CN112034160B - Circulating tumor cell detection kit based on rare earth nano material fluorescence amplification and application thereof - Google Patents
Circulating tumor cell detection kit based on rare earth nano material fluorescence amplification and application thereof Download PDFInfo
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Abstract
The invention provides a circulating tumor cell detection kit based on rare earth nano material fluorescence amplification, which comprises a capture probe, a fluorescence detection probe and an enhancement solution, wherein the fluorescence detection probe is a rare earth nano material modified by an antibody aiming at an antigenic epitope of a circulating tumor cell. The detection kit adopts the marker capable of specifically targeting the circulating tumor cell surface antigen to modify the nano rare earth material, and has good identification capability on the circulating tumor cell; various tool enzymes and complicated amplification steps are not needed; the fluorescence can be enhanced by nearly one million times by adding the enhancement solution at the end of the experiment, the detection sensitivity is greatly improved, the operation is very simple and convenient, and the circulating tumor cells can be directly detected in the whole blood; the rare earth nano material has stable property, large specific surface area, strong modifiability and low cost, each nano particle contains thousands of rare earth ions, the labeling ratio of the rare earth ions is greatly improved, the rare earth nano material is not influenced by anticoagulant, and the applicability is wider.
Description
Technical Field
The invention relates to the technical field of nano fluorescence detection, in particular to a circulating tumor cell detection kit based on rare earth nano material fluorescence amplification and application thereof.
Background
Circulating tumor cells are tumor cells that are shed from primary foci and invade the blood circulation. Since the first discovery of circulating tumor cells in the peripheral blood of cancer patients in 1869, there is increasing evidence that they are closely associated with cancer metastasis and can serve as markers of metastatic recurrence of the tumor. Therefore, the detection of circulating tumor cells has been receiving attention and has become a hot point of research. Circulating tumor cell detection refers to a method for analyzing circulating tumor cells in peripheral blood of a tumor patient. As a liquid biopsy method, the method can be used for guiding clinical medical scheme formulation, judging patient prognosis, evaluating curative effect and the like, and has unique advantages for real-time individualized treatment of tumor patients. However, the number of circulating tumor cells in the blood is very rare and can only be measured in single digits, which poses a great challenge to the detection thereof. Therefore, the establishment of a stable, highly sensitive and highly specific circulating tumor cell detection method is the key for developing related research.
The detection of substances by utilizing the nanotechnology is a cross discipline integrating biology, chemistry, pharmacy and physics, and has wide application prospect in the aspects of tumor monitoring and personalized treatment. In recent years, methods for detecting circulating tumor cells based on different signal patterns have emerged, such as electrochemistry, inductively coupled plasma mass spectrometry, raman spectroscopy, colorimetry, fluorescence, and the like. Among them, fluorescence detection is a field of great interest in circulating tumor cell detection methods due to its advantages of rapid reaction, high sensitivity, non-destructive and real-time monitoring. Currently, there are many detection methods based on fluorescent dyes to detect circulating tumors. However, the greatest difficulty in detecting circulating tumor cells with fluorescent dye labeling is the interference of the complex biochemical environment of the blood and biological background fluorescence. Because the interval between excitation and emission spectra of most fluorescent markers is very small, and most fluorescent markers are interfered by biological background fluorescence in the emission wavelength range, the detection steps are complex, and the detection sensitivity and accuracy are low.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a circulating tumor cell detection kit based on rare earth nano material fluorescence amplification, which comprises a capture probe, a fluorescence detection probe and an enhancement solution, wherein the fluorescence detection probe is a rare earth nano material modified by an antibody aiming at an antigenic epitope of a circulating tumor cell.
According to the invention, the fluorescence detection probe is in the form of nanoparticles.
According to the invention, the detection kit further comprises a solid phase carrier, a coating solution and a sealing solution.
According to the invention, the capture probe is immobilized on a solid support; the immobilization can be carried out by means of covalent coupling or physical adsorption. In one embodiment of the invention, the capture probe may be immobilized on a solid support by covalent coupling. The capture probe can be covalently coupled to the solid support by a variety of methods known in the art, including, but not limited to, covalent coupling of the capture probe to the solid support via chemically reactive groups, such as amino, carboxyl, amide, etc., attached to the 5' end of the capture probe.
According to the invention, the immobilization comprises diluting the capture antibody with a coating solution and then adding the antibody solution to a solid support for immobilization. In one embodiment of the invention, a carbonate buffer solution of pH 9.6 is used as coating solution. According to the invention, the immobilization may also comprise a blocking step after covalent coupling. In one embodiment of the present invention, after the capture probe is coated on the solid support, the blocking solution is further used to block unreacted chemical groups on the capture probe, so as to reduce the false positive that may occur in the subsequent detection. In one embodiment of the present invention, the blocking solution used is a Bovine Serum Albumin (BSA) solution, for example, a BSA solution (2% w/v) prepared with 0.1mol/L carbonate buffer.
According to the present invention, the solid phase carrier can be various solid phase carriers known in the detection field, including but not limited to a micro-porous plate, a magnetic microsphere, a gold foil, a polymer microsphere, an enzyme label plate, a nitrocellulose membrane, and the like. In one embodiment of the invention, the solid support is a microplate, such as a 96-well plate.
According to the invention, the capture probe is an antibody directed against an antigenic epitope of the circulating tumor cell, which antibody can specifically bind to a marker specific to the surface of the circulating tumor cell.
According to the invention, the fluorescence detection probe is a rare earth nano material modified by an antibody aiming at the antigenic epitope of the circulating tumor cell, and the antibody can be specifically combined with a marker on the surface of the circulating tumor cell, so that the combination specificity and stability of the detection probe and the circulating tumor cell can be improved, and the false positive and false negative of detection can be reduced. In a specific embodiment of the present invention, the capture probe and the antibody for modifying rare earth nanoparticles are the same or different, are independently selected from the group consisting of antibodies known in the art to specifically bind to an antigenic epitope of a circulating tumor cell, and can be any one of monoclonal antibodies and polyclonal antibodies; for example, independently of each other, an antibody that binds to at least one circulating tumor cell-associated antigen selected from the group consisting of: epCAM, epithelial keratins (CKs), epithelial cell membrane specific antigens, tumor cell glycoproteins (TAG) or specific markers (e.g., HER2, anti-lactoglobulin, etc.); in particular, it may be selected from anti-EpCAM antibodies, anti-EGFR antibodies, anti-cytokeratin CK-8, CK18 and CK-19 antibodies, preferably anti-EpCAM antibodies.
According to the invention, the rare earth nanomaterial is an up-conversion fluorescent nanomaterial; illustratively, the rare earth nanomaterial is XYF 4 And the X is selected from one or more of lithium, sodium, potassium and the like, and the Y is selected from one or more of europium, samarium, terbium and dysprosium. In one embodiment of the present invention, the rare earth nanomaterial is NaEuF 4 And (4) nanocrystals.
According to the present invention, the fluorescent detection probe may be synthesized by using various methods known in the art for modifying rare earth nanomaterials with antibodies, including but not limited to electrostatic adsorption, and/or chemical coordination.
For example, the rare earth nanomaterial is combined with an organic ligand, and then an antibody is labeled by a chemical coordination method, so that the rare earth nanomaterial modified by the antibody is obtained.
According to the invention, the synthesis method of the fluorescence detection probe comprises the following steps: synthesizing a water-soluble rare earth nano material, carrying out surface modification by using a hydrophilic organic ligand to obtain an organic-rare earth complex, adding an activating agent for activation, and then adding an antibody for marking to obtain the antibody-modified rare earth nano material. In one embodiment of the present invention, the organic ligand is at least one of polyacrylic acid (PAA) and Polyethyleneimine (PEI); the activating agent is selected from at least one of carbodiimide (EDC), N-hydroxysuccinimide (NHS), dimethylacetamide (DMAC).
In one embodiment of the invention, in the synthesis method of the fluorescence detection probe, the mass ratio of the water-soluble rare earth nanomaterial to the hydrophilic organic ligand is (1-3) 5, for example, 1; the mass ratio of the antibody to the organic-rare earth complex is 1 (100-1000), such as 1; the mass ratio of the activating agent to the organic-rare earth complex is 1 (0.5-5), such as 1.
In one embodiment of the present invention, the fluorescence detection probe is: naEuF 4 The nano-crystal is prepared by loading PAA (poly (acrylic acid)) by an electrostatic adsorption method and marking an antibody by a chemical coordination method; illustratively, the method comprises the following steps:
1) Mixing oil soluble NaEuF 4 Adding equal amount of absolute ethyl alcohol into the nanocrystal for precipitation, adding chloroform, absolute ethyl alcohol, ultrapure water and concentrated hydrochloric acid, removing oleic acid on the surface of the nanocrystal, and adding deionized water for dissolution to obtain water-soluble nanocrystal;
2) Taking the water-soluble nanocrystal synthesized in the step 1), adding PAA, performing ultrasonic treatment, performing centrifugal washing by using deionized water, and finally dissolving in the deionized water to obtain PAA-loaded nanocrystal;
3) And (3) adding carbodiimide (EDC) and N-hydroxysuccinimide (NHS) into the PAA loaded nanocrystalline obtained in the step 2), uniformly mixing, centrifugally washing by deionized water, dissolving in the deionized water, adding an EpCAM antibody, uniformly mixing, centrifugally washing by deionized water, dissolving in a HEPES buffer solution to obtain the target circulating tumor cell fluorescence detection probe, and storing at 4 ℃ for later use.
According to the invention, the enhancing solution contains polyethylene glycol octyl phenyl ether Triton X-100, naphthoic acid trifluoroacetone, tri-n-octyl phosphine oxide and water. In one embodiment of the present invention, the enhancing fluid consists of: each liter of the enhanced solution contains 1g of Triton X-100, 26.6mg of naphthoyl trifluoroacetone, 193mg of tri-n-octyl phosphine oxide, and the balance of water, and the pH value is 2.3.
The rare earth nano material modified by the antibody aiming at the circulating tumor cell antigenic epitope is applied to the preparation of a circulating tumor cell detection reagent.
The detection kit is applied to the preparation of a circulating tumor cell detection reagent.
The invention also provides a using method of the detection kit, which comprises the following steps:
(1) Immobilizing a capture probe to a solid support;
(2) Adding a sample containing circulating tumor cells to be detected;
(3) Adding the fluorescent detection probe;
(4) Adding the enhancing solution;
(5) The fluorescence signal is detected by time-resolved fluorescence analysis.
According to the present invention, the step (1) may be: and diluting the capture probe by the coating solution, then fixing the capture probe on a solid phase carrier, and blocking the capture probe by the blocking solution.
In the method of use of the invention, the fluorescent detection probe may be added to the sample to a final concentration of 30-50. Mu.g/mL, preferably 40. Mu.g/mL.
The invention also provides a circulating tumor cell detection method based on rare earth nano material fluorescence amplification, which comprises the following steps:
(1) Providing a capture probe that is an antibody directed against an antigenic epitope of a circulating tumor cell;
(2) Adding a sample containing circulating tumor cells to be detected;
(3) Adding a fluorescent detection probe targeting circulating tumor cells, wherein the detection probe is an antibody-modified rare earth nano material;
(4) Adding the enhancement solution;
(5) The fluorescence signal is detected by time-resolved fluorescence analysis.
According to the invention, the sample containing the circulating tumor cells to be detected is one of blood, cell suspension, pleural fluid, ascites and cerebrospinal fluid from an organism.
According to the invention, in the step of detecting the fluorescence signal by time-resolved fluorescence, the principle of signal amplification is that after the fluorescent labeled antibody containing the rare earth nano material targets circulating tumor cells existing in a binding system, an enhancement solution is added to dissolve the rare earth nano material into rare earth ions, and the rare earth ions and ligands in the enhancement solution form a chelate to generate new signal molecules, and intramolecular and intermolecular energy transfer is generated to enhance the intensity of the fluorescence signal by nearly million times, so that the fluorescence signal is detected.
According to the invention, in the time-resolved fluorescence detection step, a standard solution of the circulating tumor cells to be detected can be prepared as required for detection, and a standard curve is drawn by taking the number of the cells to be detected as a horizontal coordinate and a fluorescence intensity variation value as a vertical coordinate. And then placing the sample to be detected in a fluorescence spectrometer to read the fluorescence intensity value, and calculating the content of the circulating tumor cells in the sample to be detected according to the standard curve.
In the method, the capture probe, the fluorescent probe, the enhancing solution and the like have the meanings as described above.
The invention has the beneficial effects that:
the invention provides an ultrasensitive detection method for circulating tumor cells based on rare earth nano material fluorescence amplification and application thereof, wherein the detection method has the following advantages: 1) Markers capable of specifically targeting circulating tumor cell surface antigens, such as EpCAM antibodies and the like, are adopted as capture probes, so that the circulating tumor cells have good recognition capability; 2) Various tool enzymes and multi-step and complex signal amplification steps are not needed; the rare earth nano material is used as a marker to mark the biomolecule, and the fluorescence can be enhanced by nearly one million times by adding the enhancement solution at the end of the experiment without any treatment in advance, so that the detection sensitivity is greatly improved, the operation is very simple and convenient, and the circulating tumor cells can be directly detected in whole blood; 3) Because the rare earth nano material has stable property, large specific surface area, strong repairability and low cost, and each nano particle contains thousands of rare earth ions, the marking ratio of the rare earth ions is greatly improved, the rare earth nano material is slightly influenced by exogenous rare earth ions, is not influenced by an anticoagulant, and has wider applicability; 4) The method of the present invention has high sensitivity and may be used in the direct detection of circulating tumor cell in complicated blood system
Drawings
FIG. 1 is a schematic diagram of the detection of circulating tumor cells according to a preferred embodiment of the present invention.
FIG. 2 is a transmission electron micrograph of NaEuF4 nanocrystals prepared in example 1.
FIG. 3 is a graph showing the sensitivity of the method for detecting circulating tumor cells described in example 1 to circulating tumor cell detection.
FIG. 4 is a graphical representation of the selective recognition of circulating tumor cells by the fluorescent probe for circulating tumor cells described in example 2.
FIG. 5 is a study of the sensitivity of detection of circulating tumor cells in a sample of complex blood system as described in example 3.
FIG. 6 is a review of the potential for potential clinical use of the method for detecting circulating tumor cells described in example 4.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples. However, those skilled in the art will appreciate that the scope of the present invention is not limited to the following examples. In light of the present disclosure, those skilled in the art will recognize that many variations and modifications may be made to the embodiments described above without departing from the spirit and scope of the invention as defined by the appended claims.
In the following examples, the transmission electron microscope image is examined by JEM-2010 and JEOL.
In the following examples, the apparatus for detecting the fluorescent signal molecules was a fluorescence microplate reader, model number Synergy4, and manufacturer BioTek.
In the following examples, the composition of the PBS buffer was: KCl 0.2g per liter of buffer solution 2 PO 4 0.2g，NaCl 8g，Na 2 HPO 4 ·12H 2 O 3.14g，pH 7.4-7.6。
Example 1
Examination of detection sensitivity for circulating tumor cells
1. NaEuF required for the experiment 4 The preparation method of the circulating tumor cell fluorescence detection probe comprises the following steps:
1) 50mg of PAA was weighed and dissolved in 1mL of ultrapure water to a final concentration of 50mg/mL. Followed by 1mL of 20mg solutionNaEuF/mL 4 The nanoparticle suspension (equivalent to 20mg of nanoparticles added) was vortexed overnight after half an hour of sonication and mixing well. Centrifuging, retaining the precipitate, dissolving in 1mL ultrapure water, and formulating into NaEuF with a concentration of 20mg/mL 4 PAA suspension, kept at room temperature until use.
2) EDC 6mg, NHS 14mg were added to 1mL NaEuF at 20mg/mL 4 The PAA suspension was stirred well, centrifuged to remove the supernatant, ultrapure water was added, mixed well and centrifuged, and then 20. Mu.L of EpCAM antibody (equivalent to 0.02mg of EpCAM) at a concentration of 1mg/mL was added to 1mL of NaEuF at a concentration of 20mg/mL 4 In PAA solution, spun at 4 ℃ for 2h, centrifuged and dissolved in 1mL HEPES buffer (50mM, pH 7.4) to give NaEuF 4 The marked circulating tumor cell fluorescence detection probe is stored at 4 ℃ for later use.
FIG. 2 shows NaEuF prepared in this example 4 Transmission electron microscopy of nanocrystals.
As can be seen from the figure, the obtained nanocrystals had uniform size distribution (particle size of 19.5. + -. 0.8 nm) and good crystallinity.
2. Preparing reinforced liquid
1g of Triton X-100, 26.6mg of naphthoyl trifluoroacetone and 193mg of tri-n-octylphosphine oxide are weighed, distilled water is added to the mixture to reach a constant volume of 1L, and the pH value is adjusted to 2.3 by using diluted HCl for later use.
3. Investigation of NaEuF 4 The sensitivity of the nanocrystal fluorescence amplification system for measuring the circulating tumor cell standard solution comprises the following steps:
1) Coating: the capture probe EpCAM antibody was diluted to 10. Mu.g/mL with 0.05mol/L carbonate buffer, 100. Mu.L was added to each well in a 96-well polystyrene plate, incubated at 37 ℃ for 1 hour, the well contents were discarded, and washed 3 times with PBS wash buffer.
2) And (3) sealing: BSA solution (2% w/v) was prepared in PBS buffer, 300. Mu.L was added to each well, incubated at 37 ℃ for 1 hour, the well liquid was discarded, and washed 3 times with PBS wash buffer.
3) Sample adding: MCF-7 cells were cultured, grown to a certain number, digested with trypsin and collected, and prepared into a cell suspension with PBS. 200. Mu.L of each cell suspension containing 2-1024 MCF-7 was added to the above-mentioned microplate, incubated in a cell incubator for 2 hours, and washed three times with PBS.
4) Adding NaEuF 4 Circulating tumor cell fluorescence detection probe: circulating tumor cell fluorescent detection probe (final concentration of fluorescent detection probe in suspension 40. Mu.g/mL) in PBS was added, incubated for 1.5 hours, and washed three times with PBS.
5) Adding a reinforcing liquid: adding 200 mu L of enhancement solution into each hole, and detecting a fluorescence signal by adopting time resolution, wherein the specific parameters are as follows: the excitation wavelength of 340nm, the emission wavelength of 616nm, the delay time and the gate time were set to 200. Mu.s and 2ms.
6) Drawing a standard curve: and (3) drawing a standard curve by taking the number of circulating tumor cells as an abscissa and the fluorescence intensity corresponding to each circulating tumor number as an ordinate.
FIG. 3 shows a standard curve of the number of circulating tumor cells versus fluorescence intensity in this example.
As can be seen, the number of circulating tumor cells is linearly related to the fluorescence intensity in the range of 2-1024 cells, and the lowest detection limit is 1 circulating tumor cell in terms of blank average value plus 3 times SD.
Example 2
Examination of Selectivity in identifying circulating tumor cells
1. The reagents and equipment required for the experiment were the same as in example 1, naEuF for the experiment 4 Circulating tumor cell fluorescent detection probes example 1 is identical.
2. Investigation of NaEuF 4 The nanocrystalline fluorescence amplification system for measuring the selectivity of the circulating tumor cells comprises the following steps:
1) Coating: the capture probe EpCAM antibody was diluted to 10. Mu.g/mL with 0.05mol/L carbonate buffer, 100. Mu.L was added to each well in a 96-well polystyrene plate, incubated at 37 ℃ for 1 hour, the well contents were discarded, and washed 3 times with PBS wash buffer.
2) And (3) sealing: BSA solution (2% w/v) was prepared with PBS buffer, 300. Mu.L was added to each well, incubated at 37 ℃ for 1 hour, the liquid in the wells was discarded, and washed 3 times with PBS wash buffer.
3) Sample adding: epCAM positive MCF-7 cells and EpCAM negative HeLa cells were cultured, grown to a certain number, digested with trypsin and collected, respectively, and prepared into cell suspensions (5000/mL) with healthy human blood. 200. Mu.L of each cell suspension containing 1000 MCF-7 and HeLa cells was added to the above-mentioned microplate, incubated in a cell incubator for 2 hours, and washed three times with PBS.
4) Adding a circulating tumor cell fluorescence detection probe: circulating tumor cell fluorescent detection probe (final concentration of fluorescent detection probe in suspension 40. Mu.g/mL) in PBS was added, incubated for 1.5 hours, and washed three times with PBS.
5) Adding an enhancement solution: adding 200 mu L of enhancement solution into each hole, and detecting a fluorescence signal by adopting time-resolved fluorescence analysis, wherein the specific parameters are as follows: the excitation wavelength of 340nm, the emission wavelength of 616nm, the delay time and the gate time were set to 200. Mu.s and 2ms.
6) Plotting fluorescence intensity F-F 0 A histogram using the name of each cell as the abscissa and the fluorescence intensity obtained for each target as the ordinate, wherein F is the fluorescence measurement of the target, F 0 Is the background fluorescence value, fluorescence intensity F-F 0 And the method is used for reflecting the change of fluorescence values and background values of different targets to draw a histogram.
FIG. 4 shows the results of a selective investigation of the identification of circulating tumor cells as described in this example.
As can be seen from FIG. 4, the fluorescence intensity of the EpCAM-positive MCF-7 cell and the EpCAM-negative HeLa cell is very significantly different from each other, which indicates that the circulating tumor cell detection scheme constructed by the method has high specificity and good selectivity.
Example 3
Examination of detection sensitivity of circulating tumor cells in complex blood system sample by using method described in this example
1. The reagents and equipment required for the experiment were the same as in example 1, naEuF for the experiment 4 Circulating tumor cell fluorescent detection probes example 1 is identical.
2. The model substrate used in the complex blood system experiment is a healthy blood sample.
3. Investigation of NaEuF 4 The sensitivity of the nanocrystalline fluorescence amplification system for measuring the circulating tumor cells in the blood sample comprises the following steps:
1) Coating: the capture probe EpCAM antibody was diluted to 10. Mu.g/mL with 0.05mol/L carbonate buffer, 100. Mu.L was added to each well in a 96-well polystyrene plate, incubated at 37 ℃ for 1 hour, the well contents were discarded, and washed 3 times with PBS wash buffer.
2) And (3) sealing: BSA solution (2% w/v) was prepared in PBS buffer, 300. Mu.L was added to each well, incubated at 37 ℃ for 1 hour, the well liquid was removed, and washed 3 times with PBS wash buffer.
3) Sample adding: MCF-7 cells are cultured, and after the cells grow to a certain number, the cells are digested and collected by trypsin, and healthy blood is prepared into a cell suspension to simulate blood circulation tumor cells. 200. Mu.L of each cell suspension containing 2-1024 MCF-7 was added to the above-mentioned microplate, incubated in a cell incubator for 2 hours, and washed three times with PBS.
4) Adding a circulating tumor cell fluorescence detection probe: circulating tumor cell detection fluorescent probe (40. Mu.g/mL) in PBS was added, incubated for 1.5 hours, and washed three times with PBS.
5) Adding an enhancement solution: adding 200 mu L of enhancement solution into each hole, and detecting a fluorescence signal by adopting time resolution, wherein the specific parameters are as follows: the excitation wavelength of 340nm, the emission wavelength of 616nm, the delay time and the gate time were set to 200. Mu.s and 2ms.
6) Drawing a standard curve: and (3) drawing a standard curve by taking the number of circulating tumor cells as an abscissa and the fluorescence intensity corresponding to each circulating tumor number as an ordinate.
FIG. 5 shows a standard curve of the number of circulating tumor cells versus fluorescence intensity in this example.
The main challenges for circulating tumor cell detection are the complex physiological environment in the blood and too small number of circulating tumor cells. Therefore, we added different numbers (2-1024) of MCF-7 cells to healthy blood to mimic the complex blood environment in which circulating tumor cells are located. Then, the circulating tumor cells are directly detected by using the circulating tumor cell detection scheme without any pretreatment and enrichment. As shown in the figure, the cell number and the fluorescence intensity still show a positive relationship for MCF-7 cells in blood, and the detection limit is as low as 1 cell calculated by blank average value plus 3 times SD, and the detection linear range is good between 2 and 1024 cells. The results prove that the established circulating tumor cell detection scheme can directly detect the circulating tumor cells in blood with high sensitivity without any enrichment means and pretreatment.
Example 4
Examination of potential for potential clinical application of the method for detecting circulating tumor cells described in this example
1. The reagents and equipment required for the experiment were the same as in example 1, naEuF for the experiment 4 Circulating tumor cell fluorescent detection probes example 1 is identical.
2. The detection matrix used was a blood sample from 15 breast cancer patients.
3. The standard working curve used for the experiment was consistent with the experimental procedure of example 3.
4. Investigation of NaEuF 4 The sensitivity of the nanocrystalline fluorescence amplification system for measuring the circulating tumor cells in the blood sample comprises the following steps:
1) Coating: the capture probe EpCAM antibody was diluted to 10. Mu.g/mL with 0.05mol/L carbonate buffer, 100. Mu.L was added to each well in a 96-well polystyrene plate, incubated at 37 ℃ for 1 hour, the well contents were discarded, and washed 3 times with PBS wash buffer.
2) And (3) sealing: BSA solution (2% w/v) was prepared in PBS buffer, 300. Mu.L was added to each well, incubated at 37 ℃ for 1 hour, the well liquid was removed, and washed 3 times with PBS wash buffer.
3) Sample adding: a200. Mu.L blood sample was added to the above-mentioned Aminomicroplate, incubated in a cell incubator for 2 hours, and washed three times with PBS.
4) Adding detection probe NaEuF 4 -Ab: circulating tumor cell fluorescent detection probe (40. Mu.g/mL) in PBS was added, incubated for 1.5 hours, and washed three times with PBS.
5) Adding a reinforcing liquid: adding 200 mu L of enhancement solution into each hole, and detecting a fluorescence signal by adopting time resolution, wherein the specific parameters are as follows: the excitation wavelength of 340nm, the emission wavelength of 616nm, the delay time and the gate time were set to 200. Mu.s and 2ms.
6) Determination of the number of circulating tumor cells in a blood sample of a patient: and calculating the number of circulating tumor cells corresponding to the blood sample of the patient by taking the working curve obtained in the example 3 as a standard curve.
The calculation method comprises the following steps: the measured fluorescence values at known concentrations for the patient's blood sample are substituted into a standard curve fitting equation: the number of circulating tumor cells =2^ (fluorescence intensity + 479.13565)/482.54485) and the number of circulating tumor cells can be determined.
Through detection, 14 cancer patients in 15 patients detect circulating tumor cells, and the detection rate is 93.9%. As shown in fig. 6, we then analyzed the correlation of blood circulating tumor cell levels with cancer progression. The cancer stages determined by clinical pathology analysis in 14 cancer patients were divided into 4 groups, namely early (3), intermediate (5), late (3) and unknown pathological stages (3). We found that patients had high levels of circulating tumor cells in the metaphase (about 15-190 cells/mL) and relatively low levels of circulating tumor cells in the early (about 10 cells/mL) or late (about 15-20 cells/mL).
It is noted that heterogeneity in EpCAM expression may affect detection of circulating tumor cells, particularly in patients with advanced cancer who may down-regulate EpCAM expression. The nano probe can be functionalized by selecting other antibodies with strong specificity, so that the nano probe has a plurality of suitable antibodies, the application range of the detection kit and the detection method is expanded, and the limitation of the types of the antibodies is reduced. Nevertheless, we have successfully established a new circulating tumor detection system, which can directly detect circulating tumor cells in real blood without any conventional circulating tumor cell enrichment step, and the time-resolved photoluminescence spectroscopy TRPL detection strategy can be applied to accurate diagnosis and prognosis of various cancers.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (6)
1. A circulating tumor cell detection kit based on rare earth nano material fluorescence amplification is characterized by comprising a capture probe, a fluorescence detection probe, an enhancement solution, a solid phase carrier, a coating solution and a sealing solution;
the fluorescent detection probe is a rare earth nano material modified by an antibody aiming at antigenic epitope of circulating tumor cells, and the rare earth nano material is NaEuF 4 A nanocrystal;
the capture probe and the antibody for modifying the rare earth nanoparticles are EpCAM antibodies;
the enhancement solution contains polyethylene glycol octyl phenyl ether Triton X-100, naphthoic acid trifluoroacetone, tri-n-octyl phosphine oxide and water;
the capture probe is fixed on a solid phase carrier; the immobilization adopts a covalent coupling or physical adsorption mode; the immobilization comprises diluting a capture antibody by adopting a coating solution, and then adding the capture antibody solution into a solid phase carrier for immobilization, wherein the solid phase carrier is an ELISA plate;
the synthesis method of the fluorescent detection probe comprises the following steps: synthesis of water-soluble rare earth nano material NaEuF 4 And (3) carrying out surface modification on the nanocrystalline by using a hydrophilic organic ligand to form an organic-rare earth complex, adding an activating agent for activation, and then adding an EpCAM antibody for marking to obtain the EpCAM antibody modified rare earth nanomaterial.
2. The kit of claim 1, wherein the blocking solution is 2% w/v bovine serum albumin solution prepared with 0.1mol/L carbonate buffer.
3. The kit according to claim 1, wherein the hydrophilic organic ligand is at least one of polyacrylic acid and polyethyleneimine; the activating agent is at least one selected from carbodiimide, N-hydroxysuccinimide and dimethylacetamide.
4. The kit according to any one of claims 1 to 3, wherein the rare earth nanomaterial NaEuF 4 The mass ratio of the nanocrystalline to the hydrophilic organic ligand is (1-3) to 5;
the mass ratio of the EpCAM antibody to the organic-rare earth complex is 1 (100-1000);
the mass ratio of the activating agent to the organic-rare earth complex is 1 (0.5-5).
5. The kit of any one of claims 1 to 3, wherein the fluorescent detection probe is prepared by subjecting NaEuF 4 The nanocrystal is loaded with polyacrylic acid by electrostatic adsorption.
6. The kit according to any one of claims 1 to 3, wherein the fluorescent detection probe is prepared by a method comprising the steps of:
1) Mixing oil soluble NaEuF 4 Adding equal amount of absolute ethyl alcohol into the nanocrystal for precipitation, adding chloroform, absolute ethyl alcohol, ultrapure water and concentrated hydrochloric acid, removing oleic acid on the surface of the nanocrystal, and adding deionized water for dissolution to obtain water-soluble nanocrystal;
2) Adding polyacrylic acid into the water-soluble nanocrystal synthesized in the step 1), performing ultrasonic treatment, performing centrifugal washing by using deionized water, and finally dissolving in the deionized water to obtain polyacrylic acid loaded nanocrystal;
3) And (3) adding carbodiimide and N-hydroxysuccinimide into the polyacrylic acid loaded nanocrystal obtained in the step 2), uniformly mixing, centrifugally washing with deionized water, dissolving in the deionized water, adding an EpCAM antibody, uniformly mixing, centrifugally washing with the deionized water, dissolving in an HEPES buffer solution to obtain the target circulating tumor cell fluorescence detection probe, and storing at 4 ℃ for later use.
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