CN110376382A - The Mass Spectrometry detection method based on competitive noncovalent interaction for biomarker Sensitive Detection - Google Patents
The Mass Spectrometry detection method based on competitive noncovalent interaction for biomarker Sensitive Detection 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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- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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Abstract
The invention discloses a kind of Mass Spectrometry detection methods based on competitive noncovalent interaction for biomarker Sensitive Detection.Present invention utilizes Mass Spectrometer Method high sensitivity, detection limit is low, high-throughput and can provide the advantage of accurate molecular mass or molecular structure information, overcomes that traditional biological marker detection sensitivity is low, the disadvantage of anti-interference difference.The present invention can be used for the detection of actual patient specimens, provide reliable analysis means for the diagnoses and treatment of clinical cancer due to its good sensitivity to the responsiveness height of the micro variation of detectable substance, and the advantage high to the anti-interference of complex matrices.
Description
Technical field
The invention belongs to mass spectrum detection fields, and in particular to it is a kind of for biomarker Sensitive Detection based on competing
The Mass Spectrometer Method new method of striving property noncovalent interaction.
Background technique
Biomarker refers to can be with the change of tagging system, organ, tissue, cell and subcellular structure or function or can
The biochemical indicator for the change that can occur, has very extensive purposes.Biomarker can be used for medical diagnosis on disease, judge disease point
Phase or for evaluating the safety and efficacy of new drug or new treatment in target group.But since the complexity of its structure is more
Sample is difficult effectively delicately to be detected.
Matrix-assisted laser desorption ionization (MALDI-TOF MS), due to its high sensitivity, detection limit
It is low, it is high-throughput and accurate molecular mass or molecular structure information can be provided, to be widely used in analysis detection.
MALDI-TOF MS not only can analyze inorganic molecule, and polymer can also analyze macromolecular, such as nucleic acid and protein.When having
When a little analytes can not be detected directly by MALDI-TOF MS, it will usually which the quality tab for introducing a kind of easy desorption ionization comes
Detection indirectly.Based on the above advantages, mass spectrum has in the detection of biomarker potential as a kind of effective analysis method
Value.
Therefore, develop a kind of new detecting method based on MALDI-TOF MS, improve the detection sensitivity of biomarker
It is particularly significant.
Summary of the invention
The object of the present invention is to provide a kind of for biomarker Sensitive Detection based on competitive non-covalent phase interaction
Mass Spectrometry detection method.
Mass spectrum based on competitive noncovalent interaction provided by the present invention for biomarker Sensitive Detection
Detection method includes the following steps:
1) aptamer is designed according to biomarker to be detected；
2) aptamer is incubated with gold nano grain, obtains aptamer-gold nano grain combination；
3) by a series of aptamer-gold nano grain combination biomarker mark with concentration respectively
Quasi- product, fixed amount quality tab be incubated for jointly, separate, obtained solid is carried out respectively it is substance assistant laser desorpted ionized fly
The analysis of row time mass spectrum, obtains a series of quality mark in conjunction with gold nano grain of biomarkers corresponding to known concentration
The signal of label establishes the corresponding relationship of the concentration of the biomarker of standard and the signal of quality tab；
4) by the biomarker of unknown concentration and step 2) the aptamer-gold nano grain combination and
Quality tab is incubated for jointly, separation, is carried out Matrix-assisted laser desorption ionization analysis to obtained solid, is obtained
The signal of the quality tab combined to the gold nano grain for the biomarker for corresponding to unknown concentration, according to the mark in step 3)
The corresponding relationship of the signal of the concentration and quality tab of quasi- biomarker, obtains the concentration of biomarker.
In the above method, the biomarker concretely prostate cancer specific antigen.
Prostate cancer specific antigen is a kind of single chain polypeptide containing 237 amino acid, is belonged to tissue specificity
The serine protease race for thering is chymotrypsin sample to act on, can decompose the main colloidal carrier in sperm, there is dilution sperm
Effect.Prostate cancer specific antigen has tissue specificity, exists only in human prostate acinus and ductal epithelial cell endochylema
In, it is not expressed in other cells, is the marker of current clinically common prostate cancer diagnosis.
When the biomarker to be detected is prostate cancer specific antigen, the sequence of the aptamer is
CGT CGT ATT AAA GCT CGC CAT CAA ATA GCT TT。
In the above method, the particle diameter distribution of the gold nano grain is between 11-17nm, average-size 13nm；It is ultraviolet
Characteristic absorption maximum value is at 520nm wavelength.
The incubation temperature of the aptamer and gold nano grain can be room temperature, and the time can be 12-24h.
Carry out can be enhanced the interaction of aptamer and gold nano grain under condition of acidic pH.
Aptamer in conjunction with the interaction of gold nano grain is when buffer solution is in the environment of aqueous solution more
By force.
The interaction maximum load rate of aptamer and gold nano grain is 65%.
In the above method, the quality tab is that can go out in MALDI MS with good parsing with ionisation characteristics
The substance of existing signal.
Specifically, the quality tab can be adenine, and there are non-covalent mutual for the amino and gold nano grain on 2
Effect.
In above method step 3), a series of concentration of the biomarker standard items of known concentrations successively may be used are as follows:
0.06、0.3、0.6、3、6ng/mL。
The temperature that aptamer-gold nano grain combination and biomarker, quality tab are incubated for jointly can be room
Temperature, time can be 1-3h, concretely 2h.
In the above method, aptamer and the interaction of gold nano grain, the phase of quality tab and gold nano grain
Interaction can at least sustain the revolving speed of 12000rpm, that is, when separation, using centrifuge separation, revolving speed is not higher than 12000rpm.
In the above method, the condition of the mass spectral analysis are as follows: voltage: acceleration voltage: 19.000kV；Postpone extraction voltage:
14.920kV；Reflector voltage: 20.000kV；Lens voltage: 7.000kV；Frequency: 1.000Hz；Energy of lasers: 20%；It is tired
Add number: 100 times；Positive ion mode.
The present invention can be used for Sensitive Detection prostate cancer specific antigen, since aptamer is the single-stranded of 32 bases
DNA, so the gold nano grain surface site that an aptamer occupies, can be filled up by multiple adenine quality tabs,
Has the effect of signal amplification in Mass Spectrometer Method；
In the above method, it is specificity to prostate cancer antigen, in specific manner detects, big portion in the body fluid of people can be excluded
Divide the interference, such as human serum albumins, transferrins, immunoglobulin G etc. of albumen.
It is low to the detection of prostate-specific antigen in the above method, high sensitivity, in the dense of prostate-specific antigen
When degree is down to 0.06ng/mL, still there is apparent mass signal.
In the above method, there is apparent response to the micro variation of prostate-specific antigen, in prostate specific
When the concentration of antigen changes to 6ng/mL by 0.06ng/mL, mass signal enhances 90 times.
The present invention can detect the prostate cancer specific antigen in complicated clinical sample, such as patient's urine
Liquid, while normal human urine is not in false positive results.
In the above method, more of the invention and commercial enzyme-linked immunosorbent assay kit, the two is in actual patient specimens
The measurement of middle prostate-specific antigen is with higher to coincide.
The present invention technically overcomes that biomarker detection sensitivity is low, and complex matrices interfere big problem, pass through
A kind of Mass Spectrometer Method new method based on emulative noncovalent interaction is designed, mass signal is amplified, it is sensitive using mass spectrum
Degree is high, and detection limits low advantage, to realize the Sensitive Detection to biomarker.Anti-interference energy of the present invention to complex system
Power is strong, not the interference by other albumen in human body fluid, can be used for prostatic cancer specific in clinical practice patient's sample
The detection of antigen；The present invention is to provide a kind of general mass spectrometric analysis methods, can be used for a lot of other biomarkers
Detection.
Detailed description of the invention
Fig. 1 is the transmission electron microscope picture of gold nano grain.
Fig. 2 is the UV absorption figure of gold nano grain.
Fig. 3 is the aptamer of various concentration and the load capacity distribution map of gold nano grain.
Fig. 4 is the aptamer of various concentration and the load factor distribution map of gold nano grain.
Fig. 5 is the mass spectrum that the signal strength of adenine quality tab corresponds to the concentration variation of prostate cancer specific antigen
Figure.
Fig. 6 is interference experiment of the present invention to other albumen in human body.
Fig. 7 is that present invention introduces the linear equations after internal standard compound.
Fig. 8 is that the rate of recovery of the present invention in normal human urine tests table.
Fig. 9 is that the present invention and commercial ELISA kits compare the detection of prostate cancer specific antigen in patient's sample
Data.
Specific embodiment
The present invention will be described below by way of specific embodiments, but the present invention is not limited thereto.
Experimental method used in following embodiments is conventional method unless otherwise specified；Institute in following embodiments
Reagent, material etc., are commercially available unless otherwise specified.
Sample used in the following embodiments of the present invention is purchased from U.S. sigma company.
The model of Matrix-assisted laser desorption ionization instrument used in the following embodiments of the present invention
BIFLEXTM III(Bruker)。
Embodiment 1, synthesis gold nano grain
50mL boil water adds 2mL 1%HAuCl4(reflux) is boiled in heating, is quickly added with stirring 5% lemon of 1mL
Sour trisodium (0.2877g Na3Ct·2H2O is settled to 5mL), continue to heat, color is by Huang-colourless-blue-black-depth in 5min
Black-claret stops heating until colour stable, reheats 5min, continues stirring to room temperature, partial size is 13 nanometers.
Fig. 1 is the transmission electron microscope picture of gold nano grain.
Fig. 2 is the UV absorption figure of gold nano grain.
The detection of embodiment 2, prostate cancer specific antigen standard items
The nucleic acid that 200 μ L 1.3nM gold nano grain solution are respectively 1 μM with the concentration of 10,50,100 and 150 μ L is fitted
(sequence is CGT CGT ATT AAA GCT CGC CAT CAA ATA GCT TT to ligand, is purchased from bioengineering (Shanghai) share
Co., Ltd) mixing, it reacts at room temperature overnight, detects the aptamer of various concentration and the load capacity of gold nano grain and load
Rate distribution map.
Fig. 3 is the aptamer of various concentration and the load capacity distribution map of gold nano grain.
Fig. 4 is the aptamer of various concentration and the load factor distribution map of gold nano grain.
Final choice mixes 200 μ L 1.3nM gold nano grain solution with 1 μM of 50 μ L of aptamer, and room temperature is anti-
Should stay overnight, 12000rpm 10min centrifugation abandon supernatant, precipitating be washed with water twice, be added adenine quality tab (10 μ L1 μM) and
Prostate cancer specific antigen standard solution, ultimate density are 0.06,0.3,0.6,3,6ng/mL respectively, and room temperature reaction 2 is small
When, supernatant is abandoned in 12000rpm 10min centrifugation, after precipitating is washed with water twice, 1 μ L of precipitating is taken to mix addition with 1 μ L of matrix (CCA)
Onto MALDI target plate, enter mass spectral analysis after drying in air.
Mass Spectrometry Conditions are as follows: voltage: acceleration voltage: 19.000kV；Postpone extraction voltage: 14.920kV；Reflector voltage:
20.000kV；Lens voltage: 7.000kV；Frequency: 1.000Hz；Energy of lasers: 20%；Accumulative frequency: 100 times；Cation
Mode.
Fig. 5 is the mass spectrum that the signal strength of adenine quality tab corresponds to the concentration variation of prostate cancer specific antigen
Figure.
Embodiment 3, interference experiment
200 μ L 1.3nM gold nano grain solution are mixed with 1 μM of 50 μ L of aptamer, room temperature reaction is stayed overnight,
Supernatant is abandoned in 12000rpm 10min centrifugation, and precipitating is washed with water twice, is separately added into adenine quality tab (10 1 μM of μ L) and 10 μ
L 6ng/mL interferes albumen, and (human albumin, transferrins, immunoglobulin, cow's serum and cow's serum and prostate cancer are special
The mixture of Specific Antigen) room temperature reaction 2 hours, supernatant is abandoned in 12000rpm 10min centrifugation, and after precipitating is washed with water twice, it is heavy to take
The 1 μ L and matrix (CCA) 1 μ L that forms sediment is mixed to join on MALDI target plate, enters mass spectral analysis after drying in air.
Mass Spectrometry Conditions are as follows: voltage: acceleration voltage: 19.000kV；Postpone extraction voltage: 14.920kV；Reflector voltage:
20.000kV；Lens voltage: 7.000kV；Frequency: 1.000Hz；Energy of lasers: 20%；Accumulative frequency: 100 times；Cation
Mode.
Fig. 6 is interference experiment of the present invention to other albumen in human body.
Embodiment 4, rate of recovery test
200 μ L 1.3nM gold nano grain solution are mixed with 1 μM of 50 μ L of aptamer, room temperature reaction is stayed overnight,
Supernatant is abandoned in 12000rpm 10min centrifugation, and precipitating is washed with water twice, be added prostate cancer specific antigen (final concentration is respectively 0,
3,6,30,60ng/mL) and adenine quality tab (10 1 μM of μ L), it reacts at room temperature 2 hours, 12000rpm10min centrifugation is abandoned
Clearly, after precipitating is washed with water twice, 1 μ L of precipitating and internal standard compound 3-MA (1 1 μM of μ L) are taken, 1 μ L of matrix (CCA) mixing adds
Enter onto MALDI target plate, enters mass spectral analysis after drying in air.
Mass Spectrometry Conditions are as follows: voltage: acceleration voltage: 19.000kV；Postpone extraction voltage: 14.920kV；Reflector voltage:
20.000kV；Lens voltage: 7.000kV；Frequency: 1.000Hz；Energy of lasers: 20%；Accumulative frequency: 100 times；Cation
Mode.
With adenine quality tab with internal standard compound 3-MA mass signal intensity than corresponding prostatic cancer specific
Antigen concentration does linearity curve.
Fig. 7 is that present invention introduces the linear equations after internal standard compound.
200 μ L 1.3nM gold nano grain solution are mixed with 1 μM of 50 μ L of aptamer, room temperature reaction is stayed overnight,
Supernatant is abandoned in 12000rpm 10min centrifugation, and precipitating is washed with water twice.Prostate cancer specific antigen is added in normal human urine
Standard items, final concentration are respectively 0.8,8,80ng/mL.Take above-mentioned 10 μ L of urine and adenine quality tab 10 μ L (1 μM) room
Temperature reaction 2 hours, supernatant is abandoned in 12000rpm 10min centrifugation, after precipitating is washed with water twice, takes 1 μ L of precipitating and internal standard compound 3- methyl
Adenine (1 1 μM of μ L), 1 μ L of matrix (CCA) is mixed to join on MALDI target plate, enters mass spectral analysis after drying in air,
Above-mentioned urine is calculated according to adenine quality tab and internal standard compound 3-MA mass signal intensity ratio and standard curve
The content of prostate cancer specific antigen in liquid, as a result as shown in Figure 8.
Prostate cancer specific antigen content analysis in embodiment 5, carcinoma of prostate patient urine
The content for testing the prostate cancer specific antigen in carcinoma of prostate patient urine, by 200 μ L 1.3nM Jenners
Rice grain solution is mixed with 1 μM of 50 μ L of aptamer, and overnight, supernatant is abandoned in 12000rpm 10min centrifugation for room temperature reaction, is sunk
Shallow lake is washed with water twice, and adenine quality tab (10 1 μM of μ L) and 10 μ L of carcinoma of prostate patient urine is added, and room temperature reaction 2 is small
When, supernatant is abandoned in 12000rpm 10min centrifugation, after precipitating is washed with water twice, takes precipitating 1 μ L and internal standard compound 3-MA (1 μ
1 μM of L), 1 μ L of matrix (CCA) is mixed to join on MALDI target plate, and mass spectral analysis is entered after drying in air, fast according to gland
Purine quality tab and internal standard compound 3-MA mass signal intensity ratio and standard curve calculate carcinoma of prostate patient
The content of prostate cancer specific antigen in urine, as a result as shown in Figure 9.
Mass Spectrometry Conditions are as follows: voltage: acceleration voltage: 19.000kV；Postpone extraction voltage: 14.920kV；Reflector voltage:
20.000kV；Lens voltage: 7.000kV；Frequency: 1.000Hz；Energy of lasers: 20%；Accumulative frequency: 100 times；Cation
Mode.
Above-mentioned forefront is tested according to standard ELISA kit (being purchased from Abcam (Shanghai) trade Co., Ltd) test method
Prostate cancer specific antigen in gland cancer disease patient urine, as a result as shown in Figure 9.
<110>university, the Chinese Academy of Sciences, Institute of Chemistry, Academia Sinica
<120>it is used for the Mass Spectrometry detection method based on competitive noncovalent interaction of biomarker Sensitive Detection
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 32
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 1
cgtcgtatta aagctcgcca tcaaatagct tt 32
Claims (10)
1. a kind of Mass Spectrometry detection method based on competitive noncovalent interaction for biological marker analyte detection, including it is following
Step:
1) aptamer is designed according to biomarker to be detected；
2) aptamer is incubated with gold nano grain, obtains aptamer-gold nano grain combination；
3) by a series of aptamer-gold nano grain combination biomarker standard with concentration respectively
Product, quality tab are incubated for jointly, and separation carries out Matrix-assisted laser desorption ionization to obtained solid respectively
Analysis, obtains a series of signal of the quality tab in conjunction with gold nano grain of biomarkers corresponding to known concentration,
Establish the corresponding relationship of the concentration of the biomarker of standard and the signal of quality tab；
4) by the aptamer-gold nano grain combination and quality in the biomarker of unknown concentration and step 2)
Label is incubated for jointly, separation, carries out Matrix-assisted laser desorption ionization analysis to obtained solid, is obtained pair
The signal for the quality tab that should be combined in the gold nano grain of the biomarker of unknown concentration, according to the standard in step 3)
The corresponding relationship of the signal of the concentration and quality tab of biomarker, obtains the concentration of biomarker.
2. according to the method described in claim 1, it is characterized by: biomarker is prostate cancer specific antigen.
3. method according to claim 1 or 2, it is characterised in that: the biomarker to be detected is prostate cancer
When specific antigen, the sequence of the aptamer is CGT CGT ATT AAA GCT CGC CAT CAA ATA GCT
TT。
4. method according to any one of claim 1-3, it is characterised in that:
The particle diameter distribution of the gold nano grain is between 11-17nm, average-size 13nm；Ultraviolet characteristic absorption maximum value exists
At 520nm wavelength；
The incubation temperature of the aptamer and gold nano grain is room temperature, time 12-24；
The incubation of the aptamer and gold nano grain preferably carries out under condition of acidic pH；Or,
The incubation of the aptamer and gold nano grain carries out preferably in buffer solution.
5. method according to any of claims 1-4, it is characterised in that: the quality tab is with good solution
The substance of signal, can occur in MALDI MS in analysis and ionisation characteristics.
6. according to the method described in claim 5, it is characterized by: the quality tab is adenine.
7. method according to claim 1 to 6, it is characterised in that: aptamer-gold nano grain combines
The temperature that body and biomarker, quality tab are incubated for jointly is room temperature, time 1-3h.
8. method according to any one of claims 1-7, it is characterised in that: the condition of the mass spectral analysis are as follows: voltage:
Acceleration voltage: 19.000kV；Postpone extraction voltage: 14.920kV；Reflector voltage: 20.000kV；Lens voltage: 7.000kV；
Frequency: 1.000Hz；Energy of lasers: 20%；Accumulative frequency: 100 times；Positive ion mode.
9. a kind of for detecting the kit of biomarker, including it is gold nano grain, matched with biomarker to be detected
Aptamer, quality tab；
The quality tab is can occur the substance of signal in MALDI MS with good parsing and ionisation characteristics.
10. application of the kit as claimed in claim 9 in the product of preparation detection biomarker.
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| CN107660235A (en) * | 2015-04-08 | 2018-02-02 | 威里利生命科学有限责任公司 | Make particle labeling to carry out the method for multiplex function screening |
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| US20110136099A1 (en) * | 2007-01-16 | 2011-06-09 | Somalogic, Inc. | Multiplexed Analyses of Test Samples |
| CN107660235A (en) * | 2015-04-08 | 2018-02-02 | 威里利生命科学有限责任公司 | Make particle labeling to carry out the method for multiplex function screening |
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