CN110376382B - Mass spectrometric detection method based on competitive non-covalent interactions for sensitive detection of biomarkers - Google Patents
Mass spectrometric detection method based on competitive non-covalent interactions for sensitive detection of biomarkers Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
Abstract
The invention discloses a mass spectrometric detection method based on competitive non-covalent interactions for sensitive detection of biomarkers. The invention utilizes the advantages of high sensitivity, low detection limit, high flux and capability of providing accurate molecular mass or molecular structure information of mass spectrum detection, and overcomes the defects of low detection sensitivity and poor anti-interference performance of the traditional biomarker. The invention has the advantages of good sensitivity, high responsiveness to trace changes of a detection object and high anti-interference performance to a complex matrix, can be used for detecting samples of actual patients, and provides a reliable analysis means for diagnosis and treatment of clinical cancers.
Description
Technical Field
The invention belongs to the technical field of mass spectrometry detection, and particularly relates to a novel mass spectrometry detection method based on competitive non-covalent interaction for sensitive detection of biomarkers.
Background
Biomarkers are biochemical markers that can mark changes or likely changes in the structure or function of systems, organs, tissues, cells and subcellular cells, and have a wide range of uses. Biomarkers can be used for disease diagnosis, to determine disease stage, or to evaluate the safety and effectiveness of new drugs or therapies in a target population. But is difficult to detect effectively and sensitively due to the complex and diverse structure.
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely applied to analysis and detection due to high sensitivity, low detection limit, high flux and capability of providing accurate molecular mass or molecular structure information. MALDI-TOF MS can analyze not only inorganic molecules, polymers, but also macromolecules such as nucleic acids and proteins. When some analytes cannot be detected directly by MALDI-TOF MS, an easy desorption ionization mass label is usually introduced for indirect detection. Based on the advantages, the mass spectrum has potential value in the detection of the biomarker as an effective analysis method.
Therefore, it is important to develop a new detection method based on MALDI-TOF MS to improve the detection sensitivity of the biomarker.
Disclosure of Invention
The invention aims to provide a mass spectrometric detection method based on competitive non-covalent interactions for sensitive detection of biomarkers.
The mass spectrum detection method based on the competitive non-covalent interaction for sensitive detection of the biomarkers comprises the following steps:
1) designing a nucleic acid aptamer according to the biomarker to be detected;
2) incubating the aptamer and the gold nanoparticles together to obtain an aptamer-gold nanoparticle combination;
3) respectively incubating and separating the aptamer-gold nanoparticle combination with a series of biomarker standard substances with the existing concentration and a fixed amount of mass labels, respectively carrying out matrix-assisted laser desorption ionization time-of-flight mass spectrometry on the obtained solid to obtain a series of signals of the mass labels combined with the gold nanoparticles corresponding to the biomarkers with the known concentration, and establishing a corresponding relation between the concentration of the standard biomarkers and the signals of the mass labels;
4) incubating the biomarker with unknown concentration with the aptamer-gold nanoparticle combination and the mass label in the step 2), separating, carrying out matrix-assisted laser desorption ionization time-of-flight mass spectrometry on the obtained solid to obtain a signal of the mass label combined with the gold nanoparticle corresponding to the biomarker with unknown concentration, and obtaining the concentration of the biomarker according to the corresponding relation between the concentration of the standard biomarker and the signal of the mass label in the step 3).
In the above method, the biomarker may specifically be an antigen specific to prostate cancer.
The prostate cancer specific antigen is a single-chain polypeptide containing 237 amino acids, belongs to a serine protease family with tissue specificity and chymotrypsin-like effect, can decompose main colloidal protein in semen and has the effect of diluting semen. The prostate cancer specific antigen has tissue specificity, is only present in cytoplasm of human prostate acinus and duct epithelial cells, is not expressed in other cells, and is a common prostate cancer diagnosis marker in clinic at present.
When the biomarker to be detected is a prostate cancer specific antigen, the sequence of the aptamer is CGT CGT ATT AAA GCT CGC CAT CAA ATA GCT TT.
In the method, the particle size of the gold nanoparticles is distributed between 11 nm and 17nm, and the average size is 13 nm; the UV characteristic absorption maximum is at a wavelength of 520 nm.
The incubation temperature of the aptamer and the gold nanoparticles can be room temperature, and the incubation time can be 12-24 h.
The interaction of the nucleic acid aptamer and the gold nanoparticle can be enhanced by performing the reaction under the acidic pH condition.
The interaction of the aptamer with the gold nanoparticle is stronger in a buffer solution than in an aqueous environment.
The interaction maximum loading rate of the aptamer and the gold nanoparticle is 65%.
In the above method, the mass label is a substance having good resolving and ionizing properties and capable of generating a signal in MALDI MS.
In particular, the mass label may be adenine, the amino group at position 2 of which has a non-covalent interaction with the gold nanoparticles.
In step 3), the concentration of the series of biomarker standards with known concentrations may be: 0.06,0.3,0.6,3,6 ng/mL.
The temperature for incubating the aptamer-gold nanoparticle combination, the biomarker and the quality label can be room temperature, and the time can be 1-3h, specifically 2 h.
In the method, the interaction between the aptamer and the gold nanoparticles and the interaction between the mass label and the gold nanoparticles can at least bear the rotation speed of 12000rpm, namely, centrifugal separation is adopted during separation, and the rotation speed is not higher than 12000 rpm.
In the above method, the conditions of the mass spectrometry are as follows: voltage: acceleration voltage: 19.000 kV; delayed extraction voltage: 14.920 kV; voltage of the reflector: 20.000 kV; lens voltage: 7.000 kV; frequency: 1.000 Hz; laser energy: 20 percent; the accumulation times are as follows: 100 times; positive ion mode.
The invention can be used for sensitively detecting the prostate cancer specific antigen, and because the aptamer is single-stranded DNA with 32 bases, the gold nanoparticle surface site occupied by one aptamer can be filled by a plurality of adenine mass labels, and the invention has the effect of signal amplification in mass spectrum detection;
in the above method, the antigen for prostate cancer is specifically and specifically detected, and the interference of most proteins in human body fluid, such as human serum albumin, transferrin, immunoglobulin G, etc., can be eliminated.
In the method, the detection of the prostate specific antigen is low, the sensitivity is high, and when the concentration of the prostate specific antigen is as low as 0.06ng/mL, a mass spectrum signal is still obvious.
In the method, the micro-change of the prostate specific antigen has obvious response, and when the concentration of the prostate specific antigen is changed from 0.06ng/mL to 6ng/mL, the mass spectrum signal is enhanced by 90 times.
The invention can detect the prostate cancer specific antigen in complex clinical samples, such as patient urine, and meanwhile, the urine of normal people does not have false positive results.
In the method, compared with the commercial enzyme-linked immunosorbent assay kit, the prostate specific antigen in the actual patient sample is determined to have higher coincidence.
The invention technically overcomes the problems of low detection sensitivity and large interference of complex matrixes of the biomarkers, amplifies mass spectrum signals by designing a novel mass spectrum detection method based on competitive non-covalent interaction, and utilizes the advantages of high sensitivity and low detection limit of mass spectrum to realize sensitive detection of the biomarkers. The invention has strong anti-interference capability to a complex system, is not interfered by other proteins in human body fluid, and can be used for detecting the prostate cancer specific antigen in a clinical practical patient sample; the invention provides a universal mass spectrometry method which can be used for detecting a plurality of other biomarkers.
Drawings
FIG. 1 is a transmission electron micrograph of gold nanoparticles.
Fig. 2 is a graph of the ultraviolet absorption of gold nanoparticles.
FIG. 3 is a graph showing the loading distribution of aptamers and gold nanoparticles at different concentrations.
FIG. 4 is a graph showing the distribution of the loading rate of aptamers and gold nanoparticles at different concentrations.
FIG. 5 is a mass spectrum of the signal intensity of the adenine mass tag versus the change in concentration of prostate cancer specific antigen.
FIG. 6 shows the interference test of the present invention with other proteins in human body.
FIG. 7 is a linear equation after the introduction of an internal standard according to the present invention.
FIG. 8 is a chart of the recovery rate in normal human urine for the present invention.
FIG. 9 is a comparison of the present invention and a commercial ELISA kit for detection of prostate cancer specific antigens in patient samples.
Detailed Description
The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.
The samples used in the following examples of the present invention were purchased from sigma.
The matrix assisted laser desorption ionization time-of-flight mass spectrometer used in the following examples of the invention was model number BIFLEXTM III (Bruker).
Example 1 Synthesis of gold nanoparticles
50mL of boiling water was added with 2mL of 1% HAuCl4Boiling (by refluxing), adding 1mL 5% trisodium citrate (0.2877g Na) under rapid stirring3Ct·2H2And (4) keeping the volume of O to 5mL), continuously heating until the color is changed from yellow-colorless-blue black-deep black-wine red within 5min to be stable, heating for 5min again, stopping heating, and continuously stirring to room temperature until the particle size is 13 nanometers.
FIG. 1 is a transmission electron micrograph of gold nanoparticles.
Fig. 2 is a graph of the ultraviolet absorption of gold nanoparticles.
Example 2 detection of prostate cancer-specific antigen Standard
mu.L of 1.3nM gold nanoparticle solution was mixed with 10,50,100 and 150. mu.L of 1. mu.M aptamer (sequence CGT CGT ATT AAA GCT CGC CAT CAA ATA GCT TT, available from bioengineering (Shanghai) Co., Ltd.) respectively, reacted overnight at room temperature, and the loading and loading rate profiles of the aptamer and gold nanoparticles at different concentrations were examined.
FIG. 3 is a graph showing the loading distribution of aptamers and gold nanoparticles at different concentrations.
FIG. 4 is a graph showing the distribution of the loading rate of aptamers and gold nanoparticles at different concentrations.
Finally, 200 μ L of 1.3nM gold nanoparticle solution is mixed with 50 μ L of 1 μ M aptamer, reacted overnight at room temperature, centrifuged at 12000rpm for 10min to discard the supernatant, the pellet is washed twice with water, adenine mass tag (10 μ L1 μ M) and prostate cancer specific antigen standard solution are added to give final concentrations of 0.06,0.3,0.6,3,6ng/mL, reacted at room temperature for 2 hours, centrifuged at 12000rpm for 10min to discard the supernatant, the pellet is washed twice with water, 1 μ L of the pellet is mixed with 1 μ L of matrix (CCA) and added to a MALDI target plate, dried in air and subjected to mass spectrometry.
The mass spectrum conditions are as follows: voltage: acceleration voltage: 19.000 kV; delayed extraction voltage: 14.920 kV; voltage of the reflector: 20.000 kV; lens voltage: 7.000 kV; frequency: 1.000 Hz; laser energy: 20 percent; the accumulation times are as follows: 100 times; positive ion mode.
FIG. 5 is a mass spectrum of the signal intensity of the adenine mass tag versus the change in concentration of prostate cancer specific antigen.
Example 3 interference test
Mixing 200 mu L of 1.3nM gold nanoparticle solution with 50 mu L of 1 mu M aptamer, reacting at room temperature overnight, centrifuging at 12000rpm for 10min to remove supernatant, washing the precipitate twice, adding adenine mass tag (10 mu L of 1 mu M) and 10 mu L of 6ng/mL interference protein (human serum protein, transferrin, immunoglobulin, bovine serum and mixture of bovine serum and prostate cancer specific antigen) to react at room temperature for 2 hours, centrifuging at 12000rpm for 10min to remove supernatant, washing the precipitate twice, mixing 1 mu L of precipitate with 1 mu L of matrix (CCA), adding the mixture to a MALDI target plate, drying in air, and performing mass spectrometry.
The mass spectrum conditions are as follows: voltage: acceleration voltage: 19.000 kV; delayed extraction voltage: 14.920 kV; voltage of the reflector: 20.000 kV; lens voltage: 7.000 kV; frequency: 1.000 Hz; laser energy: 20 percent; the accumulation times are as follows: 100 times; positive ion mode.
FIG. 6 shows the interference test of the present invention with other proteins in human body.
Example 4 recovery test
Mixing 200 mu L of 1.3nM gold nanoparticle solution with 50 mu L of 1 mu M aptamer, reacting at room temperature overnight, centrifuging at 12000rpm for 10min to discard the supernatant, washing the precipitate twice with water, adding prostate cancer specific antigen (final concentration is 0, 3,6, 30, 60ng/mL respectively) and adenine mass label (10 mu L of 1 mu M), reacting at room temperature for 2 hours, centrifuging at 12000rpm for 10min to discard the supernatant, washing the precipitate twice with water, taking 1 mu L of precipitate and internal standard substance 3-methyladenine (1 mu L of 1 mu M), mixing and adding 1 mu L of matrix (CCA) to a MALDI target plate, drying in the air, and performing mass spectrometry.
The mass spectrum conditions are as follows: voltage: acceleration voltage: 19.000 kV; delayed extraction voltage: 14.920 kV; voltage of the reflector: 20.000 kV; lens voltage: 7.000 kV; frequency: 1.000 Hz; laser energy: 20 percent; the accumulation times are as follows: 100 times; positive ion mode.
And (3) making a linear curve by using the signal intensity ratio of the adenine mass label to the internal standard substance 3-methyladenine mass spectrum corresponding to the concentration of the prostate cancer specific antigen.
FIG. 7 is a linear equation after the introduction of an internal standard according to the present invention.
mu.L of 1.3nM gold nanoparticle solution was mixed with 50. mu.L of 1. mu.M aptamer, reacted overnight at room temperature, centrifuged at 12000rpm for 10min to discard the supernatant, and the precipitate was washed twice with water. Prostate cancer specific antigen standard is added into urine of normal human, and the final concentration is 0.8, 8 and 80ng/mL respectively. Taking 10 mu L of the urine and 10 mu L (1 mu M) of an adenine mass label to react for 2 hours at room temperature, centrifuging at 12000rpm for 10min, discarding supernatant, washing precipitates twice with water, taking 1 mu L of the precipitates and 3-methyladenine (1 mu L1 mu M) of an internal standard substance, mixing and adding 1 mu L of a matrix (CCA) onto a MALDI target plate, drying in the air, performing mass spectrometry, and calculating the content of the prostate cancer specific antigen in the urine according to the mass spectrum signal intensity ratio of the adenine mass label and the 3-methyladenine of the internal standard substance and a standard curve, wherein the result is shown in FIG. 8.
Example 5 assay of prostate cancer-specific antigen content in urine of prostate cancer patients
Testing the content of prostate cancer specific antigen in urine of prostate cancer patients, mixing 200 μ L of 1.3nM gold nanoparticle solution with 50 μ L of 1 μ M aptamer, reacting overnight at room temperature, centrifuging at 12000rpm for 10min to discard supernatant, washing precipitate with water twice, adding adenine mass label (10 μ L of 1 μ M) and 10 μ L of urine of prostate cancer patients, reacting for 2 hours at room temperature, centrifuging at 12000rpm for 10min to discard supernatant, washing precipitate with water twice, taking 1 μ L precipitate and internal standard substance 3-methyladenine (1 μ L of 1 μ M), mixing matrix (CCA)1 μ L, adding onto MALDI target plate, drying in air, performing mass spectrometry, and calculating the content of prostate cancer specific antigen in urine of prostate cancer patients according to the signal intensity ratio of adenine mass label to internal standard substance 3-methyladenine mass spectrometry and standard curve, the results are shown in FIG. 9.
The mass spectrum conditions are as follows: voltage: acceleration voltage: 19.000 kV; delayed extraction voltage: 14.920 kV; voltage of the reflector: 20.000 kV; lens voltage: 7.000 kV; frequency: 1.000 Hz; laser energy: 20 percent; the accumulation times are as follows: 100 times; positive ion mode.
The above-mentioned prostate cancer patient urine was tested for prostate cancer specific antigens according to the standard ELISA kit (purchased from Abcam (shanghai) trade company, ltd.) test method, and the results are shown in fig. 9.
<110> university of Chinese academy of sciences chemical research institute of Chinese academy of sciences
<120> Mass spectrometric detection method based on competitive non-covalent interactions for sensitive detection of biomarkers
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<170>PatentIn version 3.5
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<213> Artificial sequence
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cgtcgtatta aagctcgcca tcaaatagct tt 32
Claims (4)
1. A mass spectrometric detection method based on competitive non-covalent interactions for biomarker detection comprising the steps of:
1) designing a nucleic acid aptamer according to the biomarker to be detected;
2) incubating the aptamer and the gold nanoparticles together to obtain an aptamer-gold nanoparticle combination;
3) respectively incubating and separating the aptamer-gold nanoparticle combination with a series of biomarker standard substances with known concentration and mass labels, respectively carrying out matrix-assisted laser desorption ionization time-of-flight mass spectrometry on the obtained solid, obtaining a series of signals of the mass labels combined with the gold nanoparticles corresponding to the biomarkers with known concentration, and establishing a corresponding relation between the concentration of the standard biomarkers and the signals of the mass labels;
4) incubating and separating the biomarker with unknown concentration with the aptamer-gold nanoparticle combination and the mass label in the step 2), performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry on the obtained solid to obtain a signal of the mass label combined with the gold nanoparticle corresponding to the biomarker with unknown concentration, and obtaining the concentration of the biomarker according to the corresponding relation between the concentration of the standard biomarker and the signal of the mass label in the step 3);
the particle size of the gold nanoparticles is distributed between 11 nm and 17nm, and the average size is 13 nm;
the incubation temperature of the aptamer and the gold nanoparticles is room temperature, and the incubation time is 12-24%;
the mass label is adenine;
incubating the aptamer-gold nanoparticle combination with the biomarker and the quality label at room temperature for 1-3 h;
the conditions of the mass spectrometry are as follows: voltage: acceleration voltage: 19.000 kV; delayed extraction voltage: 14.920 kV; voltage of the reflector: 20.000 kV; lens voltage: 7.000 kV; frequency: 1.000 Hz; laser energy: 20 percent; the accumulation times are as follows: 100 times; positive ion mode.
2. The method of claim 1, wherein: the biomarker is a prostate cancer specific antigen.
3. The method of claim 1, wherein: when the biomarker to be detected is a prostate cancer specific antigen, the sequence of the aptamer is CGT CGT ATT AAA GCT CGC CAT CAA ATA GCT TT.
4. The method of claim 1, wherein:
the ultraviolet characteristic absorption maximum value of the gold nanoparticles is at the wavelength of 520 nm;
the incubation of the aptamer with the gold nanoparticles is performed under acidic pH conditions; or the like, or, alternatively,
the incubation of the aptamer with the gold nanoparticles is performed in a buffered solution.
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| CN110376382A (en) | 2019-10-25 |
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