SERS probe combination, kit and method for identifying and / or quantifying platelet adhesion type CTC

Abstract

The invention discloses an SERS (Surface Enhanced Raman Scattering) probe combination, a kit and a method for identifying and / or quantifying platelet adhesion type CTC (Cytotoxic T Cell). The probe combination comprises a first probe and a second probe, the first probe comprises nanoparticles, Raman reporter molecules and a first recognition element, and the first recognition element comprises a reagent for specifically recognizing circulating tumor cells (CTC); and a second probe, the second probe comprising nanoparticles, a Raman reporter molecule and a second recognition element, the second recognition element comprising a reagent for specifically recognizing platelets.

Description

Technical Field

[0001] This application relates to the field of biomedical engineering, specifically to diagnostic and screening systems, and more specifically to the identification of platelet-adherent circulating tumor cells (CTCs). Background Art

[0002] Circulating tumor cells (CTCs) play a key role in cancer metastasis. CTCs survive in the blood circulation and interact with blood cells. Among them, platelets are most likely to contact CTCs first due to their large number, triggering signal activation and functional changes. The interactions between CTCs and platelets include: (1) CTCs induce platelet activation; (2) platelets protect CTCs from shear stress and anoikis; (3) platelets mediate the immune escape of CTCs. Therefore, it is of great significance to study the biological functions and mechanisms of platelet-adherent CTCs.

[0003] Currently, the research on platelets altering the immunophenotype of CTCs to help tumor cells escape immune killing usually visualizes platelet-adherent CTCs through experimental means of immunofluorescence imaging.

[0004] However, the following problems still need to be improved in the field of immunofluorescence imaging for detecting platelet-adherent CTCs: (1) Non-specific staining: Immunofluorescence staining usually requires the use of fluorescently labeled antibodies to identify target cells, but there may be non-specific binding or cross-reactions, resulting in misjudgment or interference from background signals; (2) Limited multi-labeling ability: Immunofluorescence imaging usually can only use a limited number of fluorescent labels to simultaneously detect multiple molecules or cell subsets and the signals are easily interfered, so it may not be able to cover multiple target analytes in complex samples; (3) Signal quenching and fluorescence quenching: At high concentrations, the signals of fluorescent labels may be affected by signal quenching or fluorescence quenching, resulting in signal attenuation or distortion; (4) Photobleaching effect: Immunofluorescence imaging requires long-term illumination, which may cause the photobleaching effect of cells, leading to cell damage or even death; (5) High detection cost: The expensive equipment and fluorescent labeling reagents required for immunofluorescence imaging are costly, increasing the economic burden of the experiment.

[0005] There is still an urgent need in this field to improve the above problems and achieve a technical solution for the specific recognition and quantification of platelet-adherent CTCs in whole blood with higher sensitivity and accuracy. So far, there is no visualization technology that can replace immunofluorescence imaging for detecting platelet-adherent CTCs.

[0006] Surface-enhanced Raman spectroscopy (SERS) is an extremely sensitive analytical method. SERS can provide chemical information of samples, including chemical composition, molecular structure, and vibration information of chemical bonds, and can detect target substances at extremely low concentrations, even reaching the single-molecule level. In addition, the SERS technique combines the resolution and selectivity of Raman spectroscopy, with a large difference from the background signal, thus achieving highly selective recognition of target molecules and receiving extensive attention in various fields such as qualitative and quantitative analysis. Moreover, SERS has a fast analysis speed and real-time detection ability, can complete sample analysis in a short time, and can further realize the monitoring of real-time molecular changes. Compared with fluorescence techniques, the SERS technique reduces the complexity and cost of experiments, has stable SERS signals, does not have the defects of photobleaching and photoluminescence quenching, and avoids false positive or false negative results that may be brought by markers. Secondly, SERS also has the ability of multi-labeling. Especially the SERS probes in the Raman silent region (1800 - 2800 cm-1) show significant advantages in the fields of bioimaging, disease diagnosis, etc. by reducing endogenous interference, enhancing signal specificity and sensitivity. Developing Raman probes with high-intensity characteristic peaks in the Raman silent region (1800 - 2800 cm-1) of cells is crucial for achieving high-resolution multi-color Raman imaging of living cells.

[0007] So far, there is a lack of the design of SERS probe combinations for the recognition and quantification of platelet-adherent CTCs. Summary of the Invention

[0008] To solve the above problems existing in the prior art, the present application provides a surface-enhanced Raman spectroscopy (SERS) probe combination, which includes SERS probes based on the Raman silent region, and the probe combination can be used for specifically recognizing platelet-adherent CTCs at the single-cell level. The present application also provides a kit containing the probe combination, and a method for specifically recognizing and / or quantifying platelet-adherent CTCs using the probe combination. The new method for recognizing and imaging platelet-adherent CTCs provided by the present application can not only replace the existing immunofluorescence imaging method in the art, but also improve the detection sensitivity and accuracy, and has significant technical advantages such as high specificity, low background noise, real-time monitoring, stable signals, multi-detection, good biocompatibility, simplicity and feasibility.

[0009] On the one hand, the present application provides a surface-enhanced Raman spectroscopy (SERS) probe combination, which includes: a first probe, the first probe includes nanoparticles, Raman reporter molecules, and a first recognition element, and the first recognition element includes a reagent for specifically recognizing circulating tumor cells (CTCs); and a second probe, the second probe includes nanoparticles, Raman reporter molecules, and a second recognition element, and the second recognition element includes a reagent for specifically recognizing platelets.

[0010] On the other hand, the present application provides a kit, which comprises: a SERS probe combination and a microwell array chip for SERS imaging. The probe combination includes: a first probe, which includes nanoparticles, Raman reporter molecules and a first recognition element, and the first recognition element includes a reagent for specifically recognizing circulating tumor cells (CTCs); and a second probe, which includes nanoparticles, Raman reporter molecules and a second recognition element, and the second recognition element includes a reagent for specifically recognizing platelets.

[0011] On the other hand, the present application provides a method for identifying and / or quantifying platelet-adhered CTCs, which comprises: incubating a SERS probe combination with a cell population. The probe combination includes: a first probe, which includes nanoparticles, Raman reporter molecules and a first recognition element, and the first recognition element includes a reagent for specifically recognizing circulating tumor cells (CTCs); and a second probe, which includes nanoparticles, Raman reporter molecules and a second recognition element, and the second recognition element includes a reagent for specifically recognizing platelets; and performing SERS scanning imaging and detecting SERS signals. Description of the Drawings

[0012] The present application will be described in more detail below with reference to the drawings, in which:

[0013] Figure 1 It is a synthetic step diagram of OPE1 and OPE2.

[0014] Figure 2 It shows (A) a transmission electron microscope (TEM) image of gold nanorods; (B) a particle size distribution diagram of gold nanorods; (C) an extinction spectrum diagram of gold nanorods before and after probe modification.

[0015] Figure 3 It shows (A) a schematic diagram for the preparation of SERS imaging probe folic acid and MPBA-modified gold nanorods (GNS-MPBA), (B) a Raman spectrum diagram of GNS-MPBA.

[0016] Figure 4 It shows (A) a schematic diagram for the preparation of EpCAM antibody and OPE1-modified gold nanorods (GNS-OPE1), (B) a Raman spectrum diagram of GNS-OPE1.

[0017] Figure 5 It shows (A) a schematic diagram for the preparation of CD41 antibody and OPE2-modified gold nanorods (GNS-OPE2), (B) a Raman spectrum diagram of GNS-OPE2.

[0018] Figure 6Shows the (A) schematic diagram and (B) Raman spectrogram when GNS-OPE1 and GNS-OPE2 are co-localized.

[0019] Figure 7 Shows the (A) schematic diagram and (B) physical diagram of slide modification and loading of the PDMS array.

[0020] Figure 8 Is the flow chart of the operation steps of the present invention.

[0021] Figure 9 Shows an actual example of SERS scanning measurement, where:

[0022] (A) is the SERS scanning map at wavenumbers of 1584 cm -1 , 2126 cm -1 and 2216 cm -1 when there are only CTCs. It can be seen that when there are only CTCs, strong SERS peaks appear at 1584 cm -1 and 2126 cm -1 , while the probe GNS-OPE2 labeled with CD41 has no obvious signal at 2216 cm -1 , indicating that this CTC is a non-platelet adherent CTC.

[0023] (B) is the SERS scanning map at wavenumbers of 1584 cm -1 , 2126 cm -1 and 2216 cm -1 when there are only platelets. It can be seen that when there are only platelets, the probe GNS-OPE2 labeled with CD41 has an obvious signal at 2216 cm -1 , while there is no signal of CTC, indicating that only platelets exist alone.

[0024] (C) is the SERS scanning map at wavenumbers of 1584 cm -1 , 2126 cm -1 and 2216 cm -1 when platelet adherent CTCs exist. It can be seen that obvious intensity SERS signals appear at all three wavenumbers, indicating that this CTC is a platelet adherent CTC.

[0025] Figure 10 Shows the confocal fluorescence microscope imaging diagram of detecting CTCs or platelet adherent CTCs by multi-color immunofluorescence imaging method. Detailed implementation mode

[0026] This application relates to a surface-enhanced Raman spectroscopy (SERS) probe combination, which includes: a first probe, the first probe includes nanoparticles, Raman reporter molecules and a first recognition element, and the first recognition element includes a reagent that specifically recognizes circulating tumor cells (CTCs); and a second probe, the second probe includes nanoparticles, Raman reporter molecules and a second recognition element, and the second recognition element includes a reagent that specifically recognizes platelets.

[0027] In some embodiments, the probe combination further includes: a third probe, the third probe includes nanoparticles, Raman reporter molecules and a third recognition element, and the third recognition element includes a reagent that specifically recognizes circulating tumor cells (CTCs) and is different from the first recognition element.

[0028] In some embodiments, the reagent that specifically recognizes CTCs includes folic acid and / or epithelial marker antibodies. In some embodiments, the reagent that specifically recognizes platelets includes platelet membrane glycoprotein antibodies. In some embodiments, the epithelial marker antibody includes an EpCAM antibody or a cytokeratin antibody. In some embodiments, the cytokeratin antibody includes CK8, CK18 or CK19 antibodies. In some embodiments, the platelet membrane glycoprotein antibody includes CD41, CD42a, CD42b or CD61 antibodies.

[0029] In some embodiments, the nanoparticles include metal nanoparticles or non-metal nanoparticles. In some embodiments, the Raman reporter molecules include Raman fingerprint region reporter molecules and / or Raman silent region reporter molecules.

[0030] In some embodiments, the Raman fingerprint region reporter molecules include 4-mercaptobenzeneboronic acid, 4-mercaptobenzoic acid, 4-nitrobenzenethiol, 4-aminobenzenethiol, 4-methoxybenzyl mercaptan and / or 5,5'-dithiobis(2-nitrobenzoic acid). In a preferred embodiment, the Raman fingerprint region reporter molecule includes 4-mercaptobenzeneboronic acid.

[0031] In some embodiments, the Raman silent region reporter molecules include 4-ethynylbenzenethiol and its derivatives and / or 4-mercaptobenzonitrile. In some embodiments, the 4-ethynylbenzenethiol derivatives include 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene (OPE1) and 4,4'-bis(thioacetyl)diphenylethyne (OPE2). In a preferred embodiment, the Raman silent region reporter molecules include 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene and 4,4'-bis(thioacetyl)diphenylethyne.

[0032] In some embodiments, the Raman reporter molecule of at least one of the first probe, the second probe, and / or the third probe is a Raman silent region reporter molecule.

[0033] In some embodiments, the metal nanoparticles include gold nanoparticles, silver nanoparticles, or gold-silver composite nanoparticles. In some embodiments, the non-metal nanoparticles include graphene. In some embodiments, the nanoparticles include nanorods, nanospheres, nanorods, nanoshells, nanoparticle clusters, nanowires, nanocubes, nanopyramids, nanopolyhedrons, and / or nanocrystals. In a specific embodiment, the nanoparticles include gold nanorods. In an alternative embodiment, the nanoparticles include silver nanorods.

[0034] In some embodiments, the SERS probe combination described herein includes the following probes: a first probe, a second probe, and / or a third probe; wherein the nanoparticles of the first probe, the second probe, and / or the third probe include gold nanoparticles, silver nanoparticles, or gold-silver composite nanoparticles, and the Raman reporter molecules of the first probe, the second probe, and / or the third probe are respectively selected from 4-mercaptobenzeneboronic acid, 4-mercaptobenzoic acid, 4-nitrobenzenethiol, 4-aminobenzenethiol, 4-methoxybenzyl mercaptan, and / or 5,5'-dithiobis(2-nitrobenzoic acid), 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene, and 4,4'-bis(thioacetyl)diphenylacetylene, and the recognition elements of the first probe, the second probe, and / or the third probe are respectively selected from folic acid, CD41 antibody, CD42a antibody, CD42b antibody, CD61 antibody, EpCAM antibody, CK8 antibody, CK18 antibody, or CK19 antibody.

[0035] In some embodiments, the SERS probe combination described herein includes the following probes: a first probe, a second probe, and / or a third probe; wherein the nanoparticles of the first probe, the second probe, and / or the third probe include gold nanoparticles, and the Raman reporter molecules of the first probe, the second probe, and / or the third probe are respectively selected from 4-mercaptobenzeneboronic acid, 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene, and 4,4'-bis(thioacetyl)diphenylacetylene, and the recognition elements of the first probe, the second probe, and / or the third probe are respectively selected from folic acid, CD41 antibody, and EpCAM antibody.

[0036] In some embodiments, the SERS probe combinations described herein include the following probes: a first probe comprising gold nanoparticles, 4-mercaptobenzeneboronic acid, and folic acid; a second probe comprising gold nanoparticles, 4,4'-bis(thioacetyl)diphenylacetylene, and a CD41 antibody; and / or a third probe comprising gold nanoparticles, 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene, and an EpCAM antibody.

[0037] In some embodiments, the SERS probe combinations described herein include the following probes: a first probe comprising gold nanorods, 4-mercaptobenzeneboronic acid, and folic acid; a second probe comprising gold nanorods, 4,4'-bis(thioacetyl)diphenylacetylene, and a CD41 antibody; and / or a third probe comprising gold nanorods, 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene, and an EpCAM antibody.

[0038] In the present application, the Raman reporter molecule is attached to the metal substrate. However, in in vivo and in vitro environments, the dissociation of the reporter molecule from the metal substrate and the adsorption of other molecules by the metal substrate may reduce the reliability and effectiveness of the naked probe. In addition, when the probe is applied to live cell detection, the issue of the probe's biotoxicity also needs to be considered. Therefore, the SERS probe may further include a surface coating material and encapsulation. In some embodiments, the surface-enhanced Raman spectroscopy (SERS) probe combinations described in the present application further include a protective shell / protective layer. In some embodiments, the protective shell is a biocompatible protective shell / biocompatible protective layer. In some embodiments, the biocompatible protective shell is PAH. In some embodiments, the protective shell can improve the biocompatibility of the probe and reduce non-specific binding.

[0039] Surface-Enhanced Raman Spectroscopy (SERS) Probe

[0040] SERS probes (or SERS tags) generally consist of nanoparticles, Raman reporter molecules, and recognition elements.

[0041] Raman reporter molecule

[0042] Raman imaging can characterize molecules by detecting the vibrations of molecular bonds and providing unique molecular "fingerprints". Raman reporter molecules adsorbed on the surface of metal nanoparticles can generate extremely strong Raman signals due to the excitation of local surface plasmons. The term "Raman reporter molecule" or "Raman active molecule" used in this article refers to a molecule with characteristic Raman spectral signals, and Raman reporter molecules have the following characteristics: (1) a characteristic chemical structure that generates a signature Raman spectrum, enabling accurate identification of the target in the Raman spectra of complex biological samples; (2) a large Raman cross-section that can generate strong Raman signals, endowing the probe with high sensitivity; (3) since the SERS effect strongly depends on the distance between the reporter molecule and the nanoparticle surface, it is also required that the reporter molecule can stably and effectively adhere to the surface of the nanostructure.

[0043] Raman reporter molecules provide unique fingerprint spectra for SERS probes. According to the characteristic peaks of the Raman reporter molecules used, SERS probes can function in the "fingerprint region" or the "Raman silent region".

[0044] In the context of this application, the term "fingerprint region" refers to the 800 - 1800 cm-1 band of the Raman spectrum. The fingerprint region is the signal response range of most Raman probes, but it is also the main section where the background signals of biological substances interfere.

[0045] In some embodiments, the Raman fingerprint region reporter molecules in the SERS probe combination include 4-mercaptophenylboronic acid (4-MPBA), 4-mercaptobenzoic acid (4-MBA), 4-nitrothiophenol (4-NTP), 4-aminothiophenol (4-ATP), 4-methoxybenzyl mercaptan (MATT), and / or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Specifically, in the context of this application, 4-MPBA means 4-mercaptophenylboronic acid. In a preferred embodiment, the Raman fingerprint region reporter molecules in the SERS probe combination include 4-mercaptophenylboronic acid (4-MPBA).

[0046] In the context of this application, the term "Raman silent region" or "cellular / biological silent region of the Raman spectrum" refers to the 1800 - 2800 cm-1 band of the Raman spectrum, where there are almost no signals generated by biomolecules. The term "Raman silent region reporter molecule" usually refers to a compound with triple bonds (such as having an alkynyl group, a cyano group, an azide group) or containing isotopes (such as containing deuterium, nitrogen-15, or carbon-13). Compared with the commercially available Raman reporter molecules in the fingerprint region, the Raman silent region probes are relatively expensive and have weaker signals. Therefore, the development of novel Raman silent region reporter molecules suitable for multiplex detection is of great significance in this field.

[0047] The inventors have designed novel Raman probes with distinct vibration frequencies in the Raman silent region. Since biological samples do not have significant Raman responses in the Raman silent region, the signals of the SERS probes described in this application will not be interfered by the spontaneous Raman scattering of biomolecules, reducing background noise and enhancing the detectability of signals, which can meet the needs of multiplex detection, thereby achieving highly selective recognition of platelet-adherent CTCs.

[0048] In some embodiments, the Raman silent region reporter molecules in the SERS probe combination include 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene (OPE1), 4,4'-bis(thioacetyl)diphenylacetylene (OPE2), 4-ethynylbenzenethiol (OPE0), 4-mercaptobenzonitrile (4-MBN). Specifically, in the context of this application, OPE1 refers to 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene (4-(2-trimethylsilylethynyl)benzenethiol); OPE2 refers to 4,4'-bis(thioacetyl)diphenylacetylene (4,4'-(1,4-phenylenebis(ethyne-2,1-diyl)dibenzenethiol). In a preferred embodiment, the Raman silent region reporter molecules in the SERS probe combination include 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene (OPE1) and / or 4,4'-bis(thioacetyl)diphenylacetylene (OPE2).

[0049] Recognition element

[0050] Based on the novel Raman silent region reporter molecules, the probe combination described in this application can achieve multiplex imaging. In the context of this application, "recognition element" refers to a reagent that can specifically recognize and / or selectively bind to a target analyte, such as a ligand, aptamer, or biomarker antibody with specificity. In some embodiments, the recognition elements used in the present invention include ligands, aptamers, or biomarker antibodies that are targeted to platelet-adherent CTCs.

[0051] For specifically recognizing and / or quantifying CTCs or platelet-adherent CTCs, this application creatively designs a SERS probe combination with folic acid, epithelial marker antibodies, and / or platelet membrane glycoprotein antibodies as recognition elements. CTC epithelial markers are epithelial-derived molecular markers used to identify CTCs, including epithelial cell adhesion molecule (EpCAM) and cytokeratins (such as CK8, CK18, or CK19, etc.). Antibodies that specifically recognize epithelial markers include EpCAM antibodies or cytokeratin (such as CK8, CK18, or CK19) antibodies.

[0052] The application of EpCAM (epithelial cell adhesion molecule) in the detection of circulating tumor cells (CTCs) has a wide range of research bases, and it has been applied in the early clinical diagnosis and monitoring of various cancers (such as breast cancer, prostate cancer, and colon cancer, etc.). However, despite the important clinical value of EpCAM, there are still some deficiencies in its application in CTC detection. For example, EpCAM is a marker on the surface of most epithelial cells, so it shows a high expression in tumor cells, which enables the detection method based on EpCAM to effectively capture CTCs of epithelial origin, but there may be a phenomenon of missed detection for CTCs with low expression of EpCAM. Cytokeratin (CK) is a structural protein in epithelial cells, and there are more than 20 members in the cytokeratin family, which are often used as epithelial-derived tumor markers. In the application of CTC detection, CK8, CK18, or CK19 are commonly used. Similar to EpCAM, the method based on CK may have a phenomenon of missed detection for CTCs with low expression of epithelial-derived markers.

[0053] Folate receptor (FR) also shows a high level of expression in various tumor cells, including lung cancer, colorectal cancer, pancreatic cancer, ovarian cancer, endometrial cancer, nasopharyngeal cancer, and urinary system cancer, etc., which makes it an important target among tumor markers. Compared with EpCAM, the expression of FR in normal tissues is extremely limited, ensuring the specificity of this detection method for tumors. However, there are still FR-negative CTCs, which may cause false detection.

[0054] The technical solution of this application combines epithelial marker antibodies and FR for CTC labeling. Taking EpCAM as an example, the examples in this application prove that the specially designed SERS probe combination further enhances the sensitivity and accuracy of CTC detection, and significantly improves the effect of early tumor detection and intervention.

[0055] Platelet membrane glycoproteins include plasma membrane and granule membrane glycoproteins. Specifically, they include proteins such as CD41, CD42a, CD42b, or CD61, etc. The technical solution of this application further incorporates platelet membrane glycoprotein antibodies as recognition elements, thus providing a probe combination and detection method for identifying platelet-adherent CTCs. Platelet membrane glycoprotein CD41 is an important receptor on the surface of platelets and participates in the processes of platelet aggregation and activation. The examples in this application prove with CD41 as an example that the specially designed SERS probe combination further enhances the sensitivity and accuracy of platelet-adherent CTC detection.

[0056] The technical solution of this application performs multiplex detection on joint labeling through a specially designed SERS probe combination, cross-verifies the multi-channel results, effectively reduces the probabilities of false positives and false negatives in the detection of platelet-adherent CTCs, overcomes the problems of misdetection and missed detection, and further improves the sensitivity and accuracy of early cancer screening and diagnosis.

[0057] nanoparticle

[0058] In the context of this application, the SERS probe includes nanoparticles as the substrate material, and the nanoparticles can enhance the Raman signal of Raman reporter molecules directly bound to their surface. The nanoparticle substrate material can be a noble metal (such as gold, silver, etc.) or a non-metal nanomaterial (such as graphene, boron nitride, semiconductor, etc.), or a composite material.

[0059] In some embodiments, the metal nanoparticles described herein include gold nanoparticles, silver nanoparticles, or gold-silver composite nanoparticles. In some embodiments, the nanoparticles described herein include single-component nanoparticles and composite nanoparticles. The composite nanoparticles include core-shell structured nanoparticles. The core-shell structured nanoparticles can be composed of a variety of materials such as metals, polymers, and / or inorganic substances, and have a central core and a shell structure wrapped on the outer layer. The core-shell structured nanoparticles contain Raman reporter molecules between the two layers of metals, have a stable structure, and have obvious SERS effects and excellent biocompatibility. In some embodiments, the "shell" or "core" of the core-shell structured nanoparticles described herein is gold or silver. In some embodiments, the "gold-silver composite nanoparticles" described herein include core-shell structured nanoparticles composed of a gold nanoparticle core and a silver shell or a silver nanoparticle core and a gold shell, or other gold-silver composite structures. Core-shell structures composed of other non-metal shells and gold or silver cores can also be used to implement the present invention, and thus are also covered by the protection scope of this application. In some embodiments, the non-metal nanoparticles described herein include graphene. In some embodiments, the nanoparticles include nanoparticles of various morphologies, such as nanospheres, nanorods, nanoshells, nanoparticle clusters, nanowires, nanocubes, nanopyramids, nanopolyhedrons, and / or nanocrystals.

[0060] In some embodiments, the nanoparticles include gold nanoparticles. In some embodiments, the gold nanoparticles include nanoparticles of various morphologies, such as gold nanospheres, gold nanorods, gold nanospheres, gold nanowires, gold nanocubes, gold nanopyramids, gold nanopolyhedrons, or gold nanocrystals. In a specific embodiment, the nanoparticles include gold nanospheres. In an alternative embodiment, the nanoparticles include silver nanospheres.

[0061] Although specific embodiments of the present application prepared and characterized gold nanostars and exemplarily adopted gold nanostars to implement the technical solutions of the present application, those skilled in the art can understand that other noble metal nanoparticles capable of realizing the SERS effect besides gold nanostars are included in the protection scope of the present application. Specifically, for example, gold nanoparticles with other three-dimensional nanostructures, silver nanostars, gold-silver composite nanoparticles with other three-dimensional nanostructures, or core-shell structure nanoparticles composed of other non-metal shells and gold / silver cores all belong to the nanoparticles with tip hot spots well-known in the art and can also be used as SERS substrates to achieve signal enhancement effects, and thus are also included in the protection scope of the present application.

[0062] In the context of the present application, the term "gold nanostar (GNS)" refers to a three-dimensional gold nanostructure with multiple pointed arms, which has rich tip structures and rich plasmon hot spot regions. In some embodiments, the gold nanostars described herein have a diameter of 30 to 70 nm. In some embodiments, the gold nanostars described herein are 40 to 60 nm. In some embodiments, the gold nanostars described herein have a diameter of about 50 nm. In some embodiments, the extinction spectrum absorption peak of the gold nanostars described herein is located near 719 nm.

[0063] On the other hand, the present application also provides a kit, which includes: a SERS probe combination and a micro-well array chip for SERS imaging. The probe combination includes: a first probe, which includes nanoparticles, Raman reporter molecules, and a first recognition element, and the first recognition element includes a reagent specifically recognizing circulating tumor cells (CTCs); and a second probe, which includes nanoparticles, Raman reporter molecules, and a second recognition element, and the second recognition element includes a reagent specifically recognizing platelets.

[0064] In some embodiments, the present application also relates to a kit, which includes the SERS probe combination described in any embodiment in the context and a micro-well array chip for SERS imaging. In some embodiments, the micro-well array is on a polylysine-modified glass slide and / or a chip carrier. In some embodiments, the surface of the glass slide is modified with polylysine and loaded with a PDMS porous micro-well membrane. In some embodiments, the PDMS micro-well array adheres to the polylysine-modified glass slide to form a target cell detection device. In some embodiments, the PDMS membrane is pre-punched, and the diameter of the pre-punched holes is about 3 mm and the height is about 1 mm. In some embodiments, the diameter of the micro-holes on the micro-well array chip is 1 to 5 mm and the height is 0.2 to 5 mm. In some embodiments, the diameter of the micro-holes on the micro-well array chip is about 3 mm and the height is about 1 mm.

[0065] On the other hand, the present application also relates to a method for identifying and / or quantifying CTCs or platelet-adhered CTCs, which includes: incubating the SERS probe combinations described herein with a cell population; and performing SERS scanning imaging and detecting SERS signals. In some embodiments, the incubation is carried out in a microwell array. In some embodiments, the incubation is carried out in a microwell array on a polylysine-modified glass slide and / or chip carrier.

[0066] In some embodiments of the present application, the incubation step includes: (a) adding the cell population into the microwell array on the polylysine-modified glass slide and chip carrier, pre-incubating at 30-40 °C, preferably at about 37 °C for 1-5 hours, preferably about 2 hours; and (b) adding the SERS probe combinations described herein into the microwell array, incubating at 30-40 °C, preferably at about 37 °C for 10 minutes to 2 hours, preferably about 30 minutes.

[0067] In some embodiments of the present application, the SERS scanning imaging and detection step includes: (c) performing SERS scanning imaging of the entire well (exemplary parameters: 638 nm laser, step size of 1 micron, collection time set to 1 s); (d) detecting the SERS signal, determining the presence of different CTC cell types and evaluating their adhesion status based on the measured SERS signal; optionally, (e) picking target cells with a micromanipulator to complete subsequent related studies.

[0068] In some embodiments, the characteristic peak of the GNS-MPBA-folic acid probe is located at about 1584 cm -1 -1. In some embodiments, the characteristic peak of the GNS-OPE1-EpCAM antibody probe is located at about 2126 cm -1 -1. In some embodiments, the characteristic peak of the GNS-OPE2-CD41 antibody probe is located at about 2216 cm -1 -1.

[0069] In some embodiments, in step (d), the SERS signals at 1584 cm -1 , 2126 cm -1 and 2216 cm -1 wavenumbers are scanned. In some embodiments, in step (d), when strong SERS peaks are presented at 1584 cm -1 (folic acid) and 2126 cm -1 (EpCAM antibody), while the probe GNS-OPE2 labeled with CD41 shows no strong SERS peak at 2216 cm -1When there is no obvious signal at [a certain position], the CTC is identified as a non-platelet adherent CTC. In some embodiments, in step (d), when the probe GNS-OPE2 labeled with CD41 has an obvious signal at 2216 cm -1 and there is no signal of CTC at 1584 cm -1 (folic acid) and 2126 cm -1 (EpCAM antibody), it is identified that platelets exist alone. In some embodiments, in step (d), when SERS signals with obvious intensities are observed at 1584 cm -1 (folic acid), 2126 cm -1 (EpCAM antibody) and 2216 cm -1 (CD41 antibody) at three wavenumbers, the CTC is identified as a platelet adherent CTC.

[0070] In some embodiments, the cell population undergoes a separation step and / or an enrichment step before incubation with the SERS probe combination. In some embodiments, the separation step and / or the enrichment step includes: (i) centrifuging the anticoagulant-treated whole blood sample at 150 g for 12 minutes. After centrifugation, the lower layer is red blood cells and white blood cells, and the upper layer is platelet-rich plasma, and the upper layer is discarded; (ii) adding red blood cell lysate to remove red blood cells; (iii) adding magnetic beads modified with CD45 antibody to the cell solution obtained in step (ii), and capturing white blood cells with a magnetic field; (iv) harvesting the remaining cell population.

[0071] In the present application, the inventors developed three novel nanoparticles with surface-enhanced Raman scattering (SERS) function based on gold nanospheres (GNS, or AuNS). These nanoparticles are respectively modified with 4-MPBA (4-mercaptobenzeneboronic acid, as a Raman reporter molecule), OPE1 (1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene) and OPE2 (4,4'-bis(thioacetyl)diphenylacetylene), and the latter two have strong SERS signals in the Raman silent region; these nanoparticles are further functionalized with recognition elements for identifying platelet-adherent CTCs or CTCs. For example, the nanoparticles in the present application are conjugated with folic acid (FA, folic acid, as a targeting ligand) or modified with EpCAM antibody for accurately identifying CTCs, and / or the nanoparticles in the present application are modified with CD41 antibody for identifying platelets.

[0072] This application designs Raman silent region reporter molecules OPE1 or OPE2, and combines them with fingerprint region reporter molecule 4-MPBA to synthesize a novel SERS probe combination. The probe combination described in this application further includes EpCAM antibody, CD41 antibody or folic acid (FA) as recognition elements, thereby creatively achieving multiplex labeling imaging of platelet-adherent CTCs. In the technical solution of this application, the signal of the Raman probe located in the Raman silent region is not interfered by biological background signals, and can well meet the increasing precision requirements of precision medicine for medical imaging. The technical solution of the present invention can improve the technical defects of the existing immunofluorescence imaging method for visualizing platelet-adherent CTCs, and overcome the problems of "non-specific staining, limited multiplex labeling ability, signal quenching and fluorescence quenching, photobleaching and high detection cost" existing in immunofluorescence imaging by means of SERS imaging technology, thereby providing a brand-new imaging visualization technology for the detection of platelet-adherent CTCs.

[0073] In summary, compared with the prior art, the technical solution of this application has the following advantages:

[0074] ① High sensitivity: The SERS probe combination of this application and its corresponding immunolabeling imaging method have sensitivity at the single-cell level. The SERS effect can significantly enhance the Raman signal of the probe. When imaging in the Raman silent region, even very low concentrations of the target can be detected, thereby improving the sensitivity of detection.

[0075] ② Strong specificity: The signal of the SERS probe in the Raman silent region has high specificity, can avoid the interference of endogenous molecular signals, and provides clearer and more specific signals, thereby improving the resolution and precision of imaging.

[0076] ③ Low background noise: The SERS probe in the Raman silent region has low background noise. Since biological samples have no significant Raman response in the Raman silent region, the signal of the SERS probe will not be interfered by the spontaneous Raman scattering of biomolecules, reducing the background noise and enhancing the detectability of the signal, thereby achieving highly selective recognition of platelet-adherent CTCs.

[0077] ④ Real-time detection: The SERS immunolabeling imaging method has a fast analysis speed and real-time detection ability, can complete sample analysis in a short time, and can monitor real-time molecular changes.

[0078] ⑤ Simple and economical: Compared with fluorescence technology, the SERS immunolabeling imaging method reduces the complexity and cost of experiments.

[0079] ⑥ Stable signal: The SERS immunolabeling imaging method has a stable signal and does not have the defects of photobleaching and photoluminescence quenching.

[0080] ⑦Avoid false positive or false negative results: The SERS immunolabeling imaging method has stable signals, avoiding false positive or false negative results that may be brought by the labeling agents.

[0081] ⑧Multiplexing ability: The SERS immunolabeling imaging probes in the Raman silent region have multiplex imaging capabilities. In the Raman silent region, different probe designs with different Raman characteristic signals can be used, making multiplex imaging and the detection of multiple labeling agents possible, and enabling the simultaneous identification and tracking of multiple target molecules or cellular processes.

[0082] ⑨Good biocompatibility: The SERS probes commonly used in the Raman silent region are functionalized based on noble metal nanomaterials (such as gold or silver), and can exhibit good compatibility in biological systems, making them suitable for biomedical imaging applications in vivo or in vitro.

[0083] Examples

[0084] The technical solutions of the present invention will be described in more detail below in conjunction with specific examples. The following examples are only for illustration and do not constitute any limitation or restriction on the technical solutions of the present invention. The specific technical parameters such as materials, steps, conditions, numerical values or numerical ranges in the following examples are only for illustration, not exhaustive nor limiting.

[0085] Except for the specific methods, devices, and materials used in the examples, according to the knowledge of those skilled in the art in the technical field and the description of the present invention, any methods, devices, and materials of the prior art similar to or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0086] Table 1. Reagents / Devices Used

[0087]

[0088]

[0089] Example 1. Synthesis and Characterization of Two Raman-Silent Region Probes, OPE1 and OPE2

[0090] (1)Synthesis and Characterization of OPE1: In a 1 L round-bottom flask, 4-iodoaniline (33 g) was dissolved in a mixed solution of concentrated hydrochloric acid (88 mL) and water (280 mL), and the mixture was cooled to 0 °C in an ice bath. Subsequently, a solution of sodium nitrite (11 g) in water (42 mL) was added dropwise to the ice-cold solution over 2 hours, and after completion, the mixture was stirred for 15 minutes. Then, diethyl ether tetrafluoroborate was added to form a diazonium tetrafluoroborate precipitate. The precipitate was filtered and washed successively with ice water, methanol, and ether. The precipitate was dried under vacuum and used directly in the next reaction without further purification. Potassium thioacetate (11.93 g) was dissolved in dimethyl sulfoxide (300 mL), and then a DMSO solution of diazonium tetrafluoroborate (10.5 g dissolved in 100 mL) was added dropwise thereto over 0.5 hour, and the mixture was stirred at room temperature for 1.5 hours. After completion of the reaction, the mixture was poured into saturated brine, and then the organic phase was extracted with ether and washed successively with sodium hydroxide solution and saturated brine. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated. The residue was further purified by silica gel column chromatography to finally obtain a light yellow solid product, 1-iodo-4-thioacetylbenzene. A mixture of 1-iodo-4-thioacetylbenzene (4.44 g), tetrahydrofuran (20.0 mL), trimethylsilylacetylene (2.35 g), bis(triphenylphosphine)palladium(II) dichloride (0.56 g), copper(I) iodide (0.16 g), and N,N-diisopropylethylamine (3.32 g) was stirred under an argon atmosphere for 24 hours. After the reaction, the mixture was poured into water, and the aqueous layer was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and the solvent was removed under vacuum to obtain a crude product. The crude product was purified by silica gel column chromatography to obtain a pale white solid, 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene.

[0091] Characterization Results of 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene: 1H NMR (300 MHz, CDCl3) δ (ppm): 0.25 (s, 9H), 2.41 (s, 3H), 7.33 (d, J = 8.1 Hz, 2H), 7.47 (d, J = 8.1 Hz, 2H); 13C NMR (300 MHz, CDCl3) δ (ppm): 0.11, 30.48, 96.43, 104.38, 124.58, 128.54, 132.73, 134.29, 134.57, 193.55. GC-MS calculated value: 248.42, measured value: 248.2. In a degassed tetrahydrofuran (THF, 10 mL) solution, 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene (2.5 mg) and ammonium hydroxide (10 μL) were added dropwise. The solution was stirred at room temperature for 30 minutes under a nitrogen atmosphere. The resulting OPE1 solution could be used without further purification.

[0092] (2) Synthesis and Characterization of OPE2: At 0 °C, 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene (1.7 g) was dissolved in tetrahydrofuran (20 mL). Subsequently, glacial acetic acid (0.1 g) and acetic anhydride (0.1 g) were added successively, and then a solution of tetrabutylammonium tetrafluoride (28.8 g) in tetrahydrofuran (10 mL) was added dropwise under an argon atmosphere. The solution was warmed to room temperature and reacted for 1 hour. The reaction mixture was poured into water, and the aqueous layer was extracted with dichloromethane and washed with brine. The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under vacuum. The residue was further purified by column chromatography to obtain the yellow to orange solid target product 4-ethynyl-1-thioacetylbenzene. Under an argon atmosphere, N,N-diisopropylethylamine (0.62 g) was added to a solution of 1-iodo-4-thioacetylbenzene (0.53 g) and 4-ethynyl-1-thioacetylbenzene (0.42 g) dissolved in tetrahydrofuran (THF, 10 mL). After stirring at 23 °C for 5 minutes, bis(triphenylphosphine)palladium(II) dichloride (Pd(PPh3)2Cl2, 81 mg) and copper(I) iodide (CuI, 68 mg) were added, and tetrahydrofuran (10 mL) was added additionally. The reaction mixture was stirred at room temperature until complete conversion was detected by TLC (18 hours). Dichloromethane (CH2Cl2) and water were added, and the aqueous phase was separated. The aqueous phase was extracted with dichloromethane, the organic phases were combined, washed with water, then dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated. The residue was purified by column chromatography to obtain the pale yellow solid product 4,4'-bis(thioacetyl)diphenylacetylene.

[0093] Characterization Results of 4,4'-bis(thioacetyl)diphenylacetylene: 1H NMR (300 MHz, CDCl3): δ = 2.44 (s, 6H), 7.42 (d, J = 8.1 Hz, 4H), 7.58 (d, J = 8.1 Hz, 4H); 13C NMR (300 MHz, CDCl3): δ = 30.20, 90.14, 124.06, 128.31, 132.14, 134.15, 193.31; GCMS calculated value: 326.43, measured value: 326.2. In a degassed tetrahydrofuran (THF, 10 mL) solution, 4,4'-bis(thioacetyl)diphenylacetylene (2.5 mg) and ammonium hydroxide (10 μL) were added dropwise. The solution was stirred at room temperature for 30 minutes under a nitrogen atmosphere. The resulting OPE2 solution can be used without further purification.

[0094] Example 2. Synthesis of Gold Nanostars

[0095] (1) Synthesis of Gold Nanostars (GNS): First, under vigorous stirring, 15 mL of 1% sodium citrate solution was added to 100 mL of boiling 1 mM HAuCl 4in a solution, and after continuing to boil for 15 minutes, it was cooled and filtered through a 0.22 μm nitrocellulose membrane. The filtered seed solution was stored at 4 °C for long-term use. In a 20 mL glass vial, stirred at 700 rpm at room temperature, 100 μL of the above sodium citrate-stabilized seed solution (particle size: 12 ± 0.7 nm; A 520 : 2.81) was added to 10 mL of HAuCl 4 solution (0.25 mM, containing 10 μL of 1 M HCl). Subsequently, 100 μL of AgNO 3 solution (final concentration 0.5 mM) and 50 μL of ascorbic acid (100 mM) were quickly added. After stirring for 30 seconds, the color of the solution rapidly changed from light red to blue or greenish-black. Subsequently, centrifuged at a centrifugal force of 3000–5000 g for 15 minutes to terminate the nanoparticle nucleation process. The centrifuged product was resuspended in deionized water and filtered through a 0.22 μm nitrocellulose membrane, and stored at 4 °C for long-term use.

[0096] (2) Physicochemical property characterization of gold nanorods (GNS): Transmission electron microscopy (TEM) was used to characterize the morphology and structure of the gold nanorods, and particle size statistics were analyzed by ImageJ software. The results showed that the synthesized gold nanorods had a diameter of approximately 50 nm. In addition, the extinction spectrum of the gold nanorods was measured using a UV-visible spectrophotometer, and the results showed that its absorption peak was located near 719 nm.

[0097] Example 3. Preparation and Physicochemical Property Characterization of Three SERS Imaging Probes

[0098] (1) Preparation of SERS imaging probes and targeting molecule-modified gold nanorods: Take three portions of 10 mL gold nanorod sols, and add 10 μL of tetrahydrofuran solutions of 4-MPBA, OPE1, and OPE2 with a concentration of 1 mM to each portion, respectively. Let it stand for reaction for 3 h. After the reaction is completed, add 1 mL of an aqueous solution of polyallylamine hydrochloride (PAH) with a concentration of 0.1% to each portion, respectively, and react for 12 h. Then, add 10 μL of an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride with a concentration of 10 mM and 20 μL of an aqueous solution of N-hydroxysuccinimide (NHS) with a concentration of 1 mM simultaneously, and let it stand to activate the carboxyl group for 1 h. Then, add 20 μL of solutions of folic acid (FA), EpCAM antibody, and CD41 antibody with a concentration of 1 mM to the three portions of probes, respectively, and let it stand for reaction for 16 h. Then, use an Amicon Ultra-15 centrifugal filter unit (Millipore, MWCO 3.0 kDa) to ultrafilter the obtained GNS–MPBA, GNS–OPE1, and GNS–OPE2 conjugated with targeting molecules. The purified nanoparticles are dispersed in 2.0 mL of Milli-Q water for the identification and quantitative detection of platelet-adherent CTCs. Among them, the SERS probes modified with FA and Epcam antibody have the ability to target the CTC cell membrane, and the SERS probe modified with CD41 antibody has the ability to target platelets.

[0099] (2) Characterization of the physicochemical properties of SERS imaging probes and targeting molecule-modified gold nanorods: The extinction spectra of the three kinds of modified gold nanorods were measured using a UV-visible spectrophotometer. The results showed that their absorption peaks were located near 725 nm, with a red shift of about 6 nm compared with the absorption peak of only the gold nanorod sol, indicating the successful modification of the probes and targeting molecules ( Figure 2 C). And the appearance of peaks in the Raman spectrum further illustrated the successful modification of the probes and targeting molecules ( Figure 3 B, Figure 4 B, Figure 5 B). And the appearance of peaks in the Raman spectrum when the three kinds of probes coexisted was as shown in Figure 6 B, indicating that the presence of the three target molecules can be co-localized.

[0100] Example 4. Construction of CTC Imaging Chip

[0101] As Figure 7As shown, after cleaning the glass slide with ethanol and ultrapure water, the glass slide was then soaked in an aqueous solution of polylysine (10 μg / ml) for 10 min, washed with ultrapure water, and dried with nitrogen for standby. Holes (3 mm in diameter and 1 mm in height) were pre-punched in the PDMS membrane with a puncher, and the holes were loaded onto the polylysine-modified glass slide. Due to the electrostatic interaction between PDMS and the polylysine-modified glass slide, the two would firmly bind to form micro reaction holes. The target cells to be measured were added into the holes, and similarly, the target cells and polylysine would also adsorb on the glass slide quickly due to electrostatic interaction, preparing for subsequent SERS imaging.

[0102] Example 5. Specific Recognition of CTC or Platelet-Adherent CTC in Whole Blood

[0103] The specific recognition operation process of CTC or platelet-adherent CTC in whole blood is as Figure 8 shown.

[0104] Separate target cells: ① Put the anticoagulant-treated whole blood sample into a centrifuge and centrifuge at a low speed of 150 g for 12 minutes. This step will separate red blood cells and white blood cells to the lower layer, and the upper layer is plasma rich in platelets, and the upper layer is removed; ② Add red blood cell lysate to remove red blood cells; ③ Add CD45-modified magnetic beads to the cell lysate after lysing red blood cells, and capture white blood cells with a magnetic field; ④ Add the remaining cells to the polylysine-modified glass slide and chip carrier for standby.

[0105] SERS measure target cells: ① Add the remaining cells to the polylysine-modified glass slide and chip carrier, and incubate at 37 °C for 2 h. ② Add the synthesized SERS probe mentioned above into the micro-well array, incubate at 37 °C for 30 min, complete the SERS labeling of the target cells, and then perform SERS scanning imaging of the whole well (638 nm laser, step size of 1 μm, collection time set to 1 s) to complete the labeling of the target cells. By detecting the SERS signal, the presence of different CTC cell types can be determined and their adhesion status can be evaluated. ③ Finally, pick the target cells with a micromanipulator to complete subsequent related research.

[0106] An actual example of SERS scanning measurement is as Figure 9 shown.

[0107] Figure 9 A shows the SERS scanning images at the wavenumbers of 1584 cm -1 , 2126 cm -1 and 2216 cm -1 when there are only CTCs. It can be seen that when there are only CTCs, at 1584 cm -1 and 2126 cm -1Strong SERS peaks are presented everywhere, while the probe GNS-OPE2 labeled with CD41 has no obvious signal at 2216, indicating that this CTC is a non-platelet adhesion type CTC.

[0108] Figure 9 B shows the SERS scanning images located at 1584 cm -1 , 2126 cm -1 and 2216 cm -1 wavenumbers when only platelets are present. It can be seen that when only platelets are present, the probe GNS-OPE2 labeled with CD41 has an obvious signal at 2216, while there is no signal of CTC, indicating that only platelets exist alone.

[0109] Figure 9 C shows the SERS scanning images located at 1584 cm -1 , 2126 cm -1 and 2216 cm -1 wavenumbers when platelet adhesion type CTC is present. It can be seen that obvious intensity SERS signals appear at all three wavenumbers, indicating that this CTC is a platelet adhesion type CTC.

[0110] Currently, the method used to identify CTC or platelet adhesion type CTC is multi-color immunofluorescence imaging method. In order to compare the existing method with the method of the present invention, multi-color immunofluorescence method is used to specifically recognize and separate CTC or platelet adhesion type CTC in whole blood and compare it with the method of the present invention.

[0111] Specific recognition of CTCs or platelet-adherent CTCs by multi-color immunofluorescence method: ① Using the same separation operation process as above, separate the target cells obtained; ② Fix the cells with 4% paraformaldehyde for 10 minutes to maintain cell structure and antigen integrity; ③ Permeabilize with 0.1% Triton X-100 PBS solution for 10 minutes to increase cell membrane permeability and facilitate antibody entry; ④ Block non-specific binding sites with 5% BSA (bovine serum albumin) to reduce background signals; ⑤ Incubate overnight at 4° with EpCAM antibody labeled with phycoerythrin (PE), Pan-CK antibody labeled with Alexa Fluor 555 (detecting CTC markers), DAPI (detecting cell nuclei), and CD41 antibody labeled with Alexa Fluor 647 (detecting platelet markers) (0.1 mg / mL Pan-CK labeled with Alexa Fluor 555, 0.1 mg / mL EpCAM primary antibody mixture labeled with phycoerythrin (PE), CD41 primary antibody labeled with Alexa Fluor 647), and then wash the cells 3 times with 1×PBS for 5 minutes each time to remove unbound primary antibodies; ⑥ Stain the cell nuclei with 8.4 μM nuclear stain DAPI (4',6-diamidino-2-phenylindole) for 10 minutes and wash the chip with 1×PBS; ⑦ Mount the slide with an anti-fluorescence quenching mounting medium, cover with a coverslip, and avoid air bubbles; ⑧ Image the target cells with an inverted fluorescence microscope to identify platelet-adherent CTCs (recognition criteria: identify CTCs by staining with EpCAM, Pan-CK, and DAPI, identify platelets by staining with CD41, and only capture platelet-adherent CTCs closely adhering to the CTC cell membrane), and transfer them to PBS buffer for secondary verification with a confocal fluorescence microscope. The confocal fluorescence microscope imaging diagram is as shown in Figure 10 shown. We compared the specific parameter situations of the existing technology and the method of the present invention in terms of detection limit (LOD), false positive rate (%), false negative rate (%), detection time (min), repeatability (RSD, %), and cost (yuan / sample), and proved that the technical solution of the present invention shows significant advantages in these aspects.

[0112] Table 2. Comparison table between the present invention and multi-color immunofluorescence imaging method

[0113]

[0114]

[0115] The above are only specific application examples of this application and do not constitute any limitation to the protection scope of this application. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to list all implementation manners here. All technical solutions formed by equivalent transformation or equivalent substitution and similar to this kind are within the scope of the rights protection of this application.

Claims

1. A surface enhanced Raman spectroscopy (SERS) probe combination, the probe combination comprising: a first probe, the first probe comprising a nanoparticle, a Raman reporter molecule and a first recognition element, the first recognition element comprising a reagent that specifically recognizes circulating tumor cells (CTCs); and The second probe comprises nanoparticles, a Raman reporter molecule and a second recognition element, wherein the second recognition element comprises a reagent that specifically recognizes platelets.

2. The SERS probe assembly according to claim 1, further comprising: The third probe comprises nanoparticles, a Raman reporter molecule and a third recognition element, wherein the third recognition element comprises an agent that is different from the first recognition element and specifically recognizes circulating tumor cells (CTCs).

3. The SERS probe assembly according to claim 1 or 2, wherein: The reagent that specifically recognizes CTCs includes folic acid and / or epithelial marker antibodies; the reagent that specifically recognizes platelets includes platelet membrane glycoprotein antibodies.

4. The SERS probe assembly as claimed in claim 3, wherein: The epithelial marker antibody includes EpCAM antibody or cytokeratin (eg CK8, CK18 or CK19) antibody; and / or the platelet membrane glycoprotein antibody includes CD41, CD42a, CD42b or CD61 antibody.

5. The SERS probe assembly according to claim 1 or 2, wherein: The nanoparticles include metal nanoparticles or non-metal nanoparticles; and / or The Raman reporter molecules include Raman fingerprint region reporter molecules and / or Raman silent region reporter molecules.

6. The SERS probe assembly according to claim 5, wherein: The Raman fingerprint region reporter molecules include 4-mercaptophenylboronic acid, 4-mercaptobenzoic acid, 4-nitrobenzenethiol, 4-aminothiophenol, 4-methoxybenzyl mercaptan and / or 5,5'-dithiobis(2-nitrobenzoic acid); The Raman silent zone reporter molecules include 4-ethynylbenzenethiol and its derivatives and / or 4-mercaptobenzonitrile; The metal nanoparticles include gold nanoparticles, silver nanoparticles or gold-silver composite nanoparticles; The non-metallic nanoparticles include graphene; and / or The nanoparticles include nanostars, nanospheres, nanorods, nanoshells, nanoparticle clusters, nanowires, nanocubes, nanocones, nanopolyhedrons and / or nanocrystals. 7 . The SERS probe assembly as claimed in claim 6 , wherein the 4-ethynylbenzenethiol derivatives include 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene (OPE1) and 4,4′-di(thioacetyl)tolane (OPE2).

8. The SERS probe assembly as claimed in claim 2, comprising the following probes: A first probe comprising gold nanoparticles, 4-mercaptophenylboronic acid, and folic acid; a second probe comprising gold nanoparticles, 4,4'-di(thioacetyl)tolan, and a CD41 antibody; and / or The third probe comprises gold nanoparticles, 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene, and EpCAM antibody.

9. A kit comprising: The SERS probe combination according to any one of claims 1 to 8; and Microwell array chip for SERS imaging.

10. A method for identifying and / or quantifying platelet adhesion CTCs, comprising: Incubating the SERS probe combination according to any one of claims 1 to 8 with a cell population; and Perform SERS scanning imaging and detect SERS signals.

Images