CN108562569B - Circulating tumor cell detection method based on surface-enhanced Raman spectrum probe - Google Patents
Circulating tumor cell detection method based on surface-enhanced Raman spectrum probe Download PDFInfo
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Abstract
The invention relates to the technical field of spectroscopy, in particular to a circulating tumor cell detection method based on a surface enhanced Raman spectroscopy probe, which comprises the following steps: constructing a surface enhanced Raman spectroscopy probe which takes luteinizing hormone releasing hormone as a targeting molecule, and then using the surface enhanced Raman spectroscopy probe for circulating tumor cell detection. The surface-enhanced Raman spectrum probe provided by the invention is quicker, more convenient and faster when used for detecting circulating tumor cells, has lower cost, has sufficient specificity and sensitivity for detecting the circulating tumor cells, and can provide a feasible strategy for clinical practice.
Description
Technical Field
The invention relates to the technical field of spectroscopy, in particular to a circulating tumor cell detection method based on a surface enhanced Raman spectroscopy probe.
Background
Circulating Tumor Cells (CTCs) are trace tumor cells released from in situ tumors into blood, metastasized through the blood and colonized at other parts of the body to cause tumor metastasis, and play an important role in the process of metastasis of malignant tumors, thus being an important cause of death caused by malignant tumors. Research shows that the CTCs level of the body of a patient with breast cancer, lung cancer, pancreatic cancer, prostate cancer and ovarian cancer has close relation with the progression-free survival period and the overall survival period, so that the early, simple and rapid detection of the CTCs is important for improving the prognosis of a tumor patient. However, it is reported that the minimum content of CTCs in patients with breast cancer, lung cancer, prostate cancer, etc. is only 5cells/7.5ml, which is a low-concentration cell in blood, and therefore, the realization of detection of CTCs puts high demands on both sensitivity and specificity of the detection method.
Surface Enhanced Raman Spectroscopy (SERS) is based on the principle of Raman spectroscopy and utilizes the mechanisms of chemical enhancement and electromagnetic enhancement of noble metal nanoparticles such as gold/silver and the like to generate Raman light more than ordinary Raman lightSpectral intensity 102～1012Multiplied signal. SERS has the characteristics of narrow Raman spectrum peak, strong correspondence between peak position and corresponding chemical structure and rich spectrum peak information, and is ultrasensitive, so that the SERS can be used as an effective detection means for CTCs. However, since simple SERS techniques do not specifically recognize CTCs, they cannot be directly used for detection of CTCs in blood.
The surface enhanced Raman spectroscopy (SERS tags/probes) is based on the principle that the precious metal nanoparticles can enhance SERS signals, and the corresponding nanoparticles are modified according to research purposes, so that the problems of insufficient specificity, weak Raman signals of samples, nanoparticle agglomeration caused by the samples and the like in detection are solved. The basic structure of a SERS probe generally consists of four parts: 1. gold/silver nanoparticles, often called SERS substrates, still play a role in enhancing raman signals in SERS probes; 2. raman active substances such as p-mercaptobenzoic acid, rhodamine isothiocyanate, bipyridine and the like are used as signal sources in an indirect detection mode; 3. coating layers, such as biomolecules, high molecular polymers, liposomes, silicon and the like, mainly play a role in reducing nonspecific adsorption and enhancing the stability of nanoparticles; 4. targeting molecules, such as antibodies, aptamers, biomolecules, and the like. According to the structure, the noble metal nanoparticles are modified step by step, so that corresponding probes suitable for research purposes are constructed, and the requirements of specificity, sensitivity and the like in the CTCs detection process can be effectively met by means of the ultrasensitiveness of the SERS technology and the specificity provided by target molecules modified by the probes.
Generally, an epidermal growth factor antibody (anti-EpCAM antibody), a human growth factor antibody (anti-HER2antibody) and the like are generally adopted as targeting molecules for constructing the SERS probe, but the targeting molecules have certain binding capacity to various tumor cells, relatively wide targeting range and weak specificity.
At present, the related detection patents of the CTCs are mainly completed by methods of synthesizing corresponding magnetic materials, constructing separation devices (such as microfluidics, chips and the like), detecting kits of the CTCs biomarkers and the like. SERS has been published for CTCs detection as early as 2008 (Sha M Y, Xu H, Natan M J, et al. surface-enhanced Raman scattering tags for doped and halogenated cells in the presence of human white blood [ J ]. Journal of the American Chemical Society,2008,130(51):17214-17215.), and many researchers have devoted extensive research in this field in the following time due to the inherent ultra-high sensitivity of SERS technology, coupled with the specificity provided by SERS probes. The domestic application of SERS probe technology to CTCs detection has few patents or other documents, and SERS probes using LHRH as targeting molecules are not reported, and the field still needs continuous research and progress to provide more clinical detection means.
Disclosure of Invention
The invention aims to provide a Surface Enhanced Raman Spectroscopy (SERS) probe taking Luteinizing Hormone Releasing Hormone (LHRH) as a targeting molecule.
Luteinizing Hormone Releasing Hormone (LHRH) is a hormone which plays a role in neuroendocrine in the hypothalamic-gonadal axis of normal human body, and under normal conditions, mainly binds to corresponding surface receptors of sexual organs to play a physiological function; however, when the organs in the organism are malignant, the receptor of the polypeptide is in a high expression state on the surfaces of tumor cells of various organs, particularly reproductive endocrine organs, so that LHRH can be used as a targeting molecule with stronger specificity to reproductive endocrine CTCs.
Another objective of the present invention is to overcome the problems of specificity and sensitivity in Circulating Tumor Cell (CTCs) detection by using the SERS probe, and to establish a technology for rapidly, simply and conveniently detecting CTCs carrying LHRH target molecules in blood samples, i.e., a method for detecting Circulating Tumor Cells (CTCs) based on a Surface Enhanced Raman Spectroscopy (SERS) probe.
In order to achieve the purpose, the invention adopts the following technical scheme:
the method comprises the following steps: construction of SERS probes
After preparing gold nanoparticles by a sodium citrate reduction method, selecting signal molecules with proper concentration to modify the gold nanoparticles by comparing the stability and signal intensity of different signal molecules when the signal molecules are used for modifying the gold nanoparticles; and then, carrying out activation reaction on the used protective layer molecules to ensure that the protective layer molecules have proper groups to combine with other substances in the application process, then carrying out coupling of the protective layer molecules and the targeting molecules through peptide formation reaction between the protective layer molecules and the targeting molecules, and finally modifying the conjugates on the surfaces of the nanoparticles so as to successfully construct probe molecules.
Step two: SERS probe for CTCs detection
Preparing a cell suspension with a certain concentration, mixing and incubating the cell suspension with an SERS probe and nanoparticles without target molecule modification, and after centrifugal washing, evaluating the specificity of the constructed SERS probe through detection of Raman spectrum; preparing cell suspensions with different concentration gradients, mixing and incubating the cell suspensions with different concentrations with an SERS probe, centrifuging and washing, and evaluating the sensitivity of the constructed SERS probe through detection of Raman spectrum; by adopting a CCK8 method, the influence of SERS probes with different concentrations on the activity of cells is evaluated by mixing and incubating the SERS probes with different concentrations with the cells, and the influence of the incubation time on the activity of the cells is evaluated by incubating the SERS probes with certain concentrations and the cells for different times; rat blood samples and cells are mixed, and the specificity and the sensitivity of the SERS probe are evaluated by the method for evaluating the specificity and the sensitivity to simulate the specificity and the sensitivity of the sample detection.
The invention provides a SERS probe which can be targeted to the surface of tumor cells and can stably exist, namely a Surface Enhanced Raman Spectroscopy (SERS) probe taking Luteinizing Hormone Releasing Hormone (LHRH) as a targeting molecule, which comprises an enhancement substrate, a signal molecule, a protective layer molecule and the targeting molecule, wherein the enhancement substrate adopts 45nm gold particles, the signal molecule is p-mercaptobenzoic acid (pMBA), the protective layer molecule is reduced bovine serum albumin (rBSA), and the targeting molecule is Luteinizing Hormone Releasing Hormone (LHRH).
Preferably, the Surface Enhanced Raman Spectroscopy (SERS) probe with Luteinizing Hormone Releasing Hormone (LHRH) as a targeting molecule is prepared by the following steps: after preparing gold nanoparticles by a sodium citrate reduction method, selecting a signal molecule with the concentration of 1mM to modify the gold nanoparticles with mercaptobenzoic acid (pMBA); and then, the used protective layer molecule is subjected to an activation reaction by reducing bovine serum albumin (rBSA), and then the protective layer molecule is subjected to a peptide reaction between the reduced bovine serum albumin (rBSA) and a targeted molecule Luteinizing Hormone Releasing Hormone (LHRH) to couple the protective layer molecule and the targeted molecule, and finally the conjugate is modified on the surface of the nanoparticle to successfully construct the probe molecule.
More preferably, the Surface Enhanced Raman Spectroscopy (SERS) probe with Luteinizing Hormone Releasing Hormone (LHRH) as a targeting molecule is prepared by the following steps:
0.01% (W/W) of HAuCl4The solution was heated to slight boiling and 1ml of 1% (W/W) C was slowly added6H5Na3O7·2H2Heating the O solution for 10min to obtain 45nm gold particles; mixing 1ml of 45nm gold particle solution with 50ul of 1mM pMBA, shaking for 5min, centrifuging at 5000rpm, and resuspending to combine pMBA on the gold nanoparticles to obtain Au-pMBA; 20ml of 20mg/ml Bovine Serum Albumin (BSA) with 260ul of 1M sodium borohydride (NaBH)4) After the mixing reaction is carried out for 3h, excess hydrogen is discharged in a constant-temperature water bath at 60 ℃ to obtain rBSA, 5ml of rBSA is taken and added into 30ul of 10mM 1- (3-dimethylaminopropyl) 3-ethylcarbodiimide hydrochloride (EDC. HCl) and N-hydroxysuccinimide (NHS) activated carboxyl respectively after cooling, after the reaction is carried out for 30min, the excess EDC/NHS is removed by adopting a 3KDa ultrafiltration centrifugal tube to carry out centrifugation at 5000rpm for 10min, then 50ul of 2.5mg/ml LHRH solution is added into the activated rBSA solution, after the reaction is carried out for 4h at normal temperature, the reaction is carried out at 4 ℃ overnight, and the excess LHRH is removed by adopting a 3KDa ultrafiltration centrifugal tube to carry out centrifugation at 5000rpm for 10min to obtain a rBSA and LHRH conjugate (rBSA-LHRH; 20ul rBSA-LHRH is dripped into 1ml Au-pMBA solution, and after 5min of reaction, 6000rpm centrifugation is carried out to resuspend to obtain the SERS probe (Au-pMBA-rBSA-LHRH).
In a second aspect of the invention, an application of the Surface Enhanced Raman Spectroscopy (SERS) probe with Luteinizing Hormone Releasing Hormone (LHRH) as a targeting molecule in preparation of a Circulating Tumor Cell (CTCs) detection reagent or kit is provided.
In a third aspect of the present invention, a method for detecting Circulating Tumor Cells (CTCs) based on Surface Enhanced Raman Spectroscopy (SERS) is provided, the method comprising: a Surface Enhanced Raman Spectroscopy (SERS) probe with Luteinizing Hormone Releasing Hormone (LHRH) as a targeting molecule as described above was constructed and then used for CTCs detection.
Preferably, the method for detecting Circulating Tumor Cells (CTCs) based on Surface Enhanced Raman Spectroscopy (SERS) specifically comprises the following steps:
A) constructing a Surface Enhanced Raman Spectroscopy (SERS) probe with Luteinizing Hormone Releasing Hormone (LHRH) as a targeting molecule;
B) separating a blood sample to be detected by adopting lymphocyte separation liquid, mixing and incubating with the SERS probe constructed in the step A, centrifuging at 1350rpm for 5min after incubation is finished, washing with PBS for three times, and detecting by adopting confocal Raman; by using 1078cm in the spectrum-1Quantifying the intensity of the peak; the laser intensity is 100mW, and the integration time is 5 s.
The method for detecting Circulating Tumor Cells (CTCs) based on Surface Enhanced Raman Spectroscopy (SERS) has the characteristic of being combined with LHRH receptors on the surfaces of the tumor cells, and can overcome the specificity problem in detection. The detection limit of 5cells/ml can be realized, and the sensitivity is good. During the detection of CTCs, the probe has low cytotoxicity to tumor cells. In the CTCs detection application simulated by rat blood samples, the specificity and the sensitivity (5cells/ml) are good; good linearity (R) can be achieved in the range of 5cells/ml to 100cells/ml2＝0.9803)。
The invention has the advantages that:
1. according to the detection method of the CTCs based on the SERS probe, which is described by the invention, 45nm gold particles are used as a substrate, pMBA is used as a signal molecule, LHRH is coupled to rBSA and then modified on the surface of a nanoparticle, so that the probe molecule which stably exists and has stronger SERS signals can be obtained, and the detection method can be smoothly used for the detection of the CTCs;
2. in addition, by evaluating the specificity, sensitivity and cytotoxicity related performance of the probe molecule when used for detecting CTCs, the probe is found to have good effects in the three aspects and be suitable for detecting CTCs;
3. finally, the probe is used for detecting CTCs in rat simulated blood samples, and the fact that good specificity and sensitivity and linear response of SERS signals can be achieved in the simulated samples is found;
4. the SERS probe provided by the invention is quicker and more convenient when being used for CTCs detection, has lower cost, has sufficient specificity and sensitivity for CTCs detection, and can provide a feasible strategy for clinical practice.
Drawings
FIG. 1 is a schematic diagram of a SERS probe constructed in accordance with the present invention;
FIG. 2 is a schematic diagram of the detection process of CTCs based on SERS probe according to the present invention;
FIG. 3 is a representation of a SERS probe constructed in accordance with the present invention; wherein A is an X photoelectron energy level spectrum of Au-pMBA, B is SERS signal results of Au-pMBA and Au-pMBA-rBSA, C is a transmission electron microscope picture of 45nm Au particles, D is a transmission electron microscope picture of Au-pMBA-rBSA-LHRH, E is the particle size distribution of Au-pMBA and Au-pMBA-rBSA-LHRH, and F is an ultraviolet absorption spectrum of Au-pMBA and Au-pMBA-rBSA-LHRH;
FIG. 4 is a diagram illustrating the specificity of SERS probes constructed according to the present invention; wherein A is an SERS imaging graph after incubation and washing of Au-pMBA-rBSA-LHRH and HeLa cells, B is an SERS imaging graph after incubation and washing of Au-pMBA-rBSA and HeLa cells, C is a transmission electron microscope graph after incubation and washing of Au-pMBA-rBSA-LHRH and HeLa cells, and D is an SERS signal result after incubation and washing of Au-pMBA and Au-pMBA-rBSA-LHRH and HeLa cells;
FIG. 5 is a graph showing the sensitivity evaluation of SERS probes constructed according to the present invention;
FIG. 6 shows the effect of SERS probes constructed according to the present invention on cell viability during detection; wherein A is the survival rate of the culture medium containing SERS probes with different concentrations after incubation with HeLa cells, and B is the survival rate of the cells after the SERS probes and the HeLa cells are incubated for different times;
FIG. 7 shows the evaluation of specificity, sensitivity and linearity when the SERS probe constructed by the invention is used for rat simulated blood sample detection; wherein A is a SERS signal acquired by detecting a rat blood sample containing HeLa cells and not containing HeLa cells according to the research construction method, B is a SERS signal acquired by a rat blood sample containing HeLa cells with different concentrations, and C is a linear relation between the cell concentration and the SERS signal intensity in 5-100 cells/ml.
Detailed Description
The method for detecting CTCs by SERS probe according to the present invention will be described in detail with reference to the following examples and accompanying drawings.
Example 1
The method comprises the following steps: preparation of SERS probes
0.01% (W/W) of HAuCl4The solution was heated to slight boiling and 1ml of 1% (W/W) C was slowly added6H5Na3O7·2H2Heating the O solution for 10min to obtain 45nm gold particles; mixing 1ml of 45nm gold particle solution with 50ul of 1mM pMBA, shaking for 5min, centrifuging at 5000rpm, and resuspending to combine pMBA on the gold nanoparticles to obtain Au-pMBA; 20ml of 20mg/ml Bovine Serum Albumin (BSA) with 260ul of 1M sodium borohydride (NaBH)4) After the mixing reaction is carried out for 3h, excess hydrogen is discharged in a constant-temperature water bath at 60 ℃ to obtain rBSA, 5ml of rBSA is taken and added into 30ul of 10mM 1- (3-dimethylaminopropyl) 3-ethylcarbodiimide hydrochloride (EDC. HCl) and N-hydroxysuccinimide (NHS) activated carboxyl respectively after cooling, after the reaction is carried out for 30min, the excess EDC/NHS is removed by adopting a 3KDa ultrafiltration centrifugal tube to carry out centrifugation at 5000rpm for 10min, then 50ul of 2.5mg/ml LHRH solution is added into the activated rBSA solution, after the reaction is carried out for 4h at normal temperature, the reaction is carried out at 4 ℃ overnight, and the excess LHRH is removed by adopting a 3KDa ultrafiltration centrifugal tube to carry out centrifugation at 5000rpm for 10min to obtain a rBSA and LHRH conjugate (rBSA-LHRH; 20ul rBSA-LHRH is dripped into 1ml Au-pMBA solution, and after 5min of reaction, the SERS probe (Au-pMBA-rBSA-LHRH) can be obtained by 6000rpm centrifugal resuspension.
Step two: characterization of SERS probes
Analyzing the nano-particles after the reaction with the pMBA by adopting an X photoelectron spectrum to obtain an energy spectrum shown as a figure 3(A), wherein an ultraviolet except gold atomic peak can be found, an obvious S atomic peak can be seen, and the combination of the pMBA and the gold nano-particles can be illustrated; the SERS spectrogram of only modified pMBA and Au-pMBA after coating rBSA is collected, the SERS chart shown in figure 3(B) can be obtained, and the SERS signal intensity of pMBA after coating rBSA is seen to be slightly reduced; transmission Electron Microscopy (TEM) and particle size analysis are performed on the gold nanoparticles and the SERS probe to obtain a transmission electron micrograph as shown in fig. 3(C, D) and a particle size distribution as shown in fig. 3(E), from the comparison of fig. 3(C, D), it can be seen that the protein molecular layer is uniformly distributed around the gold nanoparticles, and from fig. 3(E), it can be seen that the coating thickness is about 5 nm; by collecting the ultraviolet absorption spectra of the gold nanoparticles and the SERS probe, the ultraviolet absorption spectrum shown in FIG. 3(F) can be obtained, and compared with the gold nanoparticles, the maximum absorption wavelength of the SERS probe is red-shifted by about 2-3 nm.
Step three: SERS probe specificity investigation
According to the culture medium: adding SERS probe (or Au-pMBA-rBSA) into different culture dishes at a ratio of 4:1, incubating for 30min, washing with Phosphate Buffer Solution (PBS) for three times, and performing confocal Raman spectroscopy at 1078cm-1The peak signals are used for imaging analysis of the two cells, so that an SERS imaging spectrum shown in fig. 4(A, B) can be obtained, and an obvious signal can be generated by the SERS probe incubation group (A) but no obvious signal is generated by the Au-pMBA-rBSA group (B), which indicates that the specificity of the SERS probe containing the targeting molecules is improved due to the combination of the SERS probe and the cells; preparing cell suspension of about 10000 HeLa cells/ml by PBS, mixing 250ul of SERS probe and Au-pMBA-rBSA with 1ml of cell suspension respectively, incubating at 37 ℃ for 30min, centrifuging and resuspending to 200ul, carrying out transmission electron microscope molecule on the cells incubated with the SERS probe group, dripping 1ul of the obtained sample on a silicon wafer for detection to obtain a transmission electron microscope image shown in a figure 4(C) and an SERS spectrum shown in a figure (D), wherein the distribution condition of the SERS probe in the cells can be seen by the figure (C), the visible SERS probe group can generate obvious signals by the figure (D), and no obvious signal is generated by the Au-pMBA-rBSA group; in summary, the SERS probe has sufficient specificity for cell detection.
Step four: SERS probe sensitivity investigation
PBS is adopted to prepare cell suspensions of 5, 10, 50, 100, 1000 and 10000cells/ml respectively, 250ul of SERS probe is taken to be mixed with 1ml of cell suspension, the mixture is incubated for 30min at 37 ℃, then the centrifugation and the resuspension are carried out until 200ul, 1ul of the obtained sample is taken to be dripped on a silicon chip for detection, and the SERS spectrogram shown in figure 5 can be obtained.
Step five: cytotoxicity investigation of SERS probes
The effect of the probe on the cell activity was evaluated by the CCK8 method. The appropriate concentration of cell suspension was transferred to a 96-well plate (approximately 5X 10)3cells/hole), incubating at 37 ℃ for 48h, and respectively arranging a blank hole (without cells), a control hole (without adding an SERS probe) and an experimental hole (with adding the SERS probe); probes in different ratios in the experimental wells: adding a culture medium (7:1, 4:1, 3:1, 2:1) to examine the influence of probes with different concentrations on cells, arranging 4 multiple holes in each proportion, detecting at the wavelength of 450nm of an enzyme labeling instrument after incubation is finished, and obtaining a cell survival rate graph shown in figure 6(A), wherein the cell survival rate graph can still keep more than 80% under different SERS probe concentrations; in addition, the following probes: adding another 96-well plate into the culture medium according to the ratio of 4:1, adopting incubation times (30 min, 45 min, 60 min and 75min) of different times to examine the influence of the probe on the cells under different incubation times, and detecting the cells at the wavelength of 450nm of an enzyme labeling instrument after the incubation is finished to obtain a cell survival rate graph shown in figure 6(B), wherein the cell survival rate is still more than 80% along with the extension of the incubation time; in conclusion, the constructed SERS probe has small influence on cell activity and has low cytotoxicity in the detection process.
Step six: SERS probe for simulating blood sample detection
Taking venous blood of rats, preparing mixed blood samples (5, 10, 50, 100, 1000 and 10000 cells) containing different numbers of cells, and fully mixing at 37 ℃; separating a rat blood sample containing tumor cells (10000 cells) by using a rat lymphocyte separation solution, mixing and incubating a low-density cell layer containing the tumor cells and the lymphocytes with an SERS probe, centrifuging at 1350rpm for 5min, washing for three times by using PBS (phosphate buffer solution), detecting by using confocal Raman, and detecting the rat blood sample which is not mixed with the tumor cells after the same steps to verify the specificity; taking mixed blood samples of different concentrationsSeparating a sample of mixed tumor cells by using lymphocyte separation liquid, then incubating with an SERS probe, centrifuging at 1350rpm for 5min after incubation is finished, flushing with PBS for three times, detecting by using confocal Raman, verifying sensitivity and linearity of a detection mode by collecting signals of cell suspensions with different concentrations to obtain figures 7(A, B and C), wherein the SERS probe still has sufficient specificity when used for simulating blood sample detection as shown in figure 7(B), the constructed SERS probe can also reach the detection limit of 5cells/ml when used for simulating blood sample detection as shown in figure 7(C), and good linearity (R) can be obtained between 5 and 100cells/ml when the constructed SERS probe is used for simulating blood sample detection as shown in figure 7(C)2＝0.9803)。
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.
Claims (4)
1. A circulating tumor cell detection method based on surface enhanced Raman spectroscopy is characterized by comprising the following steps: constructing a surface-enhanced Raman spectroscopy probe which takes luteinizing hormone releasing hormone as a targeting molecule, and then using the surface-enhanced Raman spectroscopy probe for circulating tumor cell detection; the surface-enhanced Raman spectrum probe with the luteinizing hormone-releasing hormone as the targeting molecule comprises an enhanced substrate, signal molecules, a protective layer molecule and the targeting molecule, wherein the enhanced substrate adopts 45nm gold particles, the signal molecules are p-mercaptobenzoic acid, the protective layer molecule is reducing bovine serum albumin, and the targeting molecule is the luteinizing hormone-releasing hormone; the preparation method of the surface enhanced Raman spectroscopy probe with the luteinizing hormone releasing hormone as the targeting molecule comprises the following steps: 0.01% (W/W) of HAuCl4The solution was heated to slight boiling and 1ml of 1% (W/W) C was slowly added6H5Na3O7·2H2O solution, heatingObtaining 45nm gold particles after 10 min; mixing 1ml 45nm gold particle solution with 50ul 1mM pMBA, shaking for 5min, centrifuging at 5000rpm, and resuspending to allow pMBA to bind to gold particles to obtain Au-pMBA; 20ml of 20mg/ml bovine serum albumin and 260ul of 1M sodium borohydride are mixed and reacted for 3 hours, then surplus hydrogen is discharged in a constant-temperature water bath at 60 ℃ to obtain rBSA, 5ml of rBSA is taken and added into 30ul of 10mM 1- (3-dimethylaminopropyl) 3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide activated carboxyl respectively after being cooled, after reaction for 30 minutes, a 3KDa ultrafiltration centrifugal tube is adopted for centrifugation at 5000rpm for 10 minutes to remove surplus EDC/NHS, then 50ul of 2.5mg/ml LHRH solution is added into the activated rBSA solution, after reaction for 4 hours at normal temperature, the reaction is carried out overnight at 4 ℃, and the 3KDa ultrafiltration centrifugal tube is adopted for centrifugation at 5000rpm for 10 minutes to remove surplus LHRH to obtain rBSA and LHRH conjugate rBSA-LHRH; 20ul of rBSA-LHRH is dripped into 1ml of Au-pMBA solution, and after 5min of reaction, 6000rpm is used for centrifugal resuspension to obtain the probe Au-pMBA-rBSA-LHRH.
2. The method for detecting circulating tumor cells based on surface enhanced raman spectroscopy of claim 1, comprising the steps of:
A) constructing the surface enhanced Raman spectroscopy probe which takes the luteinizing hormone releasing hormone as a targeting molecule;
B) separating a blood sample to be detected by adopting lymphocyte separation liquid, mixing and incubating the blood sample with the surface enhanced Raman spectrum probe constructed in the step A, centrifuging at 1350rpm for 5min after incubation is finished, washing the blood sample with PBS for three times, and detecting the blood sample by adopting confocal Raman; by using 1078cm in the spectrum-1Quantifying the intensity of the peak; the laser intensity is 100mW, and the integration time is 5 s.
3. A surface enhanced Raman spectroscopy probe taking luteinizing hormone-releasing hormone as a targeting molecule is characterized by comprising an enhanced substrate, a signal molecule, a protective layer molecule and a targeting molecule, wherein the enhanced substrate adopts 45nm gold particles, the signal molecule is p-mercaptobenzoic acid, the protective layer molecule is reducing bovine serum albumin, and the targeting molecule is luteinizing hormone-releasing hormone; the luteinizing hormoneThe preparation method of the surface enhanced Raman spectroscopy probe taking the hormone releasing hormone as the targeting molecule comprises the following steps: 0.01% (W/W) of HAuCl4The solution was heated to slight boiling and 1ml of 1% (W/W) C was slowly added6H5Na3O7·2H2Heating the O solution for 10min to obtain 45nm gold particles; mixing 1ml 45nm gold particle solution with 50ul 1mM pMBA, shaking for 5min, centrifuging at 5000rpm, and resuspending to allow pMBA to bind to gold particles to obtain Au-pMBA; 20ml of 20mg/ml bovine serum albumin and 260ul of 1M sodium borohydride are mixed and reacted for 3 hours, then surplus hydrogen is discharged in a constant-temperature water bath at 60 ℃ to obtain rBSA, 5ml of rBSA is taken and added into 30ul of 10mM 1- (3-dimethylaminopropyl) 3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide activated carboxyl respectively after being cooled, after reaction for 30 minutes, a 3KDa ultrafiltration centrifugal tube is adopted for centrifugation at 5000rpm for 10 minutes to remove surplus EDC/NHS, then 50ul of 2.5mg/ml LHRH solution is added into the activated rBSA solution, after reaction for 4 hours at normal temperature, the reaction is carried out overnight at 4 ℃, and the 3KDa ultrafiltration centrifugal tube is adopted for centrifugation at 5000rpm for 10 minutes to remove surplus LHRH to obtain rBSA and LHRH conjugate rBSA-LHRH; 20ul of rBSA-LHRH is dripped into 1ml of Au-pMBA solution, and after 5min of reaction, 6000rpm is used for centrifugal resuspension to obtain the probe Au-pMBA-rBSA-LHRH.
4. Use of the surface-enhanced Raman spectroscopy probe comprising luteinizing hormone-releasing hormone of claim 3 as a targeting molecule in the preparation of a circulating tumor cell detection reagent or kit.
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