CN102175649B - LSPR (Localized Surface Plasmon Resonance) sensing chip for detecting recombinant protein of carcinogene C-myc - Google Patents
LSPR (Localized Surface Plasmon Resonance) sensing chip for detecting recombinant protein of carcinogene C-myc Download PDFInfo
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Abstract
The invention provides an LSPR (Localized Surface Plasmon Resonance) sensing chip for detecting recombinant protein (6) of carcinogene C-myc based on an LSPR spectrum, wherein a DTT (Dithiothreitol) monomolecular layer (2) is assembled on the surface of a gold film (1) of the LSPR sensing chip; a gold nano particle layer (3) is connected to the DTT monomolecular layer (2); a 3-sulfydryl propionic acid monomolecular layer (4) is modified on the surface of the gold nano particle layer (3); the 3-sulfydryl propionic acid monomolecular layer (4) and a monoclonal antibody (5) of the C-myc are combined through DMAP (Dimethylamino Pyridine)-EDC (Dichloroethane) activation; and the surface of gold nano particles is caused to generate the displacement of an LSPR absorption peak through the immunoreaction combination of the monoclonal antibody (5) of the C-myc and a recombinant protein (6) of the C-myc, thereby detecting the content of the recombinant protein (6) of the C-myc in cancerous tissues. The LSPR sensing chip provided by the invention has the beneficial effects of simplicity and convenience in assembly, rapidness in quantitation, high sensitivity and good selectivity and repeatability, can be used for realizing multi-channel detection and is prior to the traditional enzyme-linked immunosorbent assay (ELISA).
Description
Technical field
The invention belongs to protein content detection technique field in the tumour cell, relate to a kind of sensing chip and method thereof that is used to detect the oncogene C-myc recombinant protein.
Background technology
C-myc is a kind of proto-oncogene, is regulating that DNA is synthetic, is playing an important role in Apoptosis, differentiation and the process of cell cycle.Not only in cell activities such as cell proliferation, cell differentiation with important role was arranged in the cell cycle, but also participated in the conversion of cell tumour nearly by the C-myc recombinant protein of C-myc gene code.The C-myc Recombinant Protein Expression is closely related with the startup and the carcinous degree of a lot of cancerous tissues and cell tumour.
To protein, antigen, detection of antibodies method Bradford method and traditional biological enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay are arranged respectively at present; ELISA), unspecial detection method to the C-myc recombinant protein.And Bradford method anti-interference, selectivity are bad; Traditional ELISA method only can be accomplished qualitative or sxemiquantitative, and said method complex operation, precision are low, and its application is restricted.Therefore need to seek a kind of method that can fast quantification ground detection C-myc recombinant protein.The invention provides the bio-sensing detection chip and the method thereof of a kind of local surface plasma resonance (LSPR) spectral technique, can detect the content of C-myc recombinant protein in the cancerous issue easy, fast, quantitatively based on golden nanometer particle.
Summary of the invention
The technical matters that the present invention will solve has provided LSPR sensing chip and the method thereof that a kind of oncogene C-myc recombinant protein detects usefulness; It is characterized in that utilizing the local surface plasma resonance spectral technique that produces based on the golden nanometer particle surface realize quick, easy, detect the content of C-myc recombinant protein in the cancerous issue quantitatively; The detection method that is said LSPR sensing chip is to combine with the immune response of C-myc recombinant protein through the C-myc monoclonal antibody that is fixed on the sensing chip; Cause the displacement that the golden nanometer particle surface produces local surface plasma resonance spectral absorption peak on the chip, reach testing goal with the linear response relation of C-myc recombinant protein content.
For addressing the above problem, the present invention adopts following technical scheme:
Adopt the magnetron sputtering embrane method, polystyrene plastics, Si oxide glass surface plate one deck gold film (30～300nm), through control plated film vacuum tightness≤1.0 * 10
-3Pa,
Make said golden film surfacing smooth.(annotate: Piranha solution is the concentrated sulphuric acid: the solution of oxydol=7: 3) drip at above-mentioned golden film surface infiltration 5～30s with Piranha solution then; Taking-up is immersed in ultrasonic 5～600s in absolute ethyl alcohol and the redistilled water with above-mentioned golden film after washing 3 processing repeatedly with redistilled water successively; Then above-mentioned golden film is immersed 1 of 10～200mmol/L, in 4-dithiothreitol (DTT) (DTT)/ethanolic solution, place 1～24h, can be at golden film surface-assembled last layer DTT unimolecule rete; After washing repeatedly with absolute ethyl alcohol, redistilled water respectively; (size is to place 1～24h in 5～50nm) to the golden film immersion nano gold sol solution that above-mentioned steps is handled; Golden nanometer particle can be fixed on the golden film surface formation golden nanometer particle layer that above-mentioned steps is handled through the Au-S key like this; Rinse well repeatedly with redistilled water, and be stored in the redistilled water subsequent use.
The golden film that above-mentioned steps is handled immerses in the 3-mercaptopropionic acid of 0.1～100mmol/L, leaves standstill 1～24h, can be on the golden nanometer particle laminar surface be modified 3-mercaptopropionic acid unimolecular layer; After washing repeatedly with redistilled water; (annotate: DMAP is the 4-dimethylamino naphthyridine to the ethanolic solution of dropping 1～200mmol/LDMAP-EDC; EDC is 1-ethyl-(3-dimethylaminopropyl) carbodiimide) activation 1～30min on the golden film that above-mentioned steps is handled, rinse well with ethanol, redistilled water respectively more repeatedly.Through the DMAP-EDC activation; On the golden film that above-mentioned steps is handled, drip 0～2.6 μ g/mL C-myc monoclonal antibody WS; Behind reaction 3～600min, make 3-mercaptopropionic acid and C-myc monoclonal antibody bonding, the adsorbance scope of C-myc monoclonal antibody is 0.01～0.4gmL
-1Mm
-2Rinse well with redistilled water again, be stored in 4 ℃ of environment subsequent usely, promptly get the LSPR sensing chip that the C-myc monoclonal antibody is modified.
When incident light irradiation sensing chip; When resonance takes place in the collective vibration of the free electron on incident photon frequency and the golden nanometer particle; Cause the internal field on golden nanometer particle surface to be enhanced; Energy of reflection light strengthens, thereby shows strong surface plasma bulk absorption, and the absorption peak scope of its local surface plasma resonance spectrum is between 200～700nm.When the C-myc monoclonal antibody with after the C-myc recombinant protein combines, the plasma resonance frequency diminishes, resonance absorbing peak moves to the long wave direction, this peak shift and C-myc recombinant protein content to be measured are 6.5 * 10
-3Linear response relation in～1.3 μ g/mL scopes detects lower limit and reaches 6.5ng/mL, can realize that online dynamic monitoring, fast quantification detect the purpose of oncogene C-myc recombinant protein molecule.Simultaneously, said LSPR sensing chip can accurately be measured C-myc recombinant protein content in the cancerous issue, and its recovery is 92.31～109.23%, has very important application prospect and economic worth at biomedical aspects such as Cancerous disease prediction and treatments.
The invention has the beneficial effects as follows that this LSPR sensing chip is compared with traditional enzyme-linked immunosorbent assay (ELISA), and is highly sensitive, selectivity and favorable reproducibility, and have simple and convenient assembly, quantitatively fast, can realize advantage such as multi-channel detection.
Description of drawings
Fig. 1 is a LSPR sensing chip sensitive membrane structural representation.Fig. 2 is a LSPR sensing chip assembling rete audio-visual picture.
Fig. 3 is based on the local surface plasma resonance spectral detection synoptic diagram of golden nanometer particle.
Fig. 4 is the local surface plasma resonance absorption peak displacement of sensing chip and the corresponding relation curve map of C-myc recombinant protein concentration, and it is 6.5 * 10
-3The typical curve of～1.3 μ g/mL scopes (seeing illustration in Fig. 4) equation is:
Δλ＝-9.5075×10
-5+1.9156C
Wherein Δ λ is expressed as maximum absorption band shift value (nm), and C representes the concentration (μ g/mL) of C-myc recombinant protein.The above-mentioned relation curve is drawn when 25 ℃ of room temperatures.
Among Fig. 1,2, the 1. uniform golden film of thickness, 2.DTT self assembled monolayer, 3. golden nanometer particle layer, 4.3-mercaptopropionic acid unimolecular layer, 5.C-myc monoclonal antibody, 6.C-myc recombinant protein.
Embodiment
The assembling preparation of LSPR sensing chip:
1) adopting the magnetron sputtering embrane method to plate the golden film of one deck 100nm on polystyrene plastics, Si oxide glass matrix surface, is 1.0 * 10 through control plated film vacuum tightness
-4Pa, coating speed does
Make said golden film surfacing smooth;
2) (annotate: Piranha solution is the concentrated sulphuric acid: the solution of oxydol=7: 3) drip at above-mentioned golden film surface infiltration 15s, take out the back and washes repeatedly 3 times with redistilled water with Piranha solution;
3) the golden film of above-mentioned steps being handled is immersed in ultrasonic 60s in absolute ethyl alcohol and the redistilled water successively;
4) the golden film of above-mentioned steps being handled immerses in the DTT solution of 50mmol/L, places 10h, forms the DTT unimolecular layer, washes 3 times repeatedly to remove the free DTT in surface with absolute ethyl alcohol, rinses well repeatedly with redistilled water again;
5) it is in the nano gold sol of 10.5nm that the golden film of above-mentioned steps being handled immerses particle diameter; Place 24h; Taking out the back rinses well with redistilled water repeatedly; Golden nanometer particle is fixed on the golden film surface formation golden nanometer particle layer that above-mentioned steps is handled through the Au-S key like this, is stored in the redistilled water subsequent use then;
6) the golden film of above-mentioned steps being handled immerses in the 3-mercaptopropionic acid of 10mmol/L, leaves standstill 6h, on the golden nanometer particle layer, forms 3-mercaptopropionic acid unimolecular layer, rinses well with redistilled water then;
7) wash repeatedly with redistilled water after; (annotate: DMAP is the 4-dimethylamino naphthyridine to the ethanolic solution of dropping 100mmol/L DMAP-EDC; EDC is 1-ethyl-(3-dimethylaminopropyl) carbodiimide) activation 10min on the golden film that above-mentioned steps is handled, rinse well with ethanol, redistilled water respectively more repeatedly;
8) on the golden film that above-mentioned steps is handled, drip the 2.4 μ g/mL C-myc monoclonal antibody WS; Behind the reaction 60min; Rinse well with redistilled water again; Be stored in 4 ℃ of environment subsequent usely, promptly make the LSPR sensing chip that the C-myc monoclonal antibody that is used to detect the oncogene C-myc recombinant protein is modified.
The mensuration of C-myc recombinant protein typical curve:
1) agents useful for same (redistilled water, PBS buffer solution) all is stored in 4 ℃ of environment subsequent use after sterilization treatment;
2) getting 1 μ LC-myc recombinant protein (antigen) and place the 1.5mL small test tube, is buffer solution with PBS, dilutes 1～100000 times respectively, is mixed with a series of solution for standby of 0～6.5 μ g/mL;
3) the LSPR sensing chip that adopts above-mentioned C-myc monoclonal antibody to modify is tested the C-myc recombinant protein sample of each concentration respectively; Combine through of the immune response of C-myc monoclonal antibody with the C-myc recombinant protein; Cause golden nanometer particle local surface plasma resonance spectral absorption peak red shift on the chip, this peak shift and the linear response relation of C-myc recombinant protein content; Concentration C (μ g/mL) with the C-myc recombinant protein is a horizontal ordinate; Maximum absorption band shift value Δ λ (nm) is an ordinate; Draw the typical curve (seeing illustration in Fig. 4) of measuring C-myc recombinant protein sample concentration; Through the LSPR absorption peak displacement signal of C-myc corresponding concentration, can determine the content of the C-myc recombinant protein in the cancerous issue sample;
4) test after, reply with 0.1mol/L HCl eluant solution, rinse well with PBS buffer solution, redistilled water respectively again, can be stored in 4 ℃ of environment subsequent use.
C-myc recombinant protein Determination on content in the liver cancer tissue:
The extraction of albumen in the tissue: get the 0.1g tissue specimen, under 4 ℃ of environment, rinse well to remove and organize foreign matter with the PBS of precooling.Be cut into 0.5mm with scissors
3Fritter also adds the 1.0mL lysate that configures, and uses the PBS diluted for use again.
C-myc recombinant protein Determination on content: the above-mentioned LSPR sensing chip for preparing is placed PBS buffer solution, earlier the absorption peak wavelength location reading λ of record PBS buffer solution
1=353.47nm.The sample solution of getting after the above-mentioned dilution is measured, record absorption peak wavelength location reading λ
2=354.68nm.The absorption peak shift value of this sample (from, nm) can draw: Δ λ=λ by following formula
2-λ
1, being updated to Δ λ in the good typical curve equation of match, the concentration value that can calculate C-myc recombinant protein in this liver cancer tissue sample is 0.632 μ g/mL.Testing result shows oncogene C-myc high expressed in the liver cancer tissue; Consistent with the testing result of ELISA, and this chip can carry out detection by quantitative, is superior to the ELISA method; Explain that this method can accurately measure the C-myc recombinant protein content in the cancerous issue, have versatility.
C-myc recombinant protein content detection in the breast cancer cell:
Gather the breast tissue sample, cultivate, obtain the tumour cell (MDA-MB-231) of C-myc sudden change in the laboratory.Condition of culture: the L-15 nutrient culture media, 15% (tire ox or calf) serum, 5%CO2 cultivates in 37 degrees centigrade of incubators.Cell growth state: epithelium appearance adherent growth.
Get the 0.1g tissue specimen, under 4 ℃ environment, rinse well to remove and organize foreign matter with the PBS of precooling.Be cut into 0.5mm with scissors
3Fritter also adds the 1.0mL lysate that configures, and uses the PBS diluted for use again.
Get the sample solution after the above-mentioned dilution, test with prepared LSPR sensing chip, peak shift is 0.23nm, is updated in the good typical curve equation of match, and the C-myc content that can calculate in the breast cancer cell dilution is 120ng/mL.Testing result shows the oncogene C-myc high expressed, and is consistent with the testing result of ELISA, and this chip can carry out detection by quantitative, is superior to the ELISA method, explains that this method can accurately measure the C-myc recombinant protein content in the cancerous issue, has versatility.
The mensuration of the recovery:
Present embodiment is measured the recovery of LSPR sensing chip to variable concentrations C-myc recombinant protein sample, and compares with the ELISA method.The C-myc sample solution that in concentration known solution, adds known quantity is measured the absorption peak of each sample, displacement calculating value Δ λ then; The contrast working curve is found out concentration, addition that relatively records and actual addition, and the gained recovery is in 92.31～109.23% scopes; Average recovery rate is 100.66%, and ELISA only can make qualitative detection, explains that the inventive method is superior to the ELISA method; And the recovery is good, has actual application value in oncogene C-myc recombinant protein context of detection.
Optionally measure:
Fixedly main response C-myc recombinant protein concentration is 1.08 μ g/mL, and increasing interfering material concentration is 100 times of main response protein concentrations, and promptly disturbing protein concentration is 108 μ g/mL.The interference albumen of being investigated is respectively: bovine serum albumin(BSA), PINPROL, BA, transferrins, human hemoglobin, fibrin ferment, lysozyme.When the concentration of C-myc recombinant protein was 1.08 μ g/mL, the displacement of LSPR absorption peak was 2.07 ± 0.07nm.When adding concentration is the chaff interference of 100 times of response protein concentration; The mobile deviation of absorption peak position is all in 5%; This shows; Above-mentioned protein substance can not produce tangible disturbing effect to the process of C-myc monoclonal antibody identification C-myc recombinant protein, explains that said LSPR sensing chip has good specific selectivity to the C-myc recombinant protein.
Reproducible mensuration:
Present embodiment is measured the reappearance of LSPR sensing chip to the absorption peak position of response variable concentrations C-myc recombinant protein solution; To the C-myc recombinant protein sample replication of 650ng/mL and 260ng/mL 12 times; The relative standard deviation that obtains is respectively 3.22% and 1.56%; Explain that said LSPR sensing chip has good reappearance, has guaranteed the accuracy of the experimental data of surveying.
Claims (8)
1. a LSPR sensing chip that is used to detect oncogene C-myc recombinant protein (6) is characterized in that the package assembly self-polystyrene plastics of said LSPR sensing chip, Si oxide glass matrix surface are followed successively by to outermost layer: the uniform golden film of thickness (1), DTT unimolecular layer (2), golden nanometer particle layer (3), 3-mercaptopropionic acid unimolecular layer (4), C-myc monoclonal antibody (5).
2. LSPR sensing chip according to claim 1 is characterized in that the preparation process of said LSPR sensing chip is following:
1) adopts the magnetron sputtering embrane method, plate one deck gold film (1) on polystyrene plastics, Si oxide glass matrix surface, through control plated film vacuum tightness≤1.0 * 10
-3Pa,
Make said golden film (1) surfacing smooth;
2) the Piranha drips of solution is added in above-mentioned golden film (1) surface infiltration 5～30s, takes out the back and wash repeatedly 3 times with redistilled water; Wherein, Piranha solution is the concentrated sulphuric acid: the solution of oxydol=7: 3;
3) the golden film of above-mentioned steps being handled (1) is immersed in ultrasonic 5～600s in absolute ethyl alcohol and the redistilled water successively;
4) the golden film of above-mentioned steps being handled (1) immerses 1 of 10～200mmol/L; In 4-dithiothreitol (DTT) (DTT)/ethanolic solution, place 1～24h, form DTT unimolecular layer (2); Wash 3 times repeatedly to remove the free DTT in surface with absolute ethyl alcohol, rinse well repeatedly with redistilled water again;
5) the golden film of above-mentioned steps being handled (1) immerses in the nano gold sol solution; Place 1～24h; Taking out the back rinses well with redistilled water repeatedly; Golden nanometer particle is fixed on golden film (1) the surface formation golden nanometer particle layer (3) that above-mentioned steps is handled through the Au-S key like this, is stored in the redistilled water subsequent use then;
6) the golden film of above-mentioned steps being handled (1) immerses in the 3-mercaptopropionic acid of 0.1～100mmol/L, leaves standstill 1～24h, goes up at golden nanometer particle layer (3) and forms 3-mercaptopropionic acid unimolecular layer (4);
7) wash repeatedly with redistilled water after, drip the golden film (1) that the ethanolic solution of 1～200mmol/L DMAP-EDC handles in above-mentioned steps and go up activation 1～30min, rinse well repeatedly with ethanol, redistilled water respectively again; Wherein, DMAP is the 4-dimethylamino naphthyridine, and EDC is 1-ethyl-(3-dimethylaminopropyl) carbodiimide;
8) the golden film of handling in above-mentioned steps (1) is gone up and is dripped 0～2.6 μ g/mL C-myc monoclonal antibody WS; Behind reaction 3～600min; Rinse well with redistilled water again, be stored in 4 ℃ of environment subsequent usely, promptly get the LSPR sensing chip that C-myc monoclonal antibody (5) is modified.
3. LSPR sensing chip according to claim 1; The detection method that it is characterized in that said LSPR sensing chip is to combine with the immune response of C-myc recombinant protein (6) through the C-myc monoclonal antibody (5) that is fixed on the sensing chip; Cause the displacement that golden nanometer particle layer (3) surface produces local surface plasma resonance spectral absorption peak on the chip, with the linear response relation of C-myc recombinant protein (6) content.
4. LSPR sensing chip according to claim 2, it is characterized in that on polystyrene plastics, Si oxide glass matrix surface the THICKNESS CONTROL of gold-plated film (1) be between 30～300nm.
5. LSPR sensing chip according to claim 2, the size that it is characterized in that said aurosol GOLD FROM PLATING SOLUTION nano particle is between 5～50nm.
6. LSPR sensing chip according to claim 2, the adsorbance that it is characterized in that being fixed in the C-myc monoclonal antibody (5) on the LSPR sensing chip is at 0.01～0.4gmL
-1Mm
-2Between.
7. LSPR sensing chip according to claim 3, the absorption peak scope that it is characterized in that the local surface plasma resonance spectrum that golden nanometer particle layer (3) surface is represented on the said LSPR sensing chip is between 200～700nm.
8. LSPR sensing chip according to claim 3 is characterized in that said LSPR sensing chip can accurately measure C-myc recombinant protein (6) content in the cancerous issue, and its recovery is 92.31～109.23%, and the range of linearity is 6.5 * 10
-3～1.3 μ g/mL detect lower limit and reach 6.5ng/mL.
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| CN104142393B (en) * | 2013-10-30 | 2016-03-30 | 郑州轻工业学院 | A kind of biological sensor sensing film and the application in detection clenobuterol hydrochloride thereof |
| CN104749379A (en) * | 2015-03-23 | 2015-07-01 | 陈志涛 | Biosensor chip for rapidly detecting salmonella typhimurium |
| CN107042092A (en) * | 2017-02-23 | 2017-08-15 | 南昌大学 | Affine sorbing material based on specific recognition c Myc label single domain heavy chain antibodies |
| CN107051397A (en) * | 2017-02-23 | 2017-08-18 | 南昌大学 | The affine in immunity sorbing material of specific recognition c Myc label nano antibodies |
| CN107042091A (en) * | 2017-02-23 | 2017-08-15 | 南昌大学 | Affine in immunity sorbing material based on specific recognition c Myc label nano antibodies |
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