CN101975854A - Single molecule force spectroscopy-based anti-cancer medicament identification method - Google Patents
Single molecule force spectroscopy-based anti-cancer medicament identification method Download PDFInfo
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Abstract
The invention discloses an anti-cancer medicament identification method for detecting membrane protein receptor-ligand interaction based on single molecule force spectroscopy. The method comprises the following steps of: modifying the ligand of a target membrane protein receptor on the needle of an atomic force microscope to obtain a modified needle; and incubating isolated cancer cells or incubating isolated cancer cells by adding a substance to be identified, putting the modified needle obtained in the step 1) and the incubated isolated cancer cells into a sample cell of the atomic force microscope after the incubation is finished, measuring the bonding force between the target membrane protein receptor and the ligand of the target membrane protein receptor in the isolated cancer cells to obtain a bonding probability A or B, and determining that the substance to be identified is a candidate anti-cancer medicament if the bonding probability B is smaller than the bonding probability A. The method has the advantages of simple operation, short period, low cost and the like, and can identify various medicaments for inhibiting the ligand-receptor interaction.
Description
Technical field
The present invention relates to cancer therapy drug authentication method based on unimolecule power spectrum.
Background technology
Cancer is the maximum killer of human health always.The hyper-proliferative of cell is that cancer takes place and reasons of development, and the hyper-proliferative of cell is general all relevant with the excessive activation of part inducing cell signal path especially growth factor signal path.Therefore suppress the excessive activation of this cell signal path, become the important channel for the treatment of cancer.The activation of growth factor cell signal path at first is to combine on cell membrane with its membrane protein receptor by part, makes acceptor produce autohemagglutination or different poly-and be activated.By the acceptor that the activates albumen of phosphorylation downstream passages again, its downstream albumen is activated, thereby activates whole signal path then.Therefore, the compound that identifies the arbitrary link that can block the activation signal path just can develop the medicine for the treatment of cancer.
One of main authentication method of cancer therapy drug molecule is at first to find out the material standed for that some can combine with the signal path associated protein by computer simulation from compound library at present, then these material standed for molecules are carried out the experiment of cellular level, detect it and whether can suppress the phosphorylation level of the downstream albumen that its part induces, reach the purpose that cancer therapy drug is identified by repeating these biochemical tests, further carry out zoopery again.But in qualification process the ubiquity cycle long, complicated operation spends high problem, is difficult to realize simple and efficient drug identification.Therefore, be necessary to develop a kind of drug identification method of simple and fast.
Unimolecule power spectral method is a kind of detection technique that develops rapidly in recent years.This method not only has high sensitivity, and the variation of the single molecular force of covering down can also show a large amount of Molecular Detection the time, be applied to the research of cellular level to the dynamic process of dynamic change, intermolecular interaction and the biochemical reaction of biomolecular structure and function increasingly extensively.But yet there are no the relevant report of utilizing this method to identify cancer therapy drug.
Summary of the invention
The purpose of this invention is to provide a kind of cancer therapy drug authentication method that detects membrane protein receptor-ligand interaction based on unimolecule power spectrum.
The authentication method of cancer therapy drug provided by the invention comprises the steps:
1) ligand modified on the needle point of atomic force microscope with the target membrane protein receptor, the needle point after obtaining modifying;
2) cancer cell that exsomatizes is hatched, hatch after finishing the needle point after the step 1) gained modified and hatch after described stripped cancer cell all place the sample cell of atomic force microscope, measure the adhesion between the part of target membrane protein receptor described in the described stripped cancer cell and described target membrane protein receptor, obtain combining probability A between the part of target membrane protein receptor described in the described stripped cancer cell and described target membrane protein receptor; Described stripped cancer cell is the cancer cell that contains described target membrane protein receptor;
3) described stripped cancer cell and material to be identified are hatched, hatch after finishing needle point after itself and described step 1) gained modified and all place the sample cell of atomic force microscope, adhesion described in the described stripped cancer cell after mensuration is hatched between the part of target membrane protein receptor and described target membrane protein receptor obtains combining probability B between the part of target membrane protein receptor described in the described stripped cancer cell after described the hatching and described target membrane protein receptor; Described stripped cancer cell is the cancer cell that contains described target membrane protein receptor;
4) if described in conjunction with probability B less than described in conjunction with probability A, then described material to be identified is candidate's a cancer therapy drug.
In the step 1) of said method, various silicon nitride atomic-force microscope needle-tips commonly used all are applicable to this method.The part of described target membrane protein receptor is preferably TGF β 1, and described stripped cancer cell is preferably the Hela cell.Described modification step is that atomic-force microscope needle-tip and part are modified by the mode of chemical crosslinking, specifically comprise the steps: a) to place the toluene solution of MPTMS to react at the needle point of described atomic force microscope, obtain the needle point of silanization after reaction finishes; B) needle point with described step a) gained silanization places ω-nitrogen hydroxysuccinimide eater poly ethylene glycol α-maleimide ((ω-N-hydroxy-succinimide-ester-poly (ethylene glycol)-α-maleimide, abbreviation NHS-PEG-MAL) reacts in the dimethyl sulphoxide solution, react the needle point that finishes after obtaining activating; C) needle point and the described cell ligand after the activation that described step b) is obtained reacts in solvent, the needle point after obtaining modifying.
In the step a) reactions steps of above-mentioned modification step, temperature is 18-25 ℃, and preferred 20 ℃, the time is 1.5-2 hour, preferred 2 hours; The concentration expressed in percentage by volume of the toluene solution of described MPTMS is 1%; After described step a), before the described step b), also the needle point to described silanization carries out following processing: the needle point of described silanization is washed with toluene, acetone and ethanol successively, and nitrogen dries up;
In the described step b) reactions steps, temperature is 18-25 ℃, and preferred 20 ℃, the time is 2.5-3 hour, preferred 3 hours; The concentration of the dimethyl sulphoxide solution of described NHS-PEG-MAL is 1mg/ml; After described step b), before the described step c), also the needle point after the described activation is carried out following processing: the needle point after the described activation is washed with dimethyl sulfoxide (DMSO), and nitrogen dries up again;
In the described step c), temperature is 18-25 ℃, and preferred 20 ℃, the time is 0.5-1 hour, preferred 0.5 hour; Described solvent is selected from least a in PBS damping fluid and the Tris-HCl damping fluid, and wherein, the pH value of PBS damping fluid is 7.2-7.6, and is preferred 7.4, and volumetric molar concentration is 0.01M, and the pH value of Tris-HCl damping fluid is 7.2-7.6, preferred 7.4, and concentration is 0.05M.Described step 2 after described step c)) before, also the needle point after the described modification is carried out following processing: wash needle point after the described modification with damping fluid; Described damping fluid is selected from least a in PBS damping fluid and the Tris-HCl damping fluid, preferred PBS damping fluid; Wherein, the pH value of PBS damping fluid is 7.2-7.6, and is preferred 7.4, and volumetric molar concentration is 0.01M, and the pH value of Tris-HCl damping fluid is 7.2-7.6, preferred 7.4, and concentration is 0.05M.
Described step 2) and step 3) hatch in the described stripped cancer cell step, temperature is 37 ℃, the time is 18-30 hour, preferably is 24 hours; Described incubation step is carried out in nutrient culture media.The various nutrient culture media that can hatch this cancer cell all are suitable for, as to can be the hyclone percent by volume be 10% DMEM nutrient culture media.
Described step 3) is hatched in the material step to be identified, and temperature is 37 ℃, and the time is 0.5-2 hour, is preferably 1 hour.
Above-mentioned steps 2) and 3) in, adhesion is to obtain as follows: make the needle point of atomic force microscope be pressed onto cell surface, and then lift and make needle point leave cell.Obtain in conjunction with the concrete grammar of probability by adhesion be: the number of calculating combinative force curve accounts for the ratio of all force curve numbers, obtains in conjunction with probability.
Described step 2) and 3) in the determination step, loading speed is 1.0 * 10
5PN/s, the time that needle point contacts with substrate is 500ms.In determination step, the needle point of used atomic force microscope is before using, capable of washing clean and dry up with nitrogen.
In the described step 4), described candidate's cancer therapy drug is for can suppress the cancer therapy drug that described stripped cancer cell combines with described part; Describedly be not less than 5% with the described difference that combines probability B, as can be 8.6% in conjunction with probability A.
In order to obtain test findings more accurately, said method step 2) but and the step 3) repeated several times, as repeating 3-5 time.
Described material to be identified is a resveratrol shown in naringenin shown in the formula I or the formula II.
The present invention utilizes the method for unimolecule power spectrum that a kind of method of identifying cancer therapy drug based on the cellular level of membrane protein receptor-ligand interaction power is provided.Whether this method can suppress combining of membrane protein receptor and part by the detection of drugs molecule, thereby obtains being expected to suppress the drug candidates of growth of tumour cell.Its ultimate principle (as shown in Figure 1) is: at first be that part is modified on the afm tip by the method for chemical crosslinking, in the presence of testing compound, combine the variation of probability with part by acceptor on the unimolecule power spectrum observation of cell film then.For example, illustrate that this medicine has the effect that suppresses to join receptors bind if acceptor diminishes with the probability that combines of part after adding testing compound.Therefore in the presence of testing compound, by detecting combining probability and whether reducing of membrane protein receptor and part, thus the evaluation of realization inhibition membrane protein receptor and the cancer therapy drug that combines of part.This method belongs to the drug identification new method, compares with the other medicines authentication method, has easy and simple to handlely, and the cycle is short, and advantage such as reduce expenses can Rapid identification be inhibited and joins the cancer therapy drug of receptors bind.
Description of drawings
Fig. 1 joins the receptors bind synoptic diagram for detecting with atomic force microscope.
When Fig. 2 is 50 μ M for contrast and naringenin and resveratrol concentration, join the variation of receptors bind probability.
At positive control, naringenin and resveratrol are observed the tumor nodule number on the mouse lungs to Fig. 3 for respectively.
Embodiment
The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.
Among the following embodiment, if no special instructions, described method is conventional method.Used 3-mercaptopropyltriethoxysilane (MPTMS) and used NHS-PEG-MAL are all available from sigma company among the following embodiment; Used naringenin is available from plant development corporation, Ltd. of Shaanxi favour section; The used double dish of incubation step is available from double dish at the bottom of the glass of the 35mm of the good Bioisystech Co., Ltd of friend of Beijing section.
All be defined as follows in conjunction with probability, loading speed described in the present invention: in conjunction with probability is the number percent that combinative force curve accounts for the number of all force curves that record; Loading speed is meant the lifting speed of needle point when the withdraw of the needle in the unimolecule power spectrum mensuration, the product of the speed of needle point rollback in power elastic constant that its size is the afm tip micro-cantilever and the scission of link process, and unit is pN/s.
Used atomic force microscope is all available from Agilent company among the following embodiment, and used needle point is the silicon nitride needle point.This atomic force microscope includes following accessory: piezoelectric scanner (Piezo-scanner), piezoelectric scanning controller (XYZ Scanner Controller), the display (Displayer) of laser instrument (Laser), afm tip (Tip), micro-cantilever (Cantilever), sample cell (Sample Substrate), raster scanning and Z axle location, accept the controller (Feedback Controller) and the detecting device (Detector) of optical feedback signal.
Utilize the principle of atomic force microscope mensuration adhesion as follows: when between needle point and the detected sample faint interaction force being arranged, cause the micro-cantilever bending, the laser that reflexes on the detecting device from micro-cantilever is offset, because optical lever action principle, even the micro-cantilever deformation less than 0.01nm also can be in the laser spots skew that produces on the photoelectric detector about 10nm, consequent change in voltage correspondence the deformation quantity of micro-cantilever, just can obtain the measured value of micro-cantilever deformation quantity by certain function.Feedback system according to the voltage signal that produces change adjust needle point or sample Z to the position, relatively moving of control needle point and sample position can be carried out pattern and force measurement.
Embodiment 1,
1) getting the afm tip that cleans up transfers to and contains in the toluene solution that concentration expressed in percentage by volume is 1.0% MPTMS, in 20 ℃ the reaction 2 hours after, clean repeatedly with toluene and to remove unreacted silylating reagent, clean 3 times with acetone and ethanol successively again, nitrogen dries up, and obtains the needle point behind the silanization.Needle point behind the silanization is immersed in the DMSO solution of 1mg/mlNHS-PEG-MAL, after 3 hours, clean 3 times to remove unreacted NHS-PEG-MAL, the needle point after obtaining activating after nitrogen dries up with DMSO in 20 ℃ of reactions.With the needle point of activation in the PBS damping fluid that the pH value is 7.4, concentration is 0.01M with TGF β 1 part (concentration of this part in the PBS damping fluid is 5 μ M) room temperature reaction 0.5h after, after the pH value is 7.4, concentration is 0.01M PBS buffer solution for cleaning 3 times, the afm tip after obtaining modifying is standby.
2) the HeLa cell is passed to hatch 24 hours (the cell coverage is 80%) in 37 ℃ of the 35mm double dish after, afm tip after the step 1) gained modified and hatch after the HeLa cell all place the sample cell of atomic force microscope, adhesion in the mensuration HeLa cell between T β RII acceptor and TGF β 1 part, same needle point and cancer cell repeat parallel laboratory test 3 times, obtain in the HeLa cell combining probability A between the T β RII acceptor and TGF β 1 part
1In this determination step, loading speed is 1.0 * 10
5PN/s, the time that needle point contacts with substrate is 500ms;
3) with step 2) after used HeLa cell passes to and hatch 24 hours (the cell coverage is 80%) in 37 ℃ of the 35mm double dish, add 37 ℃ of naringenins again and hatch 1h (the cell coverage is 80%); Hatch after finishing the afm tip after the step 1) gained modified and hatch after the HeLa cell all place the sample cell of atomic force microscope, adhesion in the mensuration HeLa cell between T β RII acceptor and TGF β 1 part, same needle point and cancer cell repeat parallel laboratory test 3 times, obtain in the HeLa cell combining probability B between the T β RII acceptor and TGF β 1 part
1In this determination step, loading speed is 1.0 * 10
5PN/s, the time that needle point contacts with substrate is 500ms;
Following blocking-up contrast is set simultaneously: with the hela cell pass to hatch in 37 ℃ of the 35mm double dish made its cell coverage be 80% in 24 hours after, with blocking agent T β RII extracellular fragment concentration is that the PBS damping fluid (the pH value is 7.4, concentration be 0.01M) of 5ug/ml adds in this hela cell, making the final concentration of this blocking agent T β RII extracellular fragment is 5ug/ml, behind the following 30min of needle point immersed in liquid level after will modifying again, begin to measure adhesion, the condition determination of this determination step and abovementioned steps 2) and 3) identical, obtaining this is 3.5 ± 0.8% in conjunction with probability.
Wherein, the preparation method of used blocking agent T β RII extracellular fragment is as follows: with pET-21b-T β RII extracellular fragment (ECD) (Crescenzo G.D., Pham P.L.; Durocher Y., O ' Connor-McCourt M.D.Transforming growthfactor-beta (TGF-beta) binding to the extracellular domain of the type TGF II-betareceptor:receptor capture on a biosensor surface using a new coiled-coil capture systemdemonstrates that avidity contributes significantly to high affinity binding.J.Mol.Biol., 2003,328,1173-1183) be converted into bacterial strain E.coli DH-5 α.The T β RII ECD albumen of the terminal histidine mark of N is induced with 1mM IPTG's, usefulness Ni-NTA agaropectin under the albuminous degeneration condition purifying (agarose, Qiagen, USA).Use list of references Wang Q. again, Bai Z., Li X., Hou L., Zhang B.The evidences of human orphan receptor COUP-TF inhibiting telomerase activity throughdecreasing hTERT II transcription.Cancer Lett., 2004,214, the method that provides among the 81-90 makes albumen folding again.Sample is at first with the Tris-HCl (20mM that contains 100mM NaCl and 0.5-2.0M urea, pH8.0) solution dilution is then successively at buffer solution I (0.5M urea, 20mM Tris-HCl, 1mM EDTA, 100mM NaCl, 25%NP-40,0.2mM PMSF) and buffering solution II (20mM Tris-HCl, 1mM EDTA, 50mM NaCl, 0.25%NP-40,0.2mM PMSF) middle dialysis 24h.
This pET-21b-T β RII extracellular fragment can obtain from Institute of Chemistry, Academia Sinica.
Above-mentioned testing result is all listed among Fig. 2.As seen from the figure, TGF β 1 part and T β RII acceptor combines probability A
1Be 25.1 ± 2.8%.After adding 50 μ M naringenins were hatched 1h, TGF β 1 part obviously reduced with the probability that combines of T β RII acceptor, in conjunction with probability B
1Be reduced to 16.5 ± 1.6%, this is in conjunction with probability A
1With combine probability B
1Difference be 8.6%, greater than 5%, illustrate that naringenin can suppress combining of TGF β 1 part and T β RII acceptor.
According to last identical step, only medicine to be identified in the step 3) is replaced with resveratrol, gained is in conjunction with probability A
2With B
2There is not marked change, in conjunction with probability A
2Be 25.1 ± 2.8%, in conjunction with probability B
2Be 24.6% ± 2.7%, this difference is 0.5%, less than 5%, makes veratryl alcohol clear and can not suppress combining of TGF β 1 part and T β RII acceptor.
The biochemical test checking:
In order to verify the validity of unimolecule power spectrum authentication method, carry out zoopery according to following steps:
As shown in Figure 3, with the 4T1 cell inoculation under Balb/c mouse the 4th fat pad, 150,000/only.Inoculate grouping in 3 days, ten every group, positive controls is irritated stomach solution (0.2%CMC-Na aqueous solution), and the administration group is gastric infusion naringenin, each 100mg/kg of resveratrol respectively, and administration is 21 days altogether, puts to death animal, observes the tumor nodule number that shifts on the lungs.
Directly allow mouse drink positive controls solution.
The gained result as shown in Figure 3.As seen from the figure, the tubercle number of handling tumour on the lungs of back through naringenin reduces and resveratrol can not significantly.This and authentication method gained experimental result provided by the invention are much at one.Therefore the method for utilizing unimolecule power spectrum to detect membrane protein receptor-ligand interaction provided by the invention can be used to identify cancer therapy drug.
Claims (9)
1. the authentication method of a cancer therapy drug comprises the steps:
1) ligand modified on the needle point of atomic force microscope with the target membrane protein receptor, the needle point after obtaining modifying;
2) cancer cell that exsomatizes is hatched, hatch after finishing the needle point after the step 1) gained modified and hatch after described stripped cancer cell all place the sample cell of atomic force microscope, measure the adhesion between the part of target membrane protein receptor described in the described stripped cancer cell and described target membrane protein receptor, obtain combining probability A between the part of target membrane protein receptor described in the described stripped cancer cell and described target membrane protein receptor; Described stripped cancer cell is the cancer cell that contains described target membrane protein receptor;
3) described stripped cancer cell and material to be identified are hatched, hatch after finishing needle point after itself and described step 1) gained modified and all place the sample cell of atomic force microscope, adhesion described in the described stripped cancer cell after mensuration is hatched between the part of target membrane protein receptor and described target membrane protein receptor obtains combining probability B between the part of target membrane protein receptor described in the described stripped cancer cell after described the hatching and described target membrane protein receptor; Described stripped cancer cell is the cancer cell that contains described target membrane protein receptor;
4) if described in conjunction with probability B less than described in conjunction with probability A, then described material to be identified is candidate's a cancer therapy drug.
2. method according to claim 1 is characterized in that: described step 1) modification step comprises the steps:
A) needle point with described atomic force microscope places the toluene solution of 3-mercaptopropyltriethoxysilane to react, and obtains the needle point of silanization after reaction finishes;
B) needle point with described step a) gained silanization places the dimethyl sulphoxide solution of ω-nitrogen hydroxysuccinimide eater poly ethylene glycol α-maleimide to react, and reacts the needle point that finishes after obtaining activating;
C) needle point after the activation that described step b) is obtained and the part of described target membrane protein receptor react in solvent, obtain the needle point after the described modification.
3. method according to claim 2 is characterized in that: in the described step a) reactions steps, temperature is 18-25 ℃, and preferred 20 ℃, the time is 1.5-2 hour, preferred 2 hours; The concentration expressed in percentage by volume of the toluene solution of described 3-mercaptopropyltriethoxysilane is 1%;
In the described step b) reactions steps, temperature is 18-25 ℃, and preferred 20 ℃, the time is 2.5-3 hour, preferred 3 hours; The concentration of the dimethyl sulphoxide solution of described ω-nitrogen hydroxysuccinimide eater poly ethylene glycol α-maleimide is 1mg/ml;
In the described step c) reactions steps, temperature is 18-25 ℃, and preferred 20 ℃, the time is 0.5-1 hour, preferred 0.5 hour; Described solvent is selected from least a in PBS damping fluid and the Tris-HCl damping fluid; Wherein, the pH value of described PBS damping fluid is 7.2-7.6, and is preferred 7.4, and volumetric molar concentration is 0.01M, and the pH value of described Tris-HCl damping fluid is 7.2-7.6, preferred 7.4, and concentration is 0.05M.
4. according to claim 2 or 3 described methods, it is characterized in that: after described step a), before the described step b), the authentication method of described cancer therapy drug also comprises the steps: the needle point of described silanization is washed with toluene, acetone and ethanol successively, and nitrogen dries up;
After described step b), before the described step c), the authentication method of described cancer therapy drug also comprises the steps: the needle point after the described activation is washed with dimethyl sulfoxide (DMSO), and nitrogen dries up again;
After described step c), described step 2) before, the authentication method of described cancer therapy drug also comprises the steps: to wash needle point after the described modification with buffer solution; Described buffer solution is selected from least a in PBS damping fluid and the Tris-HCl damping fluid, preferred PBS damping fluid; The pH value of described buffer solution is 7.2-7.4, preferred 7.2.
5. according to the arbitrary described method of claim 1-4, it is characterized in that: described step 2) and step 3) hatch in the described stripped cancer cell step, temperature is 37 ℃, the time is 18-30 hour, preferably is 24 hours.
6. according to the arbitrary described method of claim 1-5, it is characterized in that: described step 3) is hatched in the material step to be identified, and temperature is 37 ℃, and the time is 0.5-2 hour, is preferably 1 hour.
7. according to the arbitrary described method of claim 1-6, it is characterized in that: the part of described target membrane protein receptor is TGF β 1, and described stripped cancer cell is the Hela cell.
8. according to the arbitrary described method of claim 1-7, it is characterized in that: in the described step 4), described candidate's cancer therapy drug is for can suppress the cancer therapy drug that target membrane protein receptor described in the described cancer cell combines with described part; Describedly be not less than 5% with the described difference that combines probability B in conjunction with probability A.
9. according to the arbitrary described method of claim 1-8, it is characterized in that: in the described step 1), the material that constitutes the needle point of described atomic force microscope is a silicon nitride.
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| CN103575934A (en) * | 2012-07-24 | 2014-02-12 | 上海纳米技术及应用国家工程研究中心有限公司 | AFM probe for single molecular force spectrum analysis and substrate functional modification method |
| CN108508238A (en) * | 2018-03-22 | 2018-09-07 | 天津职业技术师范大学 | Single molecule force spectroscopy device and method are tested based on double drive AFM system |
| CN111521803A (en) * | 2020-05-15 | 2020-08-11 | 华南理工大学 | Protein probe and application thereof in evaluating specific binding of antigen and antibody |
| CN111693695A (en) * | 2020-05-15 | 2020-09-22 | 华南理工大学 | Signal acquisition and processing method for antigen and antibody protein combined dynamic action process |
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| WO2009001220A2 (en) * | 2007-06-26 | 2008-12-31 | Universitetet I Oslo | Functionalization of microscopy probe tips |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103575934A (en) * | 2012-07-24 | 2014-02-12 | 上海纳米技术及应用国家工程研究中心有限公司 | AFM probe for single molecular force spectrum analysis and substrate functional modification method |
| CN108508238A (en) * | 2018-03-22 | 2018-09-07 | 天津职业技术师范大学 | Single molecule force spectroscopy device and method are tested based on double drive AFM system |
| CN111521803A (en) * | 2020-05-15 | 2020-08-11 | 华南理工大学 | Protein probe and application thereof in evaluating specific binding of antigen and antibody |
| CN111693695A (en) * | 2020-05-15 | 2020-09-22 | 华南理工大学 | Signal acquisition and processing method for antigen and antibody protein combined dynamic action process |
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