and a small-segment complementary DNA chain of the C-reactive protein aptamers; mixing a plurality of C-reactive protein standard solutions with known concentrations with a biological probe, standing and incubating, detecting an emission fluorescence spectrum of the mixed solution under excitation light with a certain wavelength, taking the concentration of the C-reactive protein standard solution as an abscissa, and taking a fluorescence intensity value obtained by detection as an ordinate, and drawing a linear relation curve of the fluorescence intensity and the concentration of the C-reactive protein; finally, detecting the fluorescence intensity in the sample to be detected, and bringing the fluorescence intensity into a linear relation curve to obtain the concentration of the C-reactive protein of the sample to be detected; the invention has the advantages of short detection time, high detection precision, simple detection process and the like.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a C-reactive protein fluorescence detection method based on a nucleic acid aptamer and quantum dot quenching effect.
Background
The C-reactive protein is a cyclic pentameric protein found in human plasma, and when human tissues are stimulated by inflammation such as injury or microbial invasion, a large amount of C-reactive protein is synthesized by hepatocytes, so that the concentration of the C-reactive protein is tens of times or even hundreds of times higher than the normal level. Research shows that C-reactive protein in human serum begins to rise after about 6 to 8 hours of bacterial infection, and the C-reactive protein is quicker than the C-reactive protein in leukocyte reaction, and the concentration can reach the peak value after 24 to 48 hours. As the infective agent is removed, C-reactive protein levels decrease. Therefore, the C-reactive protein level has been used as an important index for the determination of infectious diseases such as acute respiratory infection and intestinal infection. Moreover, the C-reactive protein is also one of the important biomarkers of acute myocardial infarction at present, the C-reactive protein level is increased rapidly in the early onset of the myocardial infarction patients, and the American centers for disease control and prevention show that the C-reactive protein level in serum can be used as the basis for judging the disease risk of the cardiovascular disease patients. Because the change of the C-reactive protein level is often accompanied by acute inflammation, infection, even myocardial infarction and other sudden diseases threatening the life of a patient, and the concentration of the C-reactive protein changes violently along with the condition of the patient, the rapid and accurate detection of the C-reactive protein has great significance for clinical diagnosis and the life health of the patient.
The concentration of the C reactive protein in the serum of a normal human body is usually less than 1 mu g/mL, the C reactive protein detection method which is most widely used clinically at present is enzyme-linked immunosorbent assay, and the detection signal can be greatly amplified by utilizing the traditional double-antibody sandwich principle and the catalytic effect of enzyme, so that higher detection precision can be obtained. However, this detection method usually requires 5 to 10 hours of detection time, and the timeliness of the detection result is crucial for patients with sudden diseases. In recent years, many scientists have also been devoted to studying methods for rapid detection of C-reactive proteins, including electrochemical detection, field effect transistor, surface enhanced raman scattering, and the like. However, these methods often require complicated detection steps and complicated signal readout equipment, have high requirements on the professional skills of detection personnel and the conditions of detection equipment, and are not suitable for clinical detection.
The aptamer is a single-stranded DNA or RNA sequence with a recognition function, is used as an emerging biological recognition sensitive unit, and has incomparable advantages compared with the traditional antibody. The method has the advantages of high affinity, good specificity, wide variety of detected biomolecules, good acid-base environment compatibility, low requirement on storage conditions and far lower price than that of antibodies. Therefore, the aptamer has great application potential in the aspect of rapid and sensitive detection of the biomarker.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a C-reactive protein detection reagent and a method based on an aptamer and quantum dot quenching effect, which realize the rapid and sensitive detection of the C-reactive protein and have the advantages of short detection time, high detection precision, simple detection process and the like.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a C-reactive protein detection reagent based on aptamer and quantum dot quenching effect is composed of a biological probe formed by combining C-reactive protein aptamer with sulfydryl and amino groups respectively modified at two ends, carboxylated quantum dots and gold nanoparticles, and a small segment of complementary DNA chain of the C-reactive protein aptamer.
The DNA sequence of the C-reactive protein aptamer with both ends modified by sulfydryl and amino is 5'-C6-NH2-CGA AGG GGA TTC GAG GGG TGA TTG CGT GCT CCA TTT GGT G-SH-C6-3'; the sequence of a small segment of complementary DNA chain of the aptamer is as follows: 5'-CTC GAATCC CCT TCG-3'.
The preparation method of the biological probe formed by combining the C-reactive protein aptamer with sulfydryl and amino groups for respectively modifying two ends with the carboxylated quantum dots and the gold nanoparticles comprises the following steps:
(1) Mixing an equal volume of 100. Mu. M C reactive protein aptamer with 20mM tris (2-carboxyethyl) phosphine and incubating at room temperature for 30 minutes to reduce disulfide bonds to give a first mixed solution;
(2) Mixing the nano-gold solution with the first mixed solution to ensure that the ratio of the nano-gold to the C-reactive protein aptamer is 1:1-1, adjusting the ratio according to the size of the nano-gold, adding 1M sodium chloride solution until the concentration of sodium chloride is 50mM, oscillating for 1h at room temperature, adding 1M sodium chloride solution again until the concentration of sodium chloride is 100mM, and repeating the operation until the final concentration of sodium chloride reaches 300mM; incubating at room temperature overnight to combine the C-reactive protein aptamer with the gold nanoparticles to obtain a second mixed solution;
(3) Centrifuging the second mixed solution to remove the upper layer transparent solution, dispersing the bottom dark red precipitate in ultrapure water, and repeatedly cleaning to obtain the nanogold-modified C-reactive protein aptamer;
(4) Activating the carboxylated quantum dots by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), mixing with the nano-gold modified C-reactive protein nucleic acid aptamer, and stirring to obtain a third mixed solution;
(5) Mixing and stirring the third mixed solution and the small segment of complementary DNA chain of the C-reactive protein aptamer to ensure that the concentration ratio of the C-reactive protein aptamer to the small segment of complementary DNA chain of the aptamer is 1:1 to obtain a fourth mixed solution;
(6) And (3) centrifugally filtering the fourth mixed solution by using an ultrafiltration centrifugal tube, and then cleaning for more than three times to obtain the biological probe formed by combining the C-reactive protein aptamer, the carboxylated quantum dot and the nano-gold particle, wherein the sulfydryl and the amino are respectively used for modifying the two ends of the C-reactive protein aptamer, and the carboxylated quantum dot and the nano-gold particle are combined.
The preparation method of the nano gold solution comprises the following steps: 100mL of HAuCl 4 The solution was heated to 80 ℃ under reflux and stirred, followed by the addition of 10mL of 38.8mM sodium citrate solution and stirring until the solution became deep red.
The detection method of the C-reactive protein detection reagent based on the aptamer and quantum dot quenching effect comprises the following steps:
(1) Mixing a plurality of C-reactive protein standard solutions with known concentrations, C-reactive protein aptamers and biological probes formed after the carboxylated quantum dots are combined with the gold nanoparticles to 1mL, and standing and incubating to obtain a fifth mixed solution;
(2) Detecting an emission fluorescence spectrum of the fifth mixed solution under the irradiation of exciting light with a certain wavelength, taking the concentration of the C-reactive protein standard solution as an abscissa, and taking a detected fluorescence intensity value as an ordinate to draw a linear relation curve of the fluorescence intensity and the concentration of the C-reactive protein;
(3) And (3) detecting the concentration of the C-reactive protein in the sample to be detected by the methods in the step (1) and the step (2), and substituting the measured fluorescence intensity into the linear relation curve in the step (2) to obtain the concentration of the C-reactive protein in the sample to be detected.
The wavelength of the excitation light with the certain wavelength is the excitation wavelength of the quantum dots.
The fluorescence intensity value is the fluorescence intensity at the peak of the emission fluorescence spectrum.
The principle of the invention is as follows: the detection method of the invention is based on the Fluorescence Resonance Energy Transfer (FRET) effect, which depends on the distance between the FRET donor-acceptor pair. Quantum dots are nanoscale zero-dimensional semiconductors that emit fluorescent light at a specific frequency by applying a certain excitation light to the semiconductor material. In the invention, the quantum dot is used as a donor, the nanogold is used as an acceptor, and when the quantum dot and the acceptor are far away from each other, the quantum dot can emit fluorescence under an excitation state; when the distance between the quantum dot and the nano-gold is close enough, electron transfer can occur between the quantum dot and the nano-gold, so that fluorescence quenching of the quantum dot is caused. When the biological probe formed by combining the C-reactive protein aptamer and the carboxylated quantum dot with the gold nanoparticles meets the C-reactive protein in the detected sample, the secondary structure of the C-reactive protein aptamer is changed when the C-reactive protein aptamer is combined with the C-reactive protein, the distance between the donor quantum dot and the acceptor gold nanoparticles at two ends of the C-reactive protein aptamer is shortened, and therefore fluorescence emitted by the quantum dots is quenched and reduced; the effect of introducing the small complementary DNA strand of the C-reactive protein aptamer is to stretch the ring structure on the C-reactive protein aptamer through the complementary DNA, so that the initial distance between the quantum dot and the nanogold is maximized, and thus a larger fluorescence intensity change is obtained. After the C-reactive protein appears, the aptamer can be separated from complementary DNA and combined with the C-reactive protein due to stronger affinity between the aptamer of the C-reactive protein and the C-reactive protein; by detecting the fluorescence change of the biological probe, the concentration of the C-reactive protein can be deduced.
The detection method provided by the invention has the beneficial effects that:
(1) The invention utilizes the C-reactive protein aptamer as a biomolecule sensitive unit and a 'nano ruler' between FRET donor-acceptor pairs, and the concentration of the target is detected through the change of the fluorescence intensity of quantum dots caused by the change of secondary structures before and after the C-reactive protein aptamer is combined with the target; the detection process of the detection method can be finished within 40 minutes, and compared with the currently widely used enzyme-linked immunosorbent assay, the detection time is greatly shortened, thereby being beneficial to the rapid diagnosis of patients with sudden diseases.
(2) The preparation method of the biological probe formed by combining the C-reactive protein aptamer, the carboxylated quantum dot and the gold nanoparticles is simple, the biological compatibility is good, the detection process is completed in one step, and the requirement on the professional skill of a detector is not high.
(3) The invention adopts a fluorescence detection method, and is easy to integrate on a chip and a test strip. The signal reading device is relatively simple and has a great potential for application to point-of-care testing (POCT).
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FIG. 1 is a schematic structural diagram of a biological probe formed by combining a C-reactive protein aptamer and a carboxylated quantum dot of example 1 of the present invention with a gold nanoparticle before and after detection of the C-reactive protein.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Embodiment 1, a C-reactive protein detection reagent based on aptamer and quantum dot quenching effect, which is composed of a biological probe formed by combining a C-reactive protein aptamer with thiol and amino groups respectively modified at two ends, a carboxylated graphene quantum dot and a gold nanoparticle, and a small segment of complementary DNA strand of the aptamer.
The DNA sequence of the C-reactive protein aptamer with both ends modified by sulfydryl and amino is 5'-C6-NH2-CGA AGG GGA TTC GAG GGG TGA TTG CGT GCT CCA TTT GGT G-SH-C6-3'; the sequence of the small complementary DNA chain of the C-reactive protein aptamer is as follows: 5'-CTC GAATCC CCT TCG-3'.
The preparation method of the biological probe formed by combining the C-reactive protein aptamer with sulfydryl and amino groups for respectively modifying two ends with the carboxylated graphene quantum dot and the gold nanoparticle comprises the following steps:
(1) Preparing a nanogold-modified C-reactive protein aptamer: mixing an equal volume of 100. Mu.M of C-reactive protein aptamer with 20mM tris (2-carboxyethyl) phosphine and incubating for 30 minutes at room temperature to reduce disulfide bonds, resulting in a first mixed solution;
(2) A200 ml two-neck flask and a magnetic stirring rod were placed in aqua regia (HCl/HNO) 3 3:1) for at least 15 minutes and then rinsed with copious amounts of ultrapure water; 100ml of 1mM HAuCl was added to a two-necked flask 4 Placing the flask on a heating plate for reflux stirring, quickly adding 10ml of 38.8mM sodium citrate solution when the solution begins to reflux, continuously heating and refluxing for 20 minutes after the color changes from light yellow to dark red, stopping heating, cooling to room temperature under stirring, and storing at 4 ℃ in a dark place to obtain the nano gold solutionLiquid;
mixing 40 mu L of the first mixed solution with 1mL of nano-gold solution to ensure that the ratio of nano-gold to the C-reactive protein aptamer is about 1; after further incubation of the solution for 1 hour or more, adding again a NaCl solution, increasing the NaCl concentration by 50mM, and finally reaching 300mM; further incubating overnight to obtain a second mixed solution;
(3) Centrifuging the second mixed solution at 14000rpm for 20 minutes, removing the upper transparent solution, repeating the centrifugal cleaning for three times, and dispersing the bottom dark red precipitate into 1mL of ultrapure water to obtain the nanogold-modified C-reactive protein aptamer;
(4) Adding a mixture of 19mg of EDC and 22mg of NHS into a 0.1mg/mL graphene quantum dot solution, stirring for 1 hour to activate carboxyl, then mixing with the nanogold-modified C-reactive protein aptamer obtained in the step (3), and stirring for 2 hours to obtain a third mixed solution;
(5) Mixing the third mixed solution with 20 mu L of small-section complementary DNA of 100 mu M C-reactive protein aptamer and stirring for 30 minutes to ensure that the concentration ratio of the C-reactive protein aptamer to the small-section complementary DNA of the aptamer is 1:1 to obtain a fourth mixed solution;
(6) And (3) centrifugally filtering the fourth mixed solution by using a 30kDa ultrafiltration centrifugal tube at the rotation speed of 4000rpm, washing the fourth mixed solution for more than three times by using ultrapure water, and dispersing the supernatant into 0.1M PBS buffer solution (pH = 7.4) to obtain 2mL of biological probe formed by combining the C-reactive protein aptamer, the sulfydryl of which is modified by amino, of which two ends are modified by the amino, with the carboxylated graphene quantum dot and the gold nanoparticle.
A detection method of a C-reactive protein detection reagent based on a nucleic acid aptamer and quantum dot quenching effect is adopted, and the specific steps are as follows:
(1) Preparing standard solutions of C-reactive protein with different known concentrations by using 0.1M PBS (pH = 7.4), mixing the standard solutions with 0.1mL of biological probe reagents formed by combining aptamer of C-reactive protein with sulfydryl and amino groups respectively modified at two ends and carboxylated graphene quantum dots and gold nanoparticles, diluting the mixture with 0.1M PBS (pH = 7.4) to obtain 1mL of solution, and standing the solution for 30 minutes to obtain a fifth mixed solution;
(2) Placing the fifth mixed solution in a quartz cuvette, detecting the fluorescence spectrum of the fifth mixed solution under 355nm laser irradiation by using a spectrophotometer, taking the concentration of the C-reactive protein standard solution as a horizontal coordinate, taking the fluorescence intensity value at the position of 450nm of a peak value as a vertical coordinate, and drawing a linear relation curve of the fluorescence intensity and the concentration of the C-reactive protein;
(3) And (3) detecting the concentration of the C-reactive protein in the sample to be detected by the methods in the steps (1) and (2), and substituting the measured fluorescence intensity into the linear relation curve in the step (2) to obtain the concentration of the C-reactive protein in the sample to be detected.
Referring to fig. 1, fig. 1 is a schematic structural diagram of a biological probe formed after a C-reactive protein aptamer and a carboxylated quantum dot are combined with a gold nanoparticle according to example 1 of the present invention before and after detection of the C-reactive protein.
Example 2, a C-reactive protein detection reagent based on the quenching effect of aptamers and quantum dots, comprising a biological probe formed by combining a C-reactive protein aptamer, a carboxylated CdSe/ZnS quantum dot and a gold nanoparticle, both ends of which are modified with a thiol group and an amino group, and a small segment of complementary DNA strand of the aptamer.
The DNA sequence of the C-reactive protein aptamer with both ends modified by sulfydryl and amino is 5'-C6-NH2-CGA AGG GGA TTC GAG GGG TGA TTG CGT GCT CCA TTT GGT G-SH-C6-3'; the small-segment complementary DNA chain sequence of the C-reactive protein aptamer is as follows: 5'-CTC GAATCC CCT TCG-3'.
The preparation method of the biological probe formed by combining the C-reactive protein aptamer with sulfydryl and amino groups for respectively modifying two ends, the carboxylated CdSe/ZnS quantum dot and the gold nanoparticle comprises the following steps of:
(1) Preparing a nanogold-modified C-reactive protein aptamer: mixing an equal volume of 100. Mu.M of C-reactive protein aptamer with 20mM tris (2-carboxyethyl) phosphine and incubating at room temperature for 30 minutes to reduce disulfide bonds, resulting in a first mixed solution;
(2) Placing a 200 ml two-neck flask and a magnetic stirring rodIn aqua regia (HCl/HNO) 3 3:1) for at least 15 minutes and then rinsed with copious amounts of ultrapure water; 100ml of 1mM HAuCl was added to a two-necked flask 4 Placing the flask on a heating plate for reflux stirring, quickly adding 10ml of 38.8mM sodium citrate solution when the solution starts to reflux, continuously heating and refluxing for 20 minutes after the color is changed from light yellow to dark red, stopping heating, cooling to room temperature under stirring, and keeping the solution at 4 ℃ in a dark place to obtain a nanogold solution;
mixing 20 mu L of the first mixed solution with 1mL of a nanogold solution to ensure that the ratio of the nanogold to the C-reactive protein aptamer is about 1 to 75, incubating at room temperature for more than 1 hour, then dropwise adding 1MNaCl and gently oscillating simultaneously to ensure that the concentration of NaCl reaches 50mM; after further incubation of the solution for 1 hour or more, adding again a NaCl solution, increasing the NaCl concentration by 50mM, and finally reaching 300mM; further incubating overnight to obtain a second mixed solution;
(3) Centrifuging the second mixed solution at 14000rpm for 20 minutes, removing the upper transparent solution, repeating the centrifugal cleaning for three times, and dispersing the bottom dark red precipitate into 1mL of ultrapure water to obtain the nanogold-modified C-reactive protein aptamer;
(4) Adding a mixture of 19mg of EDC and 22mg of NHS to 1mL of 8. Mu.M CdSe/ZnS quantum dot solution, stirring for 1 hour to activate carboxyl, followed by mixing with the nanogold-modified C-reactive protein aptamer obtained in step (3) and stirring for 2 hours to obtain a third mixed solution;
(5) Mixing the third mixed solution with 20 mu L of the small segment complementary DNA of the C-reactive protein aptamer with the concentration ratio of the C-reactive protein aptamer to the small segment complementary DNA strand of the aptamer of 1:1 by stirring for 30 minutes to obtain a fourth mixed solution;
(6) And (3) centrifugally filtering the fourth mixed solution by using a 30kDa ultrafiltration centrifugal tube at the rotating speed of 4000rpm, washing the fourth mixed solution for more than three times by using ultrapure water, and dispersing the supernatant into 0.1M PBS buffer solution (pH = 7.4) to obtain 2mL of biological probe formed by combining the C-reactive protein aptamer, the sulfydryl of which and the amino of which are respectively modified at two ends, and the carboxylated CdSe/ZnS quantum dot and the nanogold particle.
A detection method of a C-reactive protein detection reagent based on a nucleic acid aptamer and quantum dot quenching effect is adopted, and the specific steps are as follows:
(1) Preparing standard solutions of C-reactive protein with different known concentrations by using 0.1M PBS buffer (pH = 7.4), mixing the standard solutions with 0.1mL of C-reactive protein aptamers of which the sulfydryl and the amino are respectively modified at two ends, biological probe reagents formed by combining carboxylated CdSe/ZnS quantum dots and gold nanoparticles, diluting the mixed solution with 0.1M PBS (pH = 7.4) to obtain 1mL of solution, and standing the solution for 30 minutes to obtain a fifth mixed solution;
(2) Placing the fifth mixed solution in a quartz cuvette, detecting the fluorescence spectrum of the fifth mixed solution under 375nm laser irradiation by using a spectrophotometer, taking the concentration of the C-reactive protein standard solution as a horizontal coordinate, taking the fluorescence intensity value at the peak value of 620nm as a vertical coordinate, and drawing a linear relation curve of the fluorescence intensity and the C-reactive protein concentration;
(3) And (3) detecting the concentration of the C-reactive protein in the sample to be detected by the methods in the steps (1) and (2), and substituting the measured fluorescence intensity into the linear relation curve in the step (2) to obtain the concentration of the C-reactive protein in the sample to be detected.
The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments, and any other changes, modifications, combinations, and simplifications which do not depart from the spirit and scope of the present invention should be construed as being equivalent substitutions and shall be included within the protection scope of the present invention.
Sequence listing
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Claims (8)
1. A C-reactive protein detection reagent based on aptamer and quantum dot quenching effect is characterized in that: the biological probe is formed by combining a C-reactive protein nucleic acid aptamer with sulfydryl and amino groups for respectively modifying two ends, a carboxylated quantum dot and a nano gold particle, and a small segment of complementary DNA chain of the C-reactive protein nucleic acid aptamer;
the preparation method of the biological probe comprises the following steps:
(1) Mixing an equal volume of 100. Mu.MC-reactive protein aptamer with 20mM tris (2-carboxyethyl) phosphine and incubating at room temperature for 30 minutes to reduce disulfide bonds, resulting in a first mixed solution;
(2) Mixing the nano-gold solution with the first mixed solution to ensure that the ratio of the nano-gold to the C-reactive protein aptamer is 1:1-1, adjusting the ratio according to the size of the nano-gold, adding 1M sodium chloride solution until the concentration of sodium chloride is 50mM, oscillating for 1h at room temperature, adding 1M sodium chloride solution again until the concentration of sodium chloride is 100mM, and repeating the operation until the final concentration of sodium chloride reaches 300mM; incubating at room temperature overnight to combine the C-reactive protein aptamer with the gold nanoparticles to obtain a second mixed solution;
(3) Centrifuging the second mixed solution to remove the upper layer transparent solution, dispersing the bottom dark red precipitate in ultrapure water, and repeatedly cleaning to obtain the nanogold-modified C-reactive protein aptamer;
(4) Activating the carboxylated quantum dots by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), mixing with the nano-gold modified C-reactive protein nucleic acid aptamer, and stirring to obtain a third mixed solution;
(5) Mixing and stirring the third mixed solution and the small segment of complementary DNA chain of the C-reactive protein aptamer to ensure that the concentration ratio of the C-reactive protein aptamer to the small segment of complementary DNA chain of the aptamer is 1:1 to obtain a fourth mixed solution;
(6) And (3) centrifugally filtering the fourth mixed solution by using an ultrafiltration centrifugal tube, and then cleaning for more than three times to obtain the biological probe formed by combining the C-reactive protein aptamer, the carboxylated quantum dot and the gold nanoparticle, wherein the sulfydryl and the amino are respectively modified at two ends of the C-reactive protein aptamer.
2. The detection reagent according to claim 1, wherein: the DNA sequence of the C-reactive protein aptamer with both ends modified by sulfydryl and amino is 5'-C6-NH2-CGA AGG GGA TTC GAG GGG TGA TTG CGT GCT CCA TTT GGT G-SH-C6-3'; the sequence of a small segment of complementary DNA chain of the aptamer is as follows: 5'-CTCGAATCCCCTTCG-3'.
3. The detection reagent according to claim 1, wherein: the preparation method of the nano gold solution comprises the following steps: 100mL of HAuCl 4 The solution was heated to 80 ℃ under reflux and stirred, followed by the addition of 10mL of 38.8mM sodium citrate solution and stirring until the solution became deep red.
4. The method for detecting the C-reactive protein detection reagent based on the aptamer and quantum dot quenching effect, which is characterized by comprising the following steps of:
(1) Mixing a plurality of C-reactive protein standard solutions with known concentrations, C-reactive protein aptamers and biological probes formed after the carboxylated quantum dots are combined with the gold nanoparticles to 1mL, and standing and incubating to obtain a fifth mixed solution;
(2) Detecting an emission fluorescence spectrum of the fifth mixed solution under the irradiation of exciting light with a certain wavelength, taking the concentration of the C-reactive protein standard solution as an abscissa, and taking a detected fluorescence intensity value as an ordinate to draw a linear relation curve of the fluorescence intensity and the concentration of the C-reactive protein;
(3) And (3) detecting the concentration of the C-reactive protein in the sample to be detected by the methods in the step (1) and the step (2), and substituting the measured fluorescence intensity into the linear relation curve in the step (2) to obtain the concentration of the C-reactive protein in the sample to be detected.
5. The detection method according to claim 4, characterized in that: the wavelength of the excitation light with the certain wavelength is the excitation wavelength of the quantum dots.
6. The detection method according to claim 4, characterized in that: the fluorescence intensity value is the fluorescence intensity at the peak of the emission fluorescence spectrum.
7. A C-reactive protein detection reagent based on aptamer and quantum dot quenching effect is characterized in that: the biological probe is formed by combining a C-reactive protein aptamer with sulfydryl and amino groups for respectively modifying two ends, a carboxylated graphene quantum dot and a gold nanoparticle, and a small segment of complementary DNA chain of the aptamer;
the DNA sequence of the C-reactive protein nucleic acid aptamer with both ends modified by sulfydryl and amino is 5'-C6-NH2-CGA AGG GGA TTC GAG GGG TGA TTG CGT GCT CCA TTT GGT G-SH-C6-3'; the small-segment complementary DNA chain sequence of the C-reactive protein aptamer is as follows: 5'-CTCGAATCCCCTTCG-3';
the preparation method of the biological probe formed by combining the C-reactive protein nucleic acid aptamer with the carboxylated graphene quantum dot and the gold nanoparticle, wherein the two ends of the C-reactive protein nucleic acid aptamer are respectively modified by sulfydryl and amino, and comprises the following steps:
(1) Preparing a nanogold-modified C-reactive protein aptamer: mixing an isometric 100 mu M C-reactive protein aptamer with 20mM tris (2-carboxyethyl) phosphine and incubating at room temperature for 30 minutes to reduce disulfide bonds to obtain a first mixed solution;
(2) A200 ml two-neck flask and a magnetic stirring rod were placed in aqua regia (HCl/HNO) 3 3:1) for at least 15 minutes and then rinsed with copious amounts of ultrapure water; adding into a two-neck flask100ml of 1mM HAuCl 4 Placing the flask on a heating plate for reflux stirring, quickly adding 10ml of 38.8mM sodium citrate solution when the solution starts to reflux, continuously heating and refluxing for 20 minutes after the color is changed from light yellow to dark red, stopping heating, cooling to room temperature under stirring, and keeping the solution at 4 ℃ in a dark place to obtain a nanogold solution;
mixing 40 mu L of the first mixed solution with 1mL of a nanogold solution to enable the ratio of the nanogold to the C-reactive protein nucleic acid aptamer to be 1; after further incubation of the solution for 1 hour or more, adding NaCl solution again, increasing the NaCl concentration by 50mM, and finally reaching 300mM; further incubating overnight to obtain a second mixed solution;
(3) Centrifuging the second mixed solution at 14000rpm for 20 minutes, removing the upper transparent solution, repeating the centrifugal cleaning for three times, and dispersing the bottom dark red precipitate into 1mL of ultrapure water to obtain the nanogold-modified C-reactive protein aptamer;
(4) Adding a mixture of 19mg EDC and 22mg NHS into 0.1mg/mL graphene quantum dot solution, stirring for 1 hour to activate carboxyl, then mixing with the nano-gold modified C-reactive protein aptamer obtained in the step (3), and stirring for 2 hours to obtain a third mixed solution;
(5) Mixing the third mixed solution with 20 μ L of the small-section complementary DNA of the 100 μ M C-reactive protein aptamer and stirring for 30 minutes to enable the concentration ratio of the C-reactive protein aptamer to the small-section complementary DNA chain of the aptamer to be 1:1, and obtaining a fourth mixed solution;
(6) And (3) centrifugally filtering the fourth mixed solution by using a 30kDa ultrafiltration centrifugal tube at the rotating speed of 4000rpm, washing the fourth mixed solution for more than three times by using ultrapure water, dispersing the supernatant into 0.1MPBS buffer solution, wherein the pH of the buffer solution is =7.4, and obtaining 2mL of biological probe formed by combining the C-reactive protein aptamer with sulfydryl and amino groups respectively modified at two ends with the carboxylated graphene quantum dots and the gold nanoparticles.
8. The detection method of the C-reactive protein detection reagent based on the aptamer and quantum dot quenching effect, which is characterized by comprising the following steps:
(1) Preparing standard solutions of C-reactive proteins with different known concentrations by using 0.1MPBS buffer solution, enabling the pH of the buffer solution to be =7.4, then mixing the standard solutions with 0.1mL of C-reactive protein nucleic acid aptamers with sulfydryl and amino groups modified at two ends respectively and biological probe reagents formed by combining carboxylated graphene quantum dots and gold nanoparticles, diluting the mixture with 0.1MPBS and pH =7.4 to obtain 1mL of solution, and standing the solution for 30 minutes to obtain a fifth mixed solution;
(2) Placing the fifth mixed solution in a quartz cuvette, detecting the fluorescence spectrum of the fifth mixed solution under 355nm laser irradiation by using a spectrophotometer, taking the concentration of the C-reactive protein standard solution as a horizontal coordinate, taking the fluorescence intensity value at the position of 450nm of a peak value as a vertical coordinate, and drawing a linear relation curve of the fluorescence intensity and the concentration of the C-reactive protein;
(3) And (3) detecting the concentration of the C-reactive protein in the sample to be detected by the methods in the steps (1) and (2), and substituting the measured fluorescence intensity into the linear relation curve in the step (2) to obtain the concentration of the C-reactive protein in the sample to be detected.
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