CN108562569A - A kind of circulating tumor cell detection method based on Surface enhanced Raman spectroscopy probe - Google Patents
A kind of circulating tumor cell detection method based on Surface enhanced Raman spectroscopy probe Download PDFInfo
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Abstract
The present invention relates to spectral technique field, specifically a kind of circulating tumor cell detection method based on Surface enhanced Raman spectroscopy probe includes the following steps：Then structure detects the Surface enhanced Raman spectroscopy probe for circulating tumor cell using luteinizing hormone releasing hormone as the Surface enhanced Raman spectroscopy probe of targeted molecular.The Surface enhanced Raman spectroscopy probe of the present invention is quicker, convenient when being detected for circulating tumor cell, and cost is lower, and has enough specificity and sensitivity for circulating tumor cell detection, and a key tactics can be provided for clinical practice.
Description
Technical field
The present invention relates to spectral technique fields, specifically, being a kind of cycle based on Surface enhanced Raman spectroscopy probe
Tumour cell detection method.
Background technology
Circulating tumor cell (Circulating tumor cells, CTCs) refers to being released into blood from situ tumor, is passed through
Blood occurs to shift and is colonized in other positions of body and causes the micro tumour cells of metastases, such cell is pernicious swollen
Key player is played during tumor metastasis, therefore is that malignant tumour causes dead major reason.Research shows that breast cancer,
Lung cancer, cancer of pancreas, prostate cancer, the CTCs levels of ovarian cancer patients body and its progression free survival phase and Overall survival have close
The relationship cut, thus the early stage of CTCs, simplicity, quickly detection for improve tumor patient prognosis it is most important.But according to
Report CTCs only has 5cells/7.5ml in the patient's bodies minimum content such as breast cancer, lung cancer, prostate cancer, is in a kind of blood
Low concentration cell, therefore realize that the detection of CTCs is proposed higher requirement to the sensitivity of detection method and specificity.
Surface enhanced Raman spectroscopy (Surface enhanced Raman scattering, SERS) is based on Raman spectrum
Principle, by the Chemical enhancement of noble metal nano particles such as gold/silver etc. and the mechanism of Electromagnetic enhancement, to generate than commonly drawing
Graceful spectrum is strong by 102~1012Signal again.SERS is narrow with Raman spectrum spectral peak, peak position and corresponding chemical structure correspondence are strong, spectrum
The characteristics of peak abundant information, its ultrasensitiveness in addition, therefore can be as a kind of effective detection means of CTCs.But due to simple
SERS technologies be unable to specific recognition CTCs, therefore the general detection that cannot be directly used to CTCs in blood.
Surface enhanced Raman spectroscopy probe (SERS tags/probes, SERS probe) is then to be based on noble metal nano particles
The principle that SERS signal can be enhanced modifies corresponding nano-particle according to research purpose, is met with overcoming in detection
The specificity that arrives is insufficient, sample itself Raman signal is faint, the relevant issues such as nanoparticle agglomerates caused by sample.SERS probes
Basic structure be generally made of four parts：1. gold medal/silver nano-grain, i.e., commonly referred SERS substrates, in SERS probes
Still the effect of enhancing Raman signal is played；2. Raman-active species, such as to mercaptobenzoic acid, rhodamine isothiocyanate, connection
Pyridine etc., as the signal source in a kind of indirect detection mode；3. clad, such as biomolecule, high molecular polymer, lipid
Body, silicon etc., it is main to play the effect for reducing non-specific adsorption and enhancing nanoparticles stable；4. targeted molecular, as antibody,
Aptamers, biomolecule etc..According to the above structure by being modified step by step noble metal nano particles, to which structure is suitable for
The correspondent probe of research purpose is carried by means of ultrasensitiveness and the modified targeted molecular of probe possessed by SERS technologies itself
The specificity of confession can effectively meet the requirements such as specificity and the sensitivity in CTCs detection process.
Usually structure SERS probes generally use epidermal growth factor antibody (anti-EpCAM antibody), life long
Factor antibody (anti-HER2antibody) etc. is used as targeted molecular, but these targeted moleculars all have kinds of tumor cells
Certain binding ability, targeting range is relatively wide, and specificity is weaker.
Currently, CTCs coherent detections patent mainly by synthesize respective magnetic material, structure separator (it is such as micro-fluidic,
Chip etc.), the methods of CTCs biomarker detection kits complete.SERS is detected for CTCs just to be had early in 2008
Document delivers (Sha M Y, Xu H, Natan M J, et al.Surface-enhanced Raman scattering tags
for rapid and homogeneous detection of circulating tumor cells in the
presence of human whole blood[J].Journal of the American Chemical Society,
2008,130(51):17214-17215.), due to the hypersensitivity that SERS technologies itself have, in addition SERS probes are carried
The specificity of confession, many researchers are dedicated to the expansion research in the field in the subsequent time.The country is by SERS probe techniques
It is less for the patent of CTCs detections or other documents, it is even more to have not been reported using LHRH as the SERS probes of targeted molecular, the neck
There is still a need for continuous research and progress in domain, to provide more clinical detection means.
Invention content
It is the table of targeted molecular that the purpose of the present invention is to provide one kind with luteinizing hormone releasing hormone (LHRH)
Face enhances Raman spectrum (SERS) probe.
Luteinizing hormone releasing hormone (LHRH) is that one kind plays in normal human's hypothalamus-gonad axis in nerve
Secretory hormone is mainly combined with correspondence organ surface receptor play physiological function under normal circumstances；But work as body
When canceration occurs for interior organ, high expression shape is presented in a variety of organs especially Reproductive Organs tumor cell surface in receptor
State, therefore LHRH can be used as a kind of stronger targeted molecular use specific to reproductive endocrine CTCs.
Another object of the present invention, by above-mentioned SERS probes, the spy in overcoming circulating tumor cell (CTCs) to detect
Anisotropic and sensitivity problem establishes a kind of technology for the CTCs for quickly and easily detecting and carrying LHRH targeted moleculars in blood sample, i.e.,
One kind being based on circulating tumor cell (CTCs) detection method of Surface enhanced Raman spectroscopy (SERS) probe.
To achieve the goals above, present invention employs following technical schemes：
Step 1：The structure of SERS probes
After preparing gold nano grain by reduction of sodium citrate method, by comparing unlike signal molecule for modifying gold nano
Stability when particle and signal strength select the signaling molecule of suitable concentration to modify gold nano grain；Then pass through
Priming reaction is carried out to used protective layer molecule, has suitable group to ensure it in application process to combine other objects
Matter then carries out the coupling of protective layer molecule and targeted molecular by peptide formation between protective layer molecule and targeted molecular,
Finally conjugate is modified in nano grain surface to successfully build probe molecule.
Step 2：SERS probes are detected for CTCs
Certain density cell suspension is configured, the nano particle that it is modified with SERS probes and without targeted molecular mixes
It closes and is incubated, the specificity of SERS probes constructed by the check and evaluation after centrifuge washing by Raman spectrum；Configure various concentration ladder
Various concentration cell suspension is mixed with SERS probes centrifuge washing after incubation, passes through the inspection of Raman spectrum by the cell suspension of degree
The sensitivity of constructed SERS probes is estimated in test and appraisal；Using CCK8 methods, by the way that the SERS probes of various concentration are incubated with mixing with cells
It educates to evaluate influence of the various concentration SERS probes to cell activity, it is different from cell incubation using certain density SERS probes
Time is to evaluate influence of the incubation time for cell activity；It is special by aforementioned evaluation using rat blood sample and mixing with cells
Property and sensitivity method assessment SERS probes for analog sample detect when specificity, sensitivity.
The first aspect of the present invention provides a kind of SERS spies that can be targeted tumor cell surface and can be stabilized
Needle, i.e., it is a kind of to be visited with the Surface enhanced Raman spectroscopy (SERS) that luteinizing hormone releasing hormone (LHRH) is targeted molecular
Needle, including enhancing substrate, signaling molecule, protective layer molecule, targeted molecular, the enhancing substrate use 45nm gold particles, institute
The signaling molecule stated is to mercaptobenzoic acid (pMBA), and the protective layer molecule is reduction bovine serum albumin(BSA) (rBSA), institute
The targeted molecular stated is luteinizing hormone-releasing hormone (LHRH).
Preferably, it is described with luteinizing hormone releasing hormone (LHRH) be targeted molecular surface-enhanced Raman light
(SERS) probe is composed, preparation method is：After preparing gold nano grain by reduction of sodium citrate method, select a concentration of 1mM's
Signaling molecule modifies gold nano grain mercaptobenzoic acid (pMBA)；Then by used protective layer molecule also
Aurochs seralbumin (rBSA) carries out priming reaction, then restores bovine serum albumin(BSA) (rBSA) and target by protective layer molecule
The idol of protective layer molecule and targeted molecular is carried out to peptide formation between molecule luteinizing hormone releasing hormone (LHRH)
Connection finally modifies conjugate in nano grain surface to successfully build probe molecule.
It is furthermore preferred that it is described with luteinizing hormone releasing hormone (LHRH) be targeted molecular surface-enhanced Raman
Spectrum (SERS) probe, preparation method specifically include following steps：
By the HAuCl of 0.01% (W/W)4Solution is heated to slightly boiling, is slowly added to 1ml 1% (W/W) C6H5Na3O7·2H2O
Solution can obtain 45nm gold particles after heating 10min；1ml 45nm gold particles solution is mixed into concussion with 50ul 1mM pMBA
5000rpm centrifuges resuspension after 5min, and Au-pMBA can be obtained by so that pMBA is incorporated on gold nano grain；20ml 20mg/ml oxen
Seralbumin (BSA) and 260ul 1M sodium borohydrides (NaBH4) after hybrid reaction 3h 60 DEG C of waters bath with thermostatic control drain surplus hydrogen
RBSA can be obtained, takes 5ml rBSA to be separately added into 30ul 10mM 1- (3- dimethylamino-propyls) 3- ethyl carbodiimide salt after cooling
Hydrochlorate (EDCHCl) and N- hydroxysuccinimides (NHS) activated carboxyl, react 30min after use 3KDa ultra-filtration centrifuge tube
5000rpm centrifuges 10min and removes extra EDC/NHS, and activation rBSA solution then is added in 50ul 2.5mg/ml LHRH solution
In, it is reacted at 4 DEG C overnight after reaction 4h under room temperature, it is extra to be removed using the ultra-filtration centrifuge tube 5000rpm centrifugations 10min of 3KDa
LHRH can obtain rBSA and LHRH conjugates (rBSA-LHRH)；20ul rBSA-LHRH are added dropwise in 1ml Au-pMBA solution,
6000rpm centrifugations resuspension can obtain the SERS probes (Au-pMBA-rBSA-LHRH) after reaction 5min.
The second aspect of the present invention provides a kind of as described above with luteinizing hormone releasing hormone (LHRH) for target
To molecule Surface enhanced Raman spectroscopy (SERS) probe in preparing circulating tumor cell (CTCs) detection reagent or kit
Application.
The third aspect of the present invention provides a kind of circulating tumor cell being based on Surface enhanced Raman spectroscopy (SERS)
(CTCs) detection method, the method include：It builds and is with luteinizing hormone releasing hormone (LHRH) as described above
Then Surface enhanced Raman spectroscopy (SERS) probe of targeted molecular detects SERS probes for CTCs.
Preferably, described circulating tumor cell (CTCs) detection method based on Surface enhanced Raman spectroscopy (SERS),
Specifically include following steps：
A) structure is drawn with the surface enhanced that luteinizing hormone releasing hormone (LHRH) is targeted molecular as described above
Graceful spectrum (SERS) probe；
B it) takes blood sample to be measured to be detached using lymphocyte separation medium, is then mixed with the SERS probes of step A structures
It is incubated, 1350rpm is centrifuged 5min and flushed three times using PBS after incubation, is detected using confocal；Using light
1078cm in spectrum-1The intensity at peak is quantified；Laser intensity is 100mW, time of integration 5s.
One kind of the present invention being based on circulating tumor cell (CTCs) detection method of Surface enhanced Raman spectroscopy (SERS), tool
There is the feature for being incorporated into tumor cell surface LHRH receptors, the specificity issues in detection can be overcome.5cells/ml can be achieved
Detection limit, have good sensitivity.In the detection process of CTCs, probe has lower cell toxicant to tumour cell
Property.In the CTCs detection applications intended using rat serum original mold, there is good specificity and sensitivity (5cells/ml)；
Good linear (R may be implemented within the scope of 5cells/ml~100cells/ml2=0.9803).
The invention has the advantages that：
1, the detection method of the CTCs having thus described the invention based on SERS probes, by using 45nm gold particles as base
Bottom, pMBA are signaling molecule, by modification after LHRH is coupled to rBSA in nano grain surface, can obtain being stabilized simultaneously
Probe molecule with stronger SERS signal can smoothly be used for the detection of CTCs；
2, in addition, by assess the probe molecule for CTCs detect when specificity, sensitivity, cytotoxicity correlation
Performance finds that probe has good effect above three aspect, is suitable for CTCs detections；
3, last, the detection by probe for CTCs in rat simulation blood sample, discovery equally can be real in analog sample
Existing good specificity, sensitivity and the linear response of SERS signal；
4, quicker, convenient when SERS probes of the invention are detected for CTCs, cost is lower, and CTCs is examined
Measuring tool has enough specificity and sensitivity, and a key tactics can be provided for clinical practice.
Description of the drawings
Fig. 1 is the schematic diagram of SERS probes constructed by the present invention；
Fig. 2 is the CTCs testing process schematic diagrames based on SERS probes of the present invention；
Fig. 3 is the phenogram of SERS probes constructed by the present invention；The X-ray electron energy level that wherein A is Au-pMBA is composed, and B is
The SERS signal of Au-pMBA and Au-pMBA-rBSA is as a result, C is the transmission electron microscope picture of 45nm Au particles, D Au-pMBA-
The transmission electron microscope picture of rBSA-LHRH, E are the particle diameter distribution of Au-pMBA and Au-pMBA-rBSA-LHRH, and F is Au-pMBA and Au-
The ultraviolet absorpting spectrum of pMBA-rBSA-LHRH；
Fig. 4 is the assessment figure of specificity when SERS probes constructed by the present invention are used to detect；Wherein A is Au-pMBA-
RBSA-LHRH and the SERS images after the washing of HeLa cell incubations, B are after Au-pMBA-rBSA is washed with HeLa cell incubations
SERS images, C is the transmission electron microscope picture after the washing of Au-pMBA-rBSA-LHRH and HeLa cell incubations, D Au-pMBA
And the SERS signal result after Au-pMBA-rBSA-LHRH and the washing of HeLa cell incubations；
Fig. 5 is the assessment figure of sensitivity when SERS probes constructed by the present invention are used to detect；
Fig. 6 is the influence to cell activity when SERS probes constructed by the present invention are used to detect；Wherein A is containing various concentration
SERS probes culture medium and HeLa cell incubations after survival rate, B is SERS probes and HeLa cell incubation different times
The survival rate of cell afterwards；
Fig. 7 be the present invention constructed by SERS probes for rat simulate blood examination when specificity, sensitivity, linearly comment
Estimate；Wherein A is acquired for the rat blood sample comprising HeLa cells and not comprising HeLa cells by the detection of this research and establishment method
SERS signal, the SERS signal that B is acquired for the rat blood sample of the cells of HeLa containing various concentration, C are thin in 5-100cells/ml
The linear relationship of born of the same parents' concentration and SERS signal intensity.
Specific implementation mode
The detection method for being carried out CTCs using SERS probes to the present invention with reference to embodiment and attached drawing is elaborated.
Embodiment 1
Step 1：The preparation of SERS probes
By the HAuCl of 0.01% (W/W)4Solution is heated to slightly boiling, is slowly added to 1ml 1% (W/W) C6H5Na3O7·2H2O
Solution can obtain 45nm gold particles after heating 10min；1ml 45nm gold particles solution is mixed into concussion with 50ul 1mM pMBA
5000rpm centrifuges resuspension after 5min, and Au-pMBA can be obtained by so that pMBA is incorporated on gold nano grain；20ml 20mg/ml oxen
Seralbumin (BSA) and 260ul 1M sodium borohydrides (NaBH4) after hybrid reaction 3h 60 DEG C of waters bath with thermostatic control drain surplus hydrogen
RBSA can be obtained, takes 5ml rBSA to be separately added into 30ul 10mM 1- (3- dimethylamino-propyls) 3- ethyl carbodiimide salt after cooling
Hydrochlorate (EDCHCl) and N- hydroxysuccinimides (NHS) activated carboxyl, react 30min after use 3KDa ultra-filtration centrifuge tube
5000rpm centrifuges 10min and removes extra EDC/NHS, and activation rBSA solution then is added in 50ul 2.5mg/ml LHRH solution
In, it is reacted at 4 DEG C overnight after reaction 4h under room temperature, it is extra to be removed using the ultra-filtration centrifuge tube 5000rpm centrifugations 10min of 3KDa
LHRH can obtain rBSA and LHRH conjugates (rBSA-LHRH)；20ul rBSA-LHRH are added dropwise in 1ml Au-pMBA solution,
6000rpm centrifugations resuspension can obtain SERS probes (Au-pMBA-rBSA-LHRH) after reaction 5min.
Step 2：The characterization of SERS probes
Nano particle after being reacted with pMBA using x-ray photoelectron spectroscopy pair is analyzed, you can is obtained as shown in Fig. 3 (A)
Energy spectrum diagram, it is possible to find except gold atom peak is ultraviolet, it is seen that an apparent S atom peak, explainable pMBA are combined with gold nano grain；
The SERS spectrograms of Au-pMBA after acquisition only modification pMBA and cladding rBSA can be obtained the SERS of Fig. 3 (B) such as and scheme, it is seen that packet
After covering rBSA, there is a fainter reduction in the SERS signal intensity of pMBA；Gold nano grain and SERS probes are used
Transmission electron microscope (TEM) and granularmetric analysis can obtain the transmission electron microscope picture such as Fig. 3 (C, D) and the particle diameter distribution such as Fig. 3 (E), from
Fig. 3 (C, D) compares visible protein molecular layer and is evenly distributed on around gold nano grain, is about from coating thickness known to Fig. 3 (E)
5nm；The ultra-violet absorption spectrum of gold nano grain and SERS probes is acquired, the ultra-violet absorption spectrum as shown in Fig. 3 (F) can be obtained,
Compared to gold nano grain, the red shift of about 2~3nm occurs for SERS probe maximum absorption wavelengths.
Step 3：SERS probe specificities are investigated
By culture medium：SERS probes (or Au-pMBA-rBSA)=4:1 ratio is separately added into different culture dishes,
It is washed three times with phosphate buffer (PBS) after being incubated 30min, uses Confocal laser-scanning microscopy with 1078cm-1Locate the signal at peak
Imaging analysis is carried out to two ware cells, you can obtain the SERS as shown in Fig. 4 (A, B) and be imaged collection of illustrative plates, it is seen that incubated with SERS probes
Apparent signal but Au-pMBA-rBSA groups (B) the then generation without clear signal can be generated by educating group (A), be illustrated containing targeted molecular
SERS probes due to and cell combination to improve its specificity；Using PBS with 10000 HeLa cells/ml's of restriction
Cell suspension takes 250ul SERS probes and Au-pMBA-rBSA to be mixed respectively with 1ml cell suspensions, after 37 DEG C are incubated 30min
Centrifugation, which is laid equal stress on, is suspended into 200ul, and the cell being incubated with SERS probe groups is carried out transmission electron microscope molecule, in addition takes gained sample
1ul is added dropwise in being detected on silicon chip, you can such as the transmission electron microscope picture of Fig. 4 (C) and the SERS spectra of (D), from (C)
Distribution situation of the SERS probes in cell can generate apparent signal and Au-pMBA-rBSA by (D) visible SERS probe groups
Group is then generated without clear signal；To sum up, with enough specificity when SERS probes are for cell detection.
Step 4：SERS probe sensitivity is investigated
The cell suspension for preparing 5,10,50,100,1000,10000cells/ml respectively using PBS, takes 250ul SERS
Probe is mixed with 1ml cell suspensions, and centrifugation, which is laid equal stress on, after 37 DEG C of incubation 30min is suspended into 200ul, takes 1ul to be added dropwise in gained sample
In being detected on silicon chip, you can SERS spectrograms as shown in Figure 5, as seen from the figure with the increase of cell concentration, gained
The trend of SERS signal also gradual image enhancement, and in the concentration of 5cells/ml, a still visible apparent SERS signal,
With enough sensitivity when illustrating SERS probes for cell detection.
Step 5：The cytotoxicity of SERS probes is investigated
Influence using CCK8 methods assessment probe to cell activity.The cell suspension of debita spissitudo is transferred in 96 orifice plates
(about 5 × 103The holes cells/), after 37 DEG C are incubated 48h, blank well (being free of cell) is respectively set, control wells (are added without SERS spies
Needle), experimental port (be added SERS probes)；Probe in experimental port in varing proportions：Culture medium (7:Isosorbide-5-Nitrae:1,3:1,2:1) it is added
To investigate influence of the various concentration probe to cell, each ratio setting multiple holes 4, in microplate reader 450nm waves after the completion of incubation
Strong point is detected, you can the cell survival rate figure as shown in Fig. 6 (A) is obtained, as seen from the figure in different SERS concentration and probe concentrations
Under, it still can keep 80% or more cell survival rate；In addition, pressing probe：Culture medium ratio 4:1 is added another piece of 96 holes
Plate, use different time incubation time (30,45,60,75min) with investigate different incubation time tests be directed to cell shadow
It rings, is detected at microplate reader 450nm wavelength after the completion of incubation, you can the cell survival rate figure as shown in Fig. 6 (B) is obtained, it can
See the extension with incubation time, cell survival rate is still 80% or more；In summary, constructed SERS probes live to cell
Property influence it is smaller, in detection process have lower cytotoxicity.
Step 6：SERS probes are for simulating blood examination
Rat vein blood is taken, it is (5,10,50,100,1000,10000 thin to configure the mixed blood sample containing different number cells
Born of the same parents), it is sufficiently mixed at 37 DEG C；Using rat lymphocyte separating liquid to containing the rat blood sample of tumour cell (10000 cells)
Detached, take the low-density cellular layer comprising tumour cell and lymphocyte mixed with SERS probes incubation after, 1350rpm from
Heart 5min, is flushed three times using PBS, is detected using confocal, while the rat that setting is not mixed with tumour cell
Blood sample is detected after using same steps to verify specificity；Various concentration mixed blood sample is taken to use lymphocyte separation medium pair
The sample of mixed rumour cell is detached, and is then incubated with SERS probes, and 1350rpm centrifuges 5min simultaneously after incubation
It is flushed three times using PBS, is detected using confocal, by acquiring the signal of various concentration cell suspension to verify inspection
It is the sensitivity of survey mode, linear, Fig. 7 (A, B, C) can be obtained, by Fig. 7 (A) it is found that when SERS probes are for simulating blood examination still
So there is enough specificity, by Fig. 7 (B) it is found that constructed SERS probes are when for simulating blood examination can equally reach
Detection to 5cells/ml limits, and by Fig. 7 (C) it is found that constructed SERS probes for when simulating blood examination 5~
A good linear (R can be obtained between 100cells/ml2=0.9803).
The preferred embodiment of the invention is illustrated above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make various equivalent under the premise of without prejudice to the invention spirit
Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.
Claims (6)
1. a kind of circulating tumor cell detection method based on Surface enhanced Raman spectroscopy, which is characterized in that including：Structure is to promote
Luteinizing hormone releasing hormone is the Surface enhanced Raman spectroscopy probe of targeted molecular, then by the surface-enhanced Raman
Spectral probe is detected for circulating tumor cell；Described increases by the surface of targeted molecular of luteinizing hormone releasing hormone
Graceful spectral probe is haled, including enhancing substrate, signaling molecule, protective layer molecule, targeted molecular, the enhancing substrate use
45nm gold particles, the signaling molecule are to mercaptobenzoic acid, and the protective layer molecule is reduction bovine serum albumin(BSA), institute
The targeted molecular stated is luteinizing hormone-releasing hormone.
2. the circulating tumor cell detection method according to claim 1 based on Surface enhanced Raman spectroscopy, feature exist
In including the following steps：
A) structure it is described using luteinizing hormone releasing hormone as the Surface enhanced Raman spectroscopy probe of targeted molecular；
B it) takes blood sample to be measured to be detached using lymphocyte separation medium, then mixes incubation with the SERS probes of step A structures,
1350rpm is centrifuged 5min and is flushed three times using PBS after incubation, is detected using confocal；Using in spectrum
1078cm-1The intensity at peak is quantified；Laser intensity is 100mW, time of integration 5s.
3. a kind of using luteinizing hormone releasing hormone as the Surface enhanced Raman spectroscopy probe of targeted molecular, feature exists
In, including enhancing substrate, signaling molecule, protective layer molecule, targeted molecular, the enhancing substrate is using 45nm gold particles, institute
The signaling molecule stated is to mercaptobenzoic acid, and the protective layer molecule is reduction bovine serum albumin(BSA), the targeted molecular
For luteinizing hormone-releasing hormone.
4. according to claim 3 using luteinizing hormone releasing hormone as the Surface enhanced Raman spectroscopy of targeted molecular
Probe, which is characterized in that preparation method is：After preparing gold nano grain by reduction of sodium citrate method, a concentration of 1mM is selected
Signaling molecule mercaptobenzoic acid modifies gold nano grain；Then by used protective layer molecule also aurochs
Seralbumin carries out priming reaction, and then restore bovine serum albumin(BSA) by protective layer molecule promotees corpus luteum generation with targeted molecular
Peptide formation carries out the coupling of protective layer molecule and targeted molecular between hormone releasing hormone, finally modifies conjugate in receiving
Rice grain surface is to successfully build probe molecule.
5. according to claim 3 using luteinizing hormone releasing hormone as the Surface enhanced Raman spectroscopy of targeted molecular
Probe, which is characterized in that preparation method includes the following steps：
By the HAuCl of 0.01% (W/W)4Solution is heated to slightly boiling, is slowly added to 1ml 1% (W/W) C6H5Na3O7·2H2O is molten
Liquid can obtain 45nm gold particles after heating 10min；1ml 45nm gold particles solution is mixed to concussion 5min with 50ul 1mM pMBA
5000rpm centrifuges resuspension afterwards, and Au-pMBA can be obtained by so that pMBA is incorporated on gold nano grain；20ml 20mg/ml cow's serums
Albumin drains surplus hydrogen with 60 DEG C of waters bath with thermostatic control after 260ul 1M sodium borohydride hybrid reactions 3h can obtain rBSA, be taken after cooling
5ml rBSA are separately added into 30ul 10mM 1- (3- dimethylamino-propyls) 3- ethyl-carbodiimide hydrochlorides and N- hydroxyls fourth two
Acid imide activated carboxyl uses the ultra-filtration centrifuge tube 5000rpm centrifugations 10min of 3KDa to remove extra EDC/ after reacting 30min
Then 50ul 2.5mg/ml LHRH solution is added in activation rBSA solution by NHS, is reacted at 4 DEG C after reaction 4h under room temperature
At night, rBSA and LHRH conjugates can be obtained by removing extra LHRH using the ultra-filtration centrifuge tube 5000rpm centrifugations 10min of 3KDa
rBSA-LHRH；20ul rBSA-LHRH are added dropwise in 1ml Au-pMBA solution, 6000rpm centrifuges resuspension after reacting 5min
The probe Au-pMBA-rBSA-LHRH can be obtained.
6. it is a kind of as described in claim 3-5 is any using luteinizing hormone releasing hormone as the surface enhanced of targeted molecular
Application of the Raman spectrum probe in preparing circulating tumor cell detection reagent or kit.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109682972A (en) * | 2018-12-19 | 2019-04-26 | 浙江卓运生物科技有限公司 | A kind of SERS reagent detecting breast cancer tumour circulating cells |
| CN109781700A (en) * | 2019-01-18 | 2019-05-21 | 拉曼兄弟(深圳)科技发展有限公司 | A kind of nanoparticle and preparation method thereof for Raman spectrum detection oral squamous cell carcinomas tumour cell |
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| CN114034679A (en) * | 2021-10-15 | 2022-02-11 | 华东师范大学 | Construction and application of high-reproducibility surface-enhanced Raman spectrum platform |
| CN114942223A (en) * | 2022-05-31 | 2022-08-26 | 南京邮电大学 | Ratio type detection kit for circulating tumor cells and preparation method and application thereof |
| CN115896026A (en) * | 2022-11-29 | 2023-04-04 | 四川大学华西医院 | Separation and purification method of circulating tumor cells |
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| CN109682972A (en) * | 2018-12-19 | 2019-04-26 | 浙江卓运生物科技有限公司 | A kind of SERS reagent detecting breast cancer tumour circulating cells |
| CN109781700A (en) * | 2019-01-18 | 2019-05-21 | 拉曼兄弟(深圳)科技发展有限公司 | A kind of nanoparticle and preparation method thereof for Raman spectrum detection oral squamous cell carcinomas tumour cell |
| CN111624189B (en) * | 2019-02-27 | 2023-06-20 | 中国科学院宁波材料技术与工程研究所 | Surface-enhanced Raman scattering magnetic composite nano material for detecting cancer cells |
| CN111624189A (en) * | 2019-02-27 | 2020-09-04 | 中国科学院宁波材料技术与工程研究所 | Surface-enhanced Raman scattering magnetic composite nano material for detecting cancer cells |
| CN109987579A (en) * | 2019-04-12 | 2019-07-09 | 东南大学 | The preparation method of multi-parameter high throughput SERS activity micropin and active micropin |
| CN109987579B (en) * | 2019-04-12 | 2021-05-11 | 东南大学 | Preparation method of multi-parameter high-flux SERS active microneedle and active microneedle |
| CN111551534A (en) * | 2020-05-18 | 2020-08-18 | 上海交通大学 | Kit based on surface enhanced Raman probe, application thereof and imaging method |
| 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 |
| CN113406053A (en) * | 2021-06-22 | 2021-09-17 | 徐州医科大学 | Detection method of tumor cell marker miRNA-21 |
| CN114034679A (en) * | 2021-10-15 | 2022-02-11 | 华东师范大学 | Construction and application of high-reproducibility surface-enhanced Raman spectrum platform |
| CN114034679B (en) * | 2021-10-15 | 2023-11-10 | 华东师范大学 | Construction and application of high-reproducibility surface-enhanced Raman spectrum platform |
| CN114942223A (en) * | 2022-05-31 | 2022-08-26 | 南京邮电大学 | Ratio type detection kit for circulating tumor cells and preparation method and application thereof |
| CN115896026A (en) * | 2022-11-29 | 2023-04-04 | 四川大学华西医院 | Separation and purification method of circulating tumor cells |
| WO2024124740A1 (en) * | 2022-12-13 | 2024-06-20 | 浙江省肿瘤医院 | Circulating tumor cell detection device based on sers technology |
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