CN110412291A - A method of building SERS spectra probe in detecting markers for breast cancer EGFR phosphorylated tyrosine - Google Patents

A method of building SERS spectra probe in detecting markers for breast cancer EGFR phosphorylated tyrosine Download PDF

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CN110412291A
CN110412291A CN201910693780.5A CN201910693780A CN110412291A CN 110412291 A CN110412291 A CN 110412291A CN 201910693780 A CN201910693780 A CN 201910693780A CN 110412291 A CN110412291 A CN 110412291A
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冯尚源
刘秀杰
卢玉栋
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Fujian Normal University
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Abstract

The invention discloses a kind of methods for constructing SERS spectra probe in detecting markers for breast cancer EGFR phosphorylated tyrosine: using golden plate as substrate, the substrate detected using core-shell structure nanometer particle as SERS develops highly selective and high sensitivity " sandwich " configuration probe molecule to detect the phosphorylation of markers for breast cancer EGFR tyrosine.It is 2221cm using biomolecule Raman quiet zone peak position is located in 4-MB Raman spectrum‑1SERS spectra intensity as internal standard to mark molecule 4-MBA spectral signal carry out intensity normalized, it can be avoided influence of the biomolecule fingerprint region Raman signal to quantitative detection, so that EGFR phosphorylated tyrosine solution concentration and the linear relationship of SERS intensity are greatly improved, the detection sensitivity of the SERS spectra probe is further increased.4-MB and 4-MBA is located at the middle layer of golden core and silver-colored shell, ensure that its property, space structure and quantity etc. are constant, and then ensure the reliability of data.The method of the present invention shows huge potentiality in terms of cancer markers super sensitivity detection.

Description

A kind of building SERS spectra probe in detecting markers for breast cancer EGFR phosphorylated tyrosine Method
Technical field
The invention belongs to biomedical cancer markers phosphorylation detection fields, and in particular to a kind of building SERS spectra spy The method of needle detection markers for breast cancer EGFR phosphorylated tyrosine.
Background technique
Breast cancer is the second common cancer in the world, the 25% of Zhan Suoyou cancer, and breast cancer is also that women is most in the world The trend of a rising is presented in the malignant tumour generally suffered from, disease incidence under the promotion of time, and occurs mainly in 45 years old Above women.EGF-R ELISA (EGFR) is one of Breast Cancer-Specific protein biomarker, is to be located at cell A kind of transmembrane glycoprotein on surface, tyrosine kinase is in conjunction with the compounds such as epidermal growth factor and transforming growth factor (TGFa) Afterwards, activation and induced cell proliferation and differentiation.The phenomenon that EGFR will appear overexpression in many epithelial origin tumours (> 75.3 µg·L-1), such as breast cancer, gastric cancer, the cancer of the esophagus, prostate cancer.
The intracorporal phosphorylated protein of breast cancer patients and its illness are closely related, and protein phosphorylation is that control cell function is several One of most important, widest molecular regulation mechanism in all aspects, during protein is translated, passes through certain side The modification of formula, the modification of certain fixations can become the biological marker of cancer or the targeting for the treatment of, abnormal protein phosphorylation It is usually related with cancer.Therefore, the state of phosphorylation event can give a clue for morbid state.Increase or remove phosphate group As the biology " ON/OFF " much reacted, it is extremely necessary for exciting a variety of biological phenomenons by protein phosphorylation, such as carefully The processes such as proliferation, the protein degradation of development, differentiation of born of the same parents.Especially tyrosine phosphorylation is cellular informatics transmitting and adjusting control enzyme One of active important form is then transduceed growth factor, thin by certain medium generally through interacting between elicitor protein Intracellular cytokine etc. adjusts the information of receptor on cell membrane.Ratio shared by tyrosine phosphorylation is lower in a variety of phosphorylation modifications, However also just because of its content degree in cell is lower, thus ensuring that biomolecule generates under the stimulation of front and back signal Sharp answers, and the phosphorylation of tyrosine has significant research during the adjusting for probing into biological information and all kinds of critical biochemicals Meaning and development potentiality.The early stage of cancer development has biggish therapy intervention potentiality.Thus, it is found that still being limited to intraorganic Malignant tumour is the key that effective treatment and raising survival rate before cancer or before shifting.The usual way that breast cancer is made a definite diagnosis have it is clinical and Auxiliary examination, such as iconography and biopsy.In biopsy, female hormone receptor and progesterone receptor point are carried out to the sample of acquisition Analysis, to determine the type of breast cancer.This technical information is abundant, is current standard selection.However, it be it is invasive, it is right Patient there are risk and price it is relatively expensive.Therefore, a noninvasive, easy diagnostic method becomes more next for people It is more important.This demonstrates the need for new diagnostic method to assess the expression of biomarker for cancer, the latest developments of molecular biology Show that Tumor biomarkers are shown and its great effect in the diagnosis, prognosis and Study of Etiology of tumour, to its into The highly selective and sensitivity Detection of row is infantile tumour detection, by stages one of monitoring and the tool of drug resistance assessment most worthy.
Surface enhanced Raman spectroscopy (SERS) technology is the detection instrument with high sensitivity, high accuracy, is being had Outside major part advantage possessed by standby normal Raman, the shortcomings that also overcoming its script.So greatly causing vast scientific work Research interest of the author to SERS.Compared to normal Raman spectrum, the 10-14 order of magnitude of SERS spectra signal enhancing is sensitive Degree is greatly improved, and just because of the high advantage of the distinctive non-destructive of SERS and sensitivity, stability, promotes it in biology Extensive development prospect is shown in medical application, the pathologic finding for medicine investigation, stubborn disease proposes novel quick nothing The method of wound and effective means.
Summary of the invention
In order to reduce existing for current breast cancer early detection it is costly, pretreatment is complicated, time-consuming and accuracy etc. Problem, the object of the present invention is to provide a kind of building SERS spectra probe in detecting markers for breast cancer EGFR phosphorylated tyrosines Method has easily operated, high sensitivity, high specificity, the advantages of capable of saving plenty of time and economic cost.
The present invention is based on above-mentioned research background, for protein phosphorylation and markers for breast cancer epidermal growth factor receptor Body (EGFR), which is associated, to be probed into.It is well known that amino acid forms polypeptide chain in the way of " dehydrating condensation ", then pass through Bending fold constitutes the matter protein for having certain space configuration, since its space structure and construction are complex, so I Research object is located in the phosphorylation of polypeptide, the main function that the phosphorylation of tyrosine is played in addition, therefore pass through building The method of Ag-Ab " sandwich " structure probe carries out the special of phosphorylation site to the tyrosine on the polypeptide chain of EGFR Property Study of recognition, so as to further carry out breast cancer non-destructive testing research.
To achieve the above object, the present invention adopts the following technical scheme:
A method of building SERS spectra probe in detecting markers for breast cancer EGFR phosphorylated tyrosine includes the following steps:
1) gold and silver core-shell nanostructure is synthesized
It 1-1) prepares gold nano colloid (Au NPs): chlorauric acid solution being transferred in conical flask, magnetic agitation is placed it in and adds It stirs and heats on hot device, 1% sodium citrate solution is added dropwise after solution boiling, observation color gradually becomes claret, keeps stirring Stop after mixing boiling a period of time, is placed under room temperature environment and cools down, for use；
1-2) Au@4-MBAWith Au@4-MBSynthesis: measure gold nano colloid in conical flask, in the continuous agitation of magnetic stir bar Under be added dropwise dropwise to mercaptobenzoic acid (4-MBA), be kept stirring 1 hour, thus in Au NPs surface modification Raman labels point Sub- 4-MBA(Au@4-MBA)；Similarly, there is mark molecule to sulfydryl benzonitrile 4-MB(Au in gold nanoparticle surface modification @4-MB)；
1-3) prepare gold and silver core-shell nano Au@4-MBA@Ag and Au@4-MB@Ag: by Au@4-MBAColloid is transferred in centrifuge tube Centrifugation, eccentric cleaning 2 times, it is therefore an objective to extra mark molecule 4-MBA be removed, then, supernatant liquor is removed, by remaining colloid It is transferred in conical flask, is resuspended in distilled water.Under magneton stirring, ascorbic acid solution is added dropwise, then, is added dropwise dropwise AgNO3 solution observes liquid color by claret and gradually becomes orange, and reaction time 1h, forming mark molecule is 4-MBA Gold and silver core-shell structure (Au@4-MBA@Ag) nanometer particle colloid.Same operation, our available internal standard molecules are 4- Gold and silver core-shell structure (the Au@of MB4-MB@Ag) nanometer particle colloid.
2) modifying DNA is single-stranded
It 2-1) prepares S1-Au plate: using milli-Q water golden plate 5 times first, then wash 3-4 again with PBS buffer solution It returns, is then immersed in single-stranded (S1) solution of DAN after drying in mild nitrogen stream, impregnate 16h, take out and use PBS Buffer solution cleans 3 times again, then dry in mild nitrogen, to modify upper DNA single-stranded (S1) in golden plate, obtains S1-Au plate。
2-2) prepare S2-Au@4-MB@Ag: by Au@4-MB@Ag nano-colloid is transferred in centrifuge tube, after eccentric cleaning 2 times, In the PBS buffer solution being resuspended in, single-stranded (S2) solution of DNA is added, 16h is reacted under stiring, thus in Au@4-MB@ The upper single-stranded S2 of DNA is modified on the core-shell structure of Ag, obtains S2-Au@4-MB@Ag；
Wherein, the sequence of the single-stranded S1 of DNA is AAAATGTCCAAGGCT-SH, and the sequence of the single-stranded S2 of DNA is TTTTACAGGTTCCGA-SH。
3) modifies anti-EGFR phosphotyrosine antibody
In S2-Au@4-MB@Ag nano-colloid surface modification EGFR phosphotyrosine antibody, will be modified with S2-Au@first4-MB@ Ag colloid eccentric cleaning 2 times, then be resuspended in PBS buffer solution, under magnetic stirring, anti-tyrosine phosphorus is added dropwise It is acidified antibody, reaction time 2h.Same operation, in the Au@of synthesis4-MBAAnti- tyrosine phosphorylation is also added in@Ag colloid Antibody, so that the core-shell structure surface modification in mark molecule 4-MBA has antibody.
Finally, bovine serum albumin(BSA) (BSA) closing is all separately added into after modification in two kinds of nucleocapsid nano-colloids.
4) is assembled
S1-Au plate and S2-Au@4-MBThe assembling of@Ag: the S2-Au@of antibody will be modified with4-MB@Ag colloid eccentric cleaning 2 After secondary, then the golden plate for modifying S1 impregnated wherein, the base of reaction time 12h, S1 and S2 by complementary pairing, thus Realize S1-Au plate and S2-Au@4-MBThe self assembly of@Ag.Next, being cleaned 3 times with PBS buffer solution, and mild It is dry in nitrogen stream, it prepares for next connection antigen.
After being completed, substrate is immersed in the solution of EGFR phosphorylated tyrosine of various concentration, the reaction time is Then 2h is cleaned 3 times with PBS buffer solution.Then it is immersed in the Au@that surface modification has antibody again4-MBA@Ag core-shell nano In colloid, reaction time 2h.It is washed 3 times with PBS buffer solution, and dry in mild nitrogen stream, is next SERS Measurement is prepared.
In the present invention, step 1-2) by Raman labels molecule to mercaptobenzoic acid (4-MBA) and internal standard molecule to sulfydryl benzene Formonitrile HCN (4-MB) modifies the middle layer in gold and silver core-shell structure, can tie to avoid next experimental procedure to its property, space Structure and the influence for connecting quantity are conducive to obtain reliable measurement result when detection.
By in golden plate and Au@in step 24-MBThe nanoparticle surface modified DNA of@Ag is single-stranded, can pass through base complementrity Pairing is connected, by Au@4-MB@Ag core-shell structure is assembled in golden plate.
In S2-Au@in step 3)4-MB@Ag and Au@4-MBAAntibody in@Ag core-shell nano surface modification, when sample is molten When containing corresponding antigen in liquid, so that it may be connected two kinds of core-shell structure nanometer particles by the specific recognition of Ag-Ab It picks up and, form " sandwich " structure, to realize the detection to breast cancer mark EGFR phosphorylated tyrosine.
The invention adopts the above technical scheme, using golden plate as substrate, using core-shell structure nanometer particle as surface The substrate for enhancing Raman scattering (SERS) detection develops a kind of highly selective and high sensitivity " sandwich " configuration probe molecule To detect the phosphorylation of markers for breast cancer EGF-R ELISA (EGFR) tyrosine.Using being located in 4-MB Raman spectrum Biomolecule Raman quiet zone peak position is 2221cm-1SERS spectra intensity as internal standard to mark molecule 4-MBA spectral signal Intensity normalized is carried out, can be avoided influence of the biomolecule fingerprint region Raman signal to quantitative detection, so that EGFR phosphorus Phosphorylated tyrosine solution concentration and the linear relationship of SERS intensity are greatly improved, and the SERS spectra probe is further increased Detection sensitivity.Internal standard molecule is located at sulfydryl benzonitrile (4-MB) and Raman labels molecule to mercaptobenzoic acid (4-MBA) The middle layer of golden core and silver-colored shell, to ensure that its property, space structure and quantity etc. are constant, and then ensure the data obtained can By property.Therefore, " sandwich " structure SERS probe constructed in experiment is shown in terms of cancer markers super sensitivity detection Huge potentiality.
Detailed description of the invention
Fig. 1 .(a) it is gold and silver core-shell structure nanometer particle (the Au@synthesized in embodiment4-MB@Ag) transmission electron microscope picture (TEM), (b) for Au NPs, Au@4-MB and Au@4-MB@Ag Nano sol UV-visible absorption spectrum.
Fig. 2 .(a) it is the detected spectrum under solid-state and liquid respectively after golden plate substrate and modification single stranded DNA (S1), (b) resulting spectrum is characterized to carry out SERS to the assembling of each step in experiment.
Fig. 3 .(a) be the concentration of EGFR phosphorylated tyrosine be the SERS spectra within the scope of 1.0 to 100 μ g/L, (b) for from The concentration and 520cm of the EGFR phosphorylated tyrosine of 1.0 to 100 μ g/L-1The standard curve of raman scattering intensity at peak position (c) is To be located at 2221cm in internal standard molecule 4-MB spectrum-1Exist after SERS spectra is normalized in the peak strength at place with concentration The standard curve of EGFR phosphorylated tyrosine within the scope of 1.0 to 100 μ g/L.
Fig. 4 is the SERS signal of " sandwich " structure under normal air environment after exposure 0 day and 3 days.
Specific embodiment
Below with reference to specific embodiment, the present invention will be further described with attached drawing.
1. reagent chlorauric acid solution (HAuCl4), sodium citrate (Na3C6H5O7), to mercaptobenzoic acid (4-MBA), right Sulfydryl benzonitrile (4-MB), ascorbic acid, silver nitrate (AgNO3), phosphate buffer (PBS, PH=7), phosphorylation serine two Peptide (PS-S), phosphorylation threonine dipeptides (PT-T), single stranded DNA (S1:AAAATGTCCAAGGCT-SH, S2: TTTTACAGGTTCCGA-SH), bovine serum albumin(BSA) (BSA), anti-EGFR phosphotyrosine antibody (Anti-EGFR (Phospho-Tyr1172) antibody), antigen EGFR phosphotyrosine peptide (DNPDY (P) QQDFFP), EGFR it is without phosphorus Phosphorylated tyrosine polypeptide (DNPDYQQDFFP).2. the preparation of solution
The preparation of 1% chlorauric acid solution, the specific steps are as follows: firstly, gold chloride solid powder is dissolved in beaker with ultrapure water In, it being then poured into 100mL volumetric flask, cleaning beaker, glass bar 2-3 are returned, cleaning solution be transferred in volumetric flask together, then At graduation mark with ultrapure water constant volume to volumetric flask, after shaking up, it is placed on dark place, for use.Similarly, using similar step come Prepare AgNO3 and ascorbic acid solution.
Prepare 4-MBA the and 4-MB solution of 1mM: weighing quality respectively on counter balance is 0.0077g and 0.00676g To mercaptobenzoic acid and to sulfydryl benzonitrile solid powder, it is poured into the volumetric flask of 50mL respectively, is all made using methanol Dissolve simultaneously constant volume for solvent, after shaking up, is put in and is protected from light place.
It prepares single-stranded (the S1 and S2) solution of DNA: being briefly explained by taking S1 as an example, PBS buffer solution is added in the DNA chain of every OD The initial concentration that 66 μ L are obtained is 100 μM, is then diluted to 50nM.
The preparation of EGFR phosphotyrosine peptide standard solution: for briefly, initial concentration is diluted with PBS buffer solution The standard solution of concentration μ g/L from 1 to 100 a series of is prepared for the stoste of 1mg/mL, then shakes up solution completely, is formed The solution of high degree of dispersion.3. synthesizing gold and silver core-shell nanostructure
1) it prepares gold nano colloid (Au NPs): 1mL chlorauric acid solution being transferred in conical flask, is then poured slowly into 99mL again Deionized water places it on magnetic agitation heater and stirs and heat, and 1% sodium citrate solution is added dropwise after solution boiling (2mL), observation color gradually become claret, are kept stirring boiling 15 minutes and stop, being placed under room temperature environment and cool down, for use.
2) Au@4-MBAWith Au@4-MBSynthesis: the gold nano colloidal sol of 12mL is measured in conical flask, in magnetic stir bar It is 1mM to mercaptobenzoic acid (4-MBA) that 10 μ L concentration are constantly added dropwise under agitation dropwise, is kept stirring 1 hour, thus in Au NPs surface modification Raman labels molecule 4-MBA(Au@4-MBA).Similarly, the gold nano of 12mL is still added in conical flask Colloidal sol, under the continuous agitation of magneton, it is 10 that concentration, which is added dropwise,-3M to 10 μ L of sulfydryl benzonitrile, be kept stirring 60min, In Gold nanoparticle surface modification has mark molecule 4-MB(Au@4-MB).
3) gold and silver core-shell nano Au@is prepared4-MBA@Ag and Au@4-MB@Ag: by Au@4-MBAColloid is transferred in centrifuge tube Centrifugation, adjustings revolving speed are 10000rpm, time 10min, eccentric cleaning 2 times, it is therefore an objective to by extra mark molecule 4-MBA shifting It removes.Then, supernatant liquor is removed, remaining colloid is transferred in conical flask, is resuspended in 10mL distilled water.Under magneton stirring, The ascorbic acid solution 2mL of 0.1M is added dropwise, then, 2.5mL AgNO3 solution (10 is added dropwise dropwise-3M), liquid color is observed Gradually become orange, reaction time 1h by claret, forms gold and silver core-shell structure (the Au@that mark molecule is 4-MBA4-MBA@ Ag) nanometer particle colloid.Same operation, gold and silver core-shell structure (the Au@that our available internal standard molecules are 4-MB4-MB@ Ag) nanometer particle colloid.4. modifying DNA is single-stranded
1) it prepares S1-Au plate: using milli-Q water golden plate 5 times first, then wash 3-4 again with PBS buffer solution and return, Then it in mild nitrogen stream after drying, is immersed in single-stranded (S1) solution of DNA that concentration is 50nM, impregnates 16h, take It cleans again 3 times out and with PBS buffer solution, it is then dry in mild nitrogen, so that it is single-stranded to modify upper DNA in golden plate (S1), S1-Au plate is obtained.
2) S2-Au@is prepared4-MB@Ag: by Au@4-MB@Ag nano-colloid is transferred in centrifuge tube, will after eccentric cleaning 2 times It is resuspended in the PBS buffer solution of 1mL, and the single-stranded S2 solution of 1nM DNA is added, 16h is reacted under stiring, thus in Au @4-MBThe upper single-stranded S2 of DNA is modified on the core-shell structure of@Ag, obtains S2-Au@4-MB@Ag.The 5 anti-EGFR phosphorylation junket ammonia of modification Sour antibody
In S2-Au@4-MB@Ag nano-colloid surface modification EGFR phosphotyrosine antibody, it will be modified with S2-Au@4- first MB@Ag colloid eccentric cleaning 2 times, then be resuspended in the PBS buffer solution of 1mL, under magnetic stirring, it is added dropwise dense Degree is the anti-tyrosine phosphorylation antibody (100 μ L) of 100 μ g/mL, reaction time 2h.Same operation, in the Au@of synthesis Also the anti-tyrosine phosphorylation antibody of 1 μ g is added in 4-MBA@Ag colloid, so that the core-shell structure surface in mark molecule 4-MBA is repaired It is decorated with antibody.Finally, the closing of 5% bovine serum albumin(BSA) is all separately added into after modification in two kinds of nucleocapsid nano-colloids (BSA).
6 assemblings
S1-Au plate and S2-Au@4-MBThe assembling of@Ag: the S2-Au@of antibody will be modified with4-MB@Ag colloid eccentric cleaning 2 times Afterwards, then by the golden plate (S1-Au plate) for modifying S1 it impregnates wherein, the base of reaction time 12h, S1 and S2 pass through complementation Pairing, to realize S1-Au plate and S2-Au@4-MBThe self assembly of@Ag.Next, cleaned 3 times with PBS buffer solution, and It is dry in mild nitrogen stream, it prepares for next connection antigen.
After being completed, substrate is immersed in the solution of EGFR phosphorylated tyrosine of various concentration, the reaction time is Then 2h is cleaned 3 times with PBS buffer solution.Then it is immersed in the Au@that surface modification has antibody again4-MBA@Ag core-shell nano In colloid, reaction time 2h.It is washed 3 times with PBS buffer solution, and dry in mild nitrogen stream, is next SERS Measurement is prepared.
The present invention restores silver-colored shell by ascorbic acid, is wrapped in golden core outer layer and forms gold and silver nano-core-shell structure, by saturating Radio mirror (TEM) its structure visible in detail, as shown in Fig. 1 (a), TEM figure shows Au@4-MB@Ag core-shell nano colloidal solid More uniform unification, partial size are about 40 ± 5nm.In addition, by the Au@of synthesis4-MB@Ag colloid passes through uv-visible absorption spectra table Sign, the black spectral line showed such as Fig. 1 (b) indicate that the maximum absorption band of Au NPs is located at 521cm-1Left and right, is repaired on the surface Au NPs After adoring internal standard molecule 4-MB, as figure red line is shown, maximum absorption band is still located at 521cm-1, illustrate repairing for 4-MB There is no have an impact gold nanoparticle to decorations.What blue line indicated is to wrap up silver-colored shell synthesis Au@4-MB@Ag core-shell glue, is opened up at this time Reveal two peak packets, wherein the maximum absorption band of Au NPs is by 521 cm-1Move on to 495cm-1Place, and in 400cm-1The peak packet at place It is the absorption peak of silver, further proves the formation of core-shell structure.
After characterizing golden plate and modification single stranded DNA (S1), whether shadow can be generated to the spectral signal of Raman dye molecule It rings, golden plate is detected with SERS technology in experiment, as shown in Figure 2 (a), black line is the Raman peaks of golden plate, can be observed It is generated to simultaneously no signal.In order to further prove, PBS buffer solution has been added dropwise in we in golden plate, and in solution and solid shape Raman detection is carried out under state respectively, as shown in spectral line red and blue in figure, is still occurred without any Raman peaks, it is possible thereby to Illustrate that the Raman background of golden plate can't generate interference to the spectrum measured is tested below.Then under liquid and solid-state, to DNA The golden plate (S1-Au plate) of single-stranded self assembly carries out Raman detection, if the green in figure is shown with pink colour spectral line, still not Have Raman peaks appearance, should the result shows that be connected in golden plate DNA it is single-stranded after, do not generate any apparent spectral signal, show Signal to subsequent experimental dye molecule 4-MBA is simultaneously noiseless.Next, we are to the modification and group in each step experimentation Row trace detection is put into, as shown in Fig. 2 (b), red line is to the nucleocapsid for being 4-MB by internal standard molecule by S1 and S2 complementary pairing Nanoparticle (Au@4-MB@Ag) it is connected to the SERS detection carried out in golden plate, it can be observed that the SERS spectra of 4-MB.Then, It is detected to the golden plate substrate after EGFR-Phospho-Tyr antigenic solution is impregnated, it is and red as shown in the blue spectral line in figure Chromatographic line is compared, and the spectral signal intensity of 4-MB is very nearly the same, is shown after having impregnated antigenic solution, to the assembling in golden plate Au@4-MBThe quantity of@Ag core-shell nano, which does not generate, to be interfered significantly with, to ensure that the reliability of measurement data.Green line Indicate the core shell nanoparticles (Au that synthesis is had to mark molecule 4-MBA4-MBA@Ag) it is connected to the SERS light detected in substrate It composes, is obviously shown other than the signal of 4-MB in figure, while also detecting the SERS signal of 4-MBA, show to be modified with The Au@of EGFR tyrosine phosphorylation antibody4-MBAThe phosphorylation that@Ag core-shell nano passes through the EGFR tyrosine in identification solution Antigen, which is connected to, is assembled with Au@4-MBIn the golden plate of@Ag.
In the present invention, using designed " sandwich " structure, specific recognition is carried out by Ag-Ab, in standard The phosphorylation of the EGFR tyrosine of various concentration is detected in solution, available EGFR tyrosine phosphorylation concentration is from 1.0 to 100 A series of SERS spectras of μ g/L.It as shown in Fig. 3 (a), can also while can detect the SERS spectral peak of internal standard molecule 4-MB To detect the spectral signal of mark molecule 4-MBA, the SERS spectra characteristic peak of 4-MBA is in 520cm-1Place, and 589 He of peak position 2221cm-1It is Raman peaks specific to 4-MB, peak position 1074,1578cm-1Etc. being two kinds of coefficient results of dye molecule. In order to accurately prove influence of the concentration of EGFR phosphorylated tyrosine to SERS intensity, we, which have monitored, is located at 520cm-1Place The intensity of SERS peak position.As shown in Figure 3 (b), increase in 100 μ g/L, 4-MBA spectrum with antigen concentration from 1.0 μ g/L 520cm-1SERS intensity at peak position has progressively increased to 6737.14 (a.u.) by 1021.04, resulting standard curve be y= 57.04x+1658.6, it should be noted that calculate resulting coefficient R2 = 0.849.In order to obtain better phase relation Number, the method that we utilize internal standard molecule normalized.In experiment, we select 4-MB as internal standard molecule, due to 4-MB With the spectral peak (2221cm for being located at biological quiet zone-1), carrying out calculating using this peak position can be to avoid the spectrum within " fingerprint region " The interference that peak generates.It as shown in Figure 3 (c), is 2221cm using the Raman peak position that 4-MB is located at biological quiet zone-1The peak value at place is strong It spends and intensity normalization is carried out to spectrum of the Raman labels molecule to mercaptobenzoic acid (4-MBA), by 520cm in 4-MBA spectrum-1Peak 2221cm in the SERS intensity and 4-MB spectrum of position-1The SERS intensity of peak position is compared, and resulting ratio is as ordinate, Ke Yiguan Observing antigen concentration increases to 100 μ g/L, ratio (I from 1.0520/I2221) variation, be important to calculate resulting phase relation Number is R2=0.990, it is compared to and directly substitutes into the resulting R of calculating using SERS intensity2, R at this time2Tool increases significantly, A good linear relationship is presented in the concentration and SERS intensity for also showing EGFR phosphorylated tyrosine.
In order to verify building " sandwich " structure probe stability, we are 100 μ assembled substrate and concentration After the EGFR phosphorylated tyrosine standard solution interaction of g/L, Au@is reconnected4-MBA@Ag nano particle, is put in air Three days in environment.Fig. 4, which shows, to be exposed to fully assembled substrate in common air environment zero day and three days In the case of, detected SERS signal, it can clearly be observed that Raman signal is not obviously reduced.Therefore, we can be with It draws a conclusion, the SERS sensor being exposed in air in short term not will lead to the obvious decaying of SERS signal.

Claims (4)

1. a kind of method for constructing SERS spectra probe in detecting markers for breast cancer EGFR phosphorylated tyrosine, it is characterised in that: Include the following steps:
1) gold and silver core-shell nanostructure is synthesized
It 1-1) prepares gold nano colloid Au NPs: using sodium citrate solution as reducing agent, chlorauric acid solution being restored, to obtain Gold nano colloid Au NPs；
1-2) Au@4-MBAWith Au@4-MBSynthesis: measure gold nano colloid Au NPs in conical flask, be added dropwise 4-MBA, keep stirring It mixes, to obtain Au@in gold nano colloid Au NPs surface modification Raman labels molecule 4-MBA4-MBA；
Gold nano colloid Au NPs is measured in conical flask, 4-MB is added dropwise, is kept stirring, thus in gold nano colloid Au NPs table Mark molecule 4-MB has been modified in face, obtains Au@4-MB；
1-3) prepare gold and silver core-shell nano Au@4-MBA@Ag and Au@4-MB@Ag: the Au@after eccentric cleaning4-MBAIn colloid by It is added dropwise to ascorbic acid solution, then, AgNO3 solution is added dropwise dropwise, forms the gold and silver core-shell structure that mark molecule is 4-MBA Au@4-MBA@Ag nanometer particle colloid；
Au@after eccentric cleaning4-MBAscorbic acid solution is added dropwise in colloid, then, AgNO3 solution is added dropwise dropwise, is formed Internal standard molecule is the gold and silver core-shell structure Au@of 4-MB4-MB@Ag nanometer particle colloid；
2) modifying DNA is single-stranded
It 2-1) prepares S1-Au plate: washing golden plate after dry with water and PBS buffer solution first and it is single-stranded to be immersed in DNA In S1 solution, takes out and cleaned again with PBS buffer solution, then re-dry, so that the upper single-stranded S1 of DNA is modified in golden plate, Obtain S1-Au plate；
2-2) prepare S2-Au@4-MB@Ag: the Au@after eccentric cleaning4-MBIt is molten that the single-stranded S2 of DNA is added in@Ag nanometer particle colloid Liquid reacts under agitation, thus in Au@4-MBThe upper single-stranded S2 of DNA is modified on the core-shell structure of@Ag nanometer particle colloid, is obtained To S2-Au@4-MB@Ag；
3) anti-EGFR phosphotyrosine antibody is modified
S2-Au@after eccentric cleaning4-MBIn@Ag, under magnetic stirring, anti-tyrosine phosphorylation antibody is added dropwise, reacts For a period of time, thus in S2-Au@4-MB@Ag surface modification EGFR phosphotyrosine antibody；
Au@after eccentric cleaning4-MBAIn@Ag, under magnetic stirring, anti-tyrosine phosphorylation antibody, reaction one is added dropwise The section time, thus in Au@4-MBA@Ag surface modification EGFR phosphotyrosine antibody；
Finally, bovine serum albumin(BSA) closing is all separately added into after modification in two kinds of nucleocapsid nano-colloids；
4) it assembles
4-1) S1-Au plate and S2-Au@4-MBThe assembling of@Ag: the S2-Au@of antibody will be modified with4-MB@Ag eccentric cleaning Afterwards, then by the golden plate S1-Au plate for modifying S1 it impregnates wherein, reaction a period of time, the base of S1 and S2 are by mutually recruiting It is right, to realize S1-Au plate and S2-Au@4-MBThe self assembly of@Ag；Next, after being cleaned with PBS buffer solution, then do It is dry, it prepares for next connection antigen；
After 4-2) being completed, substrate is immersed in the solution of EGFR phosphorylated tyrosine of various concentration, when reacting one section Between, it is then cleaned with PBS buffer solution, is then immersed in the Au@that surface modification has antibody again4-MBAIn@Ag, one section is reacted Then time is washed with PBS buffer solution, and dry in mild nitrogen stream, is prepared for next SERS measurement.
2. a kind of building SERS spectra probe in detecting markers for breast cancer EGFR phosphorylation junket ammonia according to claim 1 The method of acid, it is characterised in that: the single-stranded S2 sequence of DNA single-stranded S1 and DNA in step 2 is respectively as follows: AAAATGTCCAAGGCT- SH and TTTTACAGGTTCCGA-SH.
3. a kind of building SERS spectra probe in detecting markers for breast cancer EGFR phosphorylation junket ammonia according to claim 1 The method of acid, it is characterised in that: step 1-1) specific as follows: chlorauric acid solution is transferred in conical flask, magnetic force is placed it in It stirring and heats on agitating and heating device, 1% sodium citrate solution is added dropwise after solution boiling, observation color gradually becomes claret, Stop after being kept stirring boiling a period of time, to obtain gold nano colloid Au NPs.
4. a kind of building SERS spectra probe in detecting markers for breast cancer EGFR phosphorylated tyrosine according to claim 1 Method, it is characterised in that: step 1-2) mixing time be 1h, step 2-2) reaction time be 16h, the reaction of step 3) Time is 2h, step 4-1) reaction time be 12h, step 4-2) reaction time be 2h.
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111996186A (en) * 2020-08-10 2020-11-27 大连理工大学 Preparation method of probe positioning modified soluble nano core cap particles
CN112505017A (en) * 2020-11-19 2021-03-16 福建师范大学 Method for detecting IL-6 in blood based on SERS technology
CN112730379A (en) * 2020-12-24 2021-04-30 北京信息科技大学 Nano Au film surface enhanced Raman sensing array substrate and preparation method thereof
CN112763474A (en) * 2020-12-23 2021-05-07 中国医学科学院血液病医院(中国医学科学院血液学研究所) Biomarker for predicting or detecting acute leukemia
CN112986211A (en) * 2021-03-10 2021-06-18 福建师范大学 Method for high-precision detection of tumor marker in human blood by aptamer SERS sensor capable of being triggered in targeted mode and self-calibrated mode
CN113125411A (en) * 2021-04-29 2021-07-16 江苏大学 SERS (surface enhanced Raman Scattering) probe for detecting patulin as well as preparation method and application thereof
CN114923893A (en) * 2022-06-02 2022-08-19 西北大学 Nondestructive label-free rapid breast cancer Raman spectrum pathological grading and staging method
CN115219428A (en) * 2022-08-15 2022-10-21 新疆师范大学 Interference-free SERS probe and preparation method and application thereof
CN115356324A (en) * 2022-08-15 2022-11-18 福州汉佰康生物科技有限公司 Method for detecting lung cancer tumor marker by using surface enhanced Raman spectroscopy

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105274195A (en) * 2014-12-11 2016-01-27 临沂大学 Kit for detection of cancer marker microRNAs
US20160077090A1 (en) * 2013-10-15 2016-03-17 Board Of Trustees Of The University Of Arkansas Nanocomposites and methods of making same
US20160153975A1 (en) * 2013-10-15 2016-06-02 Board Of Trustees Of The University Of Arkansas Nanocomposites, methods of making same, and applications of same for multicolor surface enhanced raman spectroscopy (sers) detections
CN109612977A (en) * 2018-12-27 2019-04-12 华东理工大学 Amplify biological marker object detecting method without enzyme signal based on Surface enhanced Raman spectroscopy
CN109991207A (en) * 2019-04-25 2019-07-09 福建师范大学 A kind of SERS sensor and its preparation and detection method for detecting the sandwich structure of tyrosinase

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160077090A1 (en) * 2013-10-15 2016-03-17 Board Of Trustees Of The University Of Arkansas Nanocomposites and methods of making same
US20160153975A1 (en) * 2013-10-15 2016-06-02 Board Of Trustees Of The University Of Arkansas Nanocomposites, methods of making same, and applications of same for multicolor surface enhanced raman spectroscopy (sers) detections
CN105274195A (en) * 2014-12-11 2016-01-27 临沂大学 Kit for detection of cancer marker microRNAs
CN109612977A (en) * 2018-12-27 2019-04-12 华东理工大学 Amplify biological marker object detecting method without enzyme signal based on Surface enhanced Raman spectroscopy
CN109991207A (en) * 2019-04-25 2019-07-09 福建师范大学 A kind of SERS sensor and its preparation and detection method for detecting the sandwich structure of tyrosinase

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
KYUNG JIN YOON等: "Bioanalytical Application of SERS Immunoassay for Detection of Prostate-Specific Antigen", 《BIOANALYTICAL APPLICATION OF SERS IMMUNOASSAY》 *
SISI MA等: "Interference-Free Surface-Enhanced Raman Scattering Tags for Single-Cell Molecular Imaging with a High Signal-to-Background Ratio", 《ADVANCED SCIENCE NEWS》 *
YA-FEI TIAN等: "Highly sensitive detection of exosomes by SERS using gold nanostar@Raman reporter@nanoshell structures modified with a bivalent cholesterol-labeled DNA anchor", 《ANALYST》 *
YANJIAOZHANG等: "A preliminary study of surface enhanced Raman scattering immunoassay based on graphene oxide substrate", 《OPTIK》 *
葛明等: "SERS标记免疫检测研究进展", 《光谱学与光谱分析》 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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CN112730379B (en) * 2020-12-24 2023-07-14 北京信息科技大学 Nano Au film surface enhanced Raman sensing array substrate and preparation method thereof
CN112730379A (en) * 2020-12-24 2021-04-30 北京信息科技大学 Nano Au film surface enhanced Raman sensing array substrate and preparation method thereof
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