A sers probe combination and kit and method of identifying and / or quantifying platelet-adherent ctc

Abstract

The present application discloses a SERS probe combination and kit and a method for identifying and / or quantifying platelet-adherent CTC. The probe combination comprises: a first probe comprising a nanoparticle, a Raman reporter molecule and a first recognition element comprising an agent that specifically recognizes a circulating tumor cell (CTC); and a second probe comprising a nanoparticle, a Raman reporter molecule and a second recognition element comprising an agent that specifically recognizes a platelet.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical engineering, in particular to a diagnosis and screening system, more particularly to the identification of platelet-adherent circulating tumor cells (CTCs). BACKGROUND

[0002] Circulating tumor cells (CTCs) play a key role in cancer metastasis. CTCs survive in the blood circulation and interact with blood cells, among which platelets are most likely to contact CTCs first and trigger signal activation and functional changes due to their numerical advantage. The interaction of CTCs with platelets includes: (1) CTCs induce platelet activation; (2) platelets protect CTCs from shear stress and anoikis; (3) platelets mediate immune escape of CTCs. Therefore, it is of great significance to study the biological function and mechanism of platelet-adherent CTCs.

[0003] Currently, research on the changes of CTCs in immune phenotype by platelets to help tumor cells escape immune killing is usually visualized by experimental means of immunofluorescence imaging to detect platelet-adherent CTCs.

[0004] However, the field of immunofluorescence imaging detection of platelet-adherent CTCs still has the following problems that need to be improved: (1) non-specific staining: immunofluorescence staining usually needs to use fluorescently labeled antibodies to recognize target cells, but there may be non-specific binding or cross-reactions, leading to misjudgment or interference of background signals; (2) limited multi-labeling capability: immunofluorescence imaging usually can only use a limited number of fluorescent labels to simultaneously detect multiple molecules or cell subpopulations and the signal is easily disturbed, so it may not be able to cover multiple target analytes in complex samples; (3) signal quenching and fluorescence quenching: at high concentrations, the signal of fluorescent labels may be affected by signal quenching or fluorescence quenching, leading to attenuation or distortion of the signal; (4) photobleaching effect: immunofluorescence imaging requires long-time light exposure, which may cause photobleaching effect of cells, leading to cell damage or even death; (5) high detection cost: the expensive equipment and fluorescently labeled reagents required for immunofluorescence imaging have high cost, increasing the economic burden of experiments.

[0005] There is still an urgent need in the art for a technical solution for specific identification and quantification of platelet-adherent CTCs in whole blood that can improve the above problems and achieve 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 a sample, including chemical composition, molecular structure and vibration information of chemical bonds, and can detect extremely low concentrations of target objects, even reaching the single molecule level. In addition, SERS technology combines the resolution and selectivity of Raman spectroscopy, and the difference from the background signal is large, thereby achieving highly selective recognition of target molecules, and has been widely concerned in various fields such as qualitative and quantitative analysis. And SERS has a fast analysis speed and real-time detection capability, which can complete sample analysis in a short time, and thus can realize real-time monitoring of molecular changes. Compared with fluorescence technology, SERS technology reduces the complexity and cost of experiments, and the SERS signal is stable, without the defects of photobleaching and photobleaching, and avoids the false positive or false negative results that may be caused by the marker. Secondly, SERS also has the ability of multi-labeling. Especially in the SERS probe in the Raman silent region (1800-2800 cm-1), by reducing endogenous interference, enhancing signal specificity and sensitivity, it shows significant advantages in the fields of biological imaging and disease diagnosis. The development of a Raman probe with high-intensity characteristic peaks in the cell Raman silent region (1800-2800 cm-1) is crucial for realizing high-resolution multicolor Raman imaging of living cells.

[0007] So far, there is still a lack of SERS probe combination design for platelet-adherent CTC recognition and quantification. SUMMARY

[0008] To solve the above problems existing in the prior art, the present application provides a surface-enhanced Raman spectroscopy (SERS) probe combination, which comprises a SERS probe based on a Raman silent region, and the probe combination can be used for specific recognition of platelet-adherent CTC at a single cell level. The present application also provides a kit comprising the probe combination, and a method for specifically recognizing and / or quantifying platelet-adherent CTC using the probe combination. The new method for recognizing and imaging platelet-adherent CTC provided by the present application not only can replace the existing immunofluorescence imaging method in the art, but also can improve the detection sensitivity and accuracy, and has the significant technical advantages of high specificity, low background noise, real-time monitoring, signal stability, multiple detection, good biocompatibility, and simple operation.

[0009] In one aspect, the present application provides a surface-enhanced Raman spectroscopy (SERS) probe combination, which comprises: a first probe comprising a nanoparticle, a Raman reporter molecule and a first recognition element, wherein the first recognition element comprises a reagent specifically recognizing a circulating tumor cell (CTC); and a second probe comprising a nanoparticle, a Raman reporter molecule and a second recognition element, wherein the second recognition element comprises a reagent specifically recognizing a platelet.

[0010] In another aspect, the present application provides a kit comprising: a SERS probe combination and a microwell array chip for SERS imaging, the probe combination comprising: a first probe comprising a nanoparticle, a Raman reporter molecule and a first recognition element comprising an agent that specifically recognizes circulating tumor cells (CTCs); and a second probe comprising a nanoparticle, a Raman reporter molecule and a second recognition element comprising an agent that specifically recognizes platelets.

[0011] In another aspect, the present application provides a method for identifying and / or quantifying platelet-adherent CTCs, comprising: incubating a SERS probe combination with a cell population, the probe combination comprising: a first probe comprising a nanoparticle, a Raman reporter molecule and a first recognition element comprising an agent that specifically recognizes circulating tumor cells (CTCs); and a second probe comprising a nanoparticle, a Raman reporter molecule and a second recognition element comprising an agent that specifically recognizes platelets; and performing SERS scanning imaging and detecting SERS signals. BRIEF DESCRIPTION OF DRAWINGS

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

[0013] Figure 1 Synthetic procedure chart for OPE1 and OPE2.

[0014] Figure 2 (A) Transmission electron microscopy (TEM) images of gold nanostars; (B) Particle size distribution of gold nanostars; (C) Extinction spectra of gold nanostars before and after probe modification are shown.

[0015] Figure 3 (A) Preparation schematic of SERS imaging probe folate and MPBA modified gold nanostars (GNS-MPBA) is shown, (B) Raman spectrum of GNS-MPBA.

[0016] Figure 4 (A) Preparation schematic of EpCAM antibody and OPE1 modified gold nanostars (GNS-OPE1) is shown, (B) Raman spectrum of GNS-OPE1.

[0017] Figure 5 (A) Preparation schematic of CD41 antibody and OPE2 modified gold nanostars (GNS-OPE2) is shown, (B) Raman spectrum of GNS-OPE2.

[0018] Figure 6(A) a schematic diagram; and (B) a Raman spectrum, when GNS-OPE1 and GNS-OPE2 co-localize.

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

[0020] Figure 8 An operational procedure flow chart for the present application.

[0021] Figure 9 (A) a schematic diagram; and (B) a Raman spectrum, when GNS-OPE1 and GNS-OPE2 co-localize.

[0022] (A) is a SERS scanning spectrum locating at 1584 cm -1 , 2126 cm -1 and 2216 cm -1 , respectively, when only CTCs are present. As can be seen, both 1584 cm -1 and 2126 cm -1 present strong SERS peaks when only CTCs are present, while the CD41-labeled probe GNS-OPE2 has no obvious signal at 2216 cm -1 , indicating that the CTCs are non-platelet-adhesion CTCs.

[0023] (B) is a SERS scanning spectrum locating at 1584 cm -1 , 2126 cm -1 and 2216 cm -1 , respectively, when only platelets are present. As can be seen, the CD41-labeled probe GNS-OPE2 has obvious signal at 2216 cm -1 when only platelets are present, while there is no signal of CTCs, indicating that only platelets are present.

[0024] (C) is a SERS scanning spectrum locating at 1584 cm -1 , 2126 cm -1 and 2216 cm -1 , respectively, when platelet-adhesion CTCs are present. As can be seen, obvious SERS signals of strong intensity are present at the three wavenumbers, indicating that the CTCs are platelet-adhesion CTCs.

[0025] Figure 10 (A) a schematic diagram; and (B) a Raman spectrum, when GNS-OPE1 and GNS-OPE2 co-localize. DETAILED DESCRIPTION

[0026] The present application relates to a surface enhanced Raman spectroscopy (SERS) probe combination, comprising: a first probe comprising a nanoparticle, a Raman reporter molecule, and a first recognition element comprising an agent that specifically recognizes a circulating tumor cell (CTC); and a second probe comprising a nanoparticle, a Raman reporter molecule, and a second recognition element comprising an agent that specifically recognizes a platelet.

[0027] In some embodiments, the probe combination further comprises: a third probe comprising a nanoparticle, a Raman reporter molecule, and a third recognition element comprising an agent that specifically recognizes a circulating tumor cell (CTC) different from the first recognition element.

[0028] In some embodiments, the agent that specifically recognizes a CTC comprises a folate and / or an epithelial marker antibody. In some embodiments, the agent that specifically recognizes a platelet comprises a platelet membrane glycoprotein antibody. In some embodiments, the epithelial marker antibody comprises an EpCAM antibody or a cytokeratin antibody. In some embodiments, the cytokeratin antibody comprises a CK8, CK18, or CK19 antibody. In some embodiments, the platelet membrane glycoprotein antibody comprises a CD41, CD42a, CD42b, or CD61 antibody.

[0029] In some embodiments, the nanoparticle comprises a metal nanoparticle or a non-metal nanoparticle. In some embodiments, the Raman reporter molecule comprises a Raman fingerprint region reporter molecule and / or a Raman silent region reporter molecule.

[0030] In some embodiments, the Raman fingerprint region reporter molecule comprises 4- mercaptobenzoic acid, 4-mercaptobenzoic acid, 4-nitrothiophenol, 4-aminothiophenol, 4- methoxybenzyl mercaptan, and / or 5,5'-dithiobis(2-nitrobenzoic acid). In preferred embodiments, the Raman fingerprint region reporter molecule comprises 4- mercaptobenzoic acid.

[0031] In some embodiments, the Raman silent region reporter molecule comprises 4-ethynylthiophenol and derivatives thereof and / or 4-mercaptobenzonitrile. In some embodiments, the 4- ethynylthiophenol derivatives comprise l-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene (OPE1) and 4,4'-di(thioacetyl)diphenylacetylene (OPE2). In preferred embodiments, the Raman silent region reporter molecule comprises l-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene, 4,4'-di(thioacetyl)diphenylacetylene.

[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 zone reporter molecule.

[0033] In some embodiments, the metal nanoparticle comprises a gold nanoparticle, a silver nanoparticle, or a gold-silver composite nanoparticle. In some embodiments, the non-metal nanoparticle comprises graphene. In some embodiments, the nanoparticle comprises a nanostar, a nanosphere, a nanorod, a nanoshell, a nanoparticle cluster, a nanowire, a nanocube, a nanopyramid, a nanopolyhedron, and / or a nanocrystal. In specific embodiments, the nanoparticle comprises a gold nanostar. In alternative embodiments, the nanoparticle comprises a silver nanostar.

[0034] In some embodiments, the SERS probe combination described herein comprises the following probes: a first probe, a second probe, and / or a third probe; wherein the nanoparticle of the first probe, the second probe, and / or the third probe comprises a gold nanoparticle, a silver nanoparticle, or a gold-silver composite nanoparticle, the Raman reporter molecule of the first probe, the second probe, and / or the third probe is selected from 4-mercaptobenzoic acid, 4-mercaptobenzoic acid, 4-nitrothiophenol, 4-aminothiophenol, 4-methoxybenzyl mercaptan, and / or 5,5'-dithiobis(2-nitrobenzoic acid), 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene, and 4,4'-bis(thioacetyl)stilbene, respectively, and the recognition element of the first probe, the second probe, and / or the third probe is selected from folate, CD41 antibody, CD42a antibody, CD42b antibody, CD61 antibody, EpCAM antibody, CK8 antibody, CK18 antibody, or CK19 antibody, respectively.

[0035] In some embodiments, the SERS probe combination described herein comprises the following probes: a first probe, a second probe, and / or a third probe; wherein the nanoparticle of the first probe, the second probe, and / or the third probe comprises a gold nanoparticle, the Raman reporter molecule of the first probe, the second probe, and / or the third probe is selected from 4-mercaptobenzoic acid, 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene, and 4,4'-bis(thioacetyl)stilbene, respectively, and the recognition element of the first probe, the second probe, and / or the third probe is selected from folate, CD41 antibody, and EpCAM antibody, respectively.

[0036] In some embodiments, the SERS probe combination described herein includes the following probes: a first probe including a gold nanoparticle, 4-mercaptobenzoic acid, and folate; a second probe including a gold nanoparticle, 4,4'-bis(thioacetyl)stilbene, and CD41 antibody; and / or a third probe including a gold nanoparticle, l-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene, and EpCAM antibody.

[0037] In some embodiments, the SERS probe combination described herein includes the following probes: a first probe including a gold nanostar, 4-mercaptobenzoic acid, and folate; a second probe including a gold nanostar, 4,4'-bis(thioacetyl)stilbene, and CD41 antibody; and / or a third probe including a gold nanostar, l-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene, and EpCAM antibody.

[0038] In this application, the Raman reporter molecule is attached to the metal substrate, however, in vivo and in vitro environments, the dissociation of the reporter molecule from the metal substrate, the adsorption of other molecules to the metal substrate can reduce the reliability and effectiveness of the naked probe. In addition, when the probe is applied to live cell detection, the problem of biological toxicity of the probe also needs to be considered. Therefore, the SERS probe can also include a surface coating material and encapsulation. In some embodiments, the surface-enhanced Raman spectroscopy (SERS) probe combination described in this application also includes 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) probes

[0040] A SERS probe (or SERS tag) is generally composed of a nanoparticle, a Raman reporter molecule, and a recognition element.

[0041] Raman reporter molecule

[0042] Raman imaging can provide a unique molecular "fingerprint" by detecting the vibrations of molecular bonds, thus characterizing the molecule. Raman reporters adsorbed on the surface of metal nanoparticles can generate extremely strong Raman signals due to the excitation of localized surface plasmons. The term "Raman reporter" or "Raman-active molecule" as used herein is a molecule that has characteristic Raman spectral signals. A Raman reporter has the following characteristics: (1) a characteristic chemical structure that generates a signature Raman spectrum so that the target can be accurately identified in a complex biological sample Raman spectrum; (2) a large Raman cross-section that can generate a strong Raman signal, thus conferring high sensitivity to the probe; (3) the reporter must be able to stably and effectively attach to the surface of the nanostructure because SERS effect strongly depends on the distance between the reporter and the nanoparticle surface.

[0043] The Raman reporter provides a unique fingerprint for the SERS probe. Depending on the characteristic peaks of the Raman reporter used, the SERS probe can function in the "fingerprint region" or the "Raman silent region".

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

[0045] In some embodiments, the Raman fingerprint region reporter in the SERS probe combination comprises 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 the present application, 4-MPBA means 4-mercaptophenylboronic acid. In preferred embodiments, the Raman fingerprint region reporter in the SERS probe combination comprises 4-mercaptophenylboronic acid (4-MPBA).

[0046] In the context of the present application, the term "Raman silent region" or "cell / biological silent region of the Raman spectrum" refers to the 1800-2800 cm"1band of the Raman spectrum, which is almost free of signals generated by biological molecules. The term "Raman silent region reporter" is generally a compound with a triple bond (e.g., with an alkyne group, a cyano group, an azido group) or containing an isotope (e.g., containing deuterium, nitrogen-15, or carbon-13). Compared with commercial Raman reporters in the fingerprint region, silent region Raman probes are relatively expensive and have weaker signals. Therefore, it is of great significance to the art to develop novel Raman silent region reporters suitable for multiplex detection.

[0047] The inventors designed novel Raman probes with distinct vibrational frequencies in the Raman silent region. Since biological samples have no significant Raman response in the Raman silent region, the signal of the SERS probes described in the present application will not be disturbed by the spontaneous Raman scattering of biomolecules, reducing background noise and enhancing the detectability of the signal, thus meeting the needs of multiplex detection, thereby achieving highly selective recognition of platelet-adherent CTCs.

[0048] In some embodiments, the Raman silent region reporter molecule in the SERS probe combination comprises 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 the present application, OPE1 means 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzenethiol; OPE2 means 4,4'-bis(thioacetyl)diphenylacetylene (4,4'-(1,4-phenylenebis(ethyne-2,1-diyl)dibenzenethiol). In preferred embodiments, the Raman silent region reporter molecule in the SERS probe combination comprises 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene (OPE1) and / or 4,4'-bis(thioacetyl)diphenylacetylene (OPE2).

[0049] Recognition element

[0050] On the basis of the novel Raman silent region reporter molecules, the probe combination described in the present application can achieve multiplex imaging. In the context of the present application, "recognition element" means a reagent capable of specifically recognizing and / or selectively binding to a target analyte, such as a specific ligand, aptamer or biomarker antibody. In some embodiments, the recognition element used by the present application comprises a ligand, aptamer or biomarker antibody that is targeted to platelet-adherent CTCs.

[0051] To specifically recognize and / or quantify CTCs or platelet-adherent CTCs, the present application creatively designed SERS probe combinations with folate, epithelial marker antibodies and / or platelet membrane glycoprotein antibodies as recognition elements. CTC epithelial markers refer to epithelial-derived molecular markers for recognizing CTCs, including epithelial cell adhesion molecule (EpCAM) and cytokeratin (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] EpCAM (epithelial cell adhesion molecule) has a wide research basis in the application of circulating tumor cell (CTC) detection, and is applied in the early clinical diagnosis and monitoring of various cancers (such as breast cancer, prostate cancer and colon cancer, etc.). However, although EpCAM has important clinical value, there are still some defects in its current application in CTC detection. For example, EpCAM is a marker on the surface of most epithelial cells, and therefore shows high expression in tumor cells, which makes the detection method based on EpCAM be able to effectively capture CTCs of epithelial origin, but there may be a missed detection phenomenon 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 tumor markers. In the application of CTC detection, CK8, CK18 or CK19 are often used. Similar to EpCAM, the method based on CK may have a missed detection phenomenon for CTCs with low expression of epithelial markers.

[0053] Folate receptor (FR) also shows high expression in a variety of 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 in tumor markers. Compared with EpCAM, the expression of FR in normal tissues is extremely limited, which ensures the specificity of the detection method for tumors. However, there are still FR-negative CTCs, which will cause a false detection phenomenon.

[0054] The technical solution of the present application combines epithelial marker antibodies and FR for CTC labeling. Taking EpCAM as an example, the examples in the present 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 detection and intervention of tumors.

[0055] Platelet membrane glycoproteins include plasma membrane and granule membrane glycoproteins, specifically, including proteins such as CD41, CD42a, CD42b or CD61. The technical solution of the present application further incorporates platelet membrane glycoprotein antibodies as recognition elements, thereby providing a probe combination and detection method for recognizing platelet-adherent CTCs. Platelet membrane glycoprotein CD41 is an important receptor on the surface of platelets, which participates in the process of platelet aggregation and activation. The examples in the present application take CD41 as an example to prove that the specially designed SERS probe combination further enhances the sensitivity and accuracy of platelet-adherent CTC detection.

[0056] The technical solution of the present application effectively reduces the probability of false positives and false negatives in the detection of platelet-adherent CTCs by combining multiple detections of the combined labels through specially designed SERS probes, cross-verification between multiple results, overcoming the problems of false detection and missed detection, and further improving the sensitivity and accuracy of early cancer screening and diagnosis.

[0057] Nanoparticle

[0058] In the context of the present application, the SERS probe includes a nanoparticle as a base material, which can enhance the Raman signal of the Raman reporter molecule directly combined with its surface. The nanoparticle base material can be a noble metal (gold, silver, etc.) or a non-metallic nanomaterial (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 structure nanoparticles. The core-shell structure nanoparticles can be composed of multiple materials such as metals, polymers, and / or inorganic materials, having a central core and a shell structure wrapped in the outer layer. The core-shell structure nanoparticles contain Raman reporter molecules between the two layers of metal, have stable structure, and have obvious SERS effect and excellent biocompatibility. In some embodiments, the "shell" or "core" of the core-shell structure nanoparticles described herein is gold or silver. In some embodiments, the "gold-silver composite nanoparticles" described herein include core-shell structure nanoparticles composed of a gold core and a silver shell, or core-shell structure nanoparticles composed of a silver core and a gold shell, or other gold-silver composite structures. Other non-metallic shell layers with gold core or silver core to form core-shell structure can also be used to implement the present application, and therefore are also covered by the protection scope of the present application. In some embodiments, the non-metallic nanoparticles described herein include graphene. In some embodiments, the nanoparticles include nanoparticles of various morphologies, such as nanostars, nanospheres, nanorods, nanoshells, nanoparticle clusters, nanowires, nanocubes, nanotapers, nanopolyhedra, 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 nanostars, gold nanorods, gold nanospheres, gold nanowires, gold nanocubes, gold nanotapers, gold nanopolyhedra, or gold nanocrystals. In a specific embodiment, the nanoparticles include gold nanostars. In an alternative embodiment, the nanoparticles include silver nanostars.

[0061] Although the specific embodiments of the present application prepare and characterize gold nanostars, and exemplarily use gold nanostars to implement the technical solutions of the present application, it is understood by those skilled in the art that other noble metal nanoparticles capable of realizing SERS effect, in addition to gold nanostars, are also 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 other non-metallic shell and gold / silver core composed of core-shell structure nanoparticles, all belong to the nanoparticles containing sharp hot spots known in the art, and can also realize signal enhancement effect as SERS substrate, 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 angular arms, which has abundant sharp end structures and abundant plasmonic hot spot regions. In some embodiments, the gold nanostar described herein has a diameter of 30-70 nm. In some embodiments, the gold nanostar described herein has a diameter of 40-60 nm. In some embodiments, the gold nanostar described herein has a diameter of about 50 nm. In some embodiments, the extinction spectrum of the gold nanostar described herein has an absorption peak near 719 nm.

[0063] In another aspect, the present application also provides a kit comprising: a SERS probe combination and a microwell array chip for SERS imaging, the probe combination comprising: a first probe comprising a nanoparticle, a Raman reporter molecule and a first recognition element comprising an agent specifically recognizing circulating tumor cells (CTC); and a second probe comprising a nanoparticle, a Raman reporter molecule and a second recognition element comprising an agent specifically recognizing platelets.

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

[0065] In another aspect, the present application also relates to a method of identifying and / or quantifying CTCs or platelet-adherent CTCs, comprising: incubating a SERS probe combination described herein with a cell population; and performing SERS scanning imaging and detecting SERS signals. In some embodiments, the incubation is performed in a microwell array. In some embodiments, the incubation is performed in a microwell array on a poly-lysine modified glass slide and / or a chip carrier.

[0066] In some embodiments of the present application, the incubation step comprises: (a) adding a cell population to a microwell array on a poly-lysine modified glass slide and chip carrier, pre-incubating at 30-40°C, preferably about 37°C for 1-5 hours, preferably about 2 hours; and (b) adding a SERS probe combination described herein to the microwell array, incubating at 30-40°C, preferably 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 detecting step comprises: (c) performing SERS scanning imaging of the whole well (exemplary parameters: 638 nm laser, step size of 1 micron, collection time set to 1 s); (d) detecting SERS signals, determining the presence of different CTC cell types and assessing their adhesion status by the measured SERS signals; and optionally, (e) picking target cells with a micromanipulator for subsequent related studies.

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

[0069] In some embodiments, in the step (d), SERS signals at 1584 cm -1 , 2126 cm -1 , and 2216 cm -1 are scanned. In some embodiments, in the step (d), when both 1584 cm -1 (folate) and 2126 cm -1 (EpCAM antibody) show strong SERS peaks, while the CD41 labeled probe GNS-OPE2 shows a weak SERS peak at 2216 cm -1When no obvious signal is detected, the CTC is identified as a non-platelet-adhesive CTC. In some embodiments, in step (d), when the CD41-labeled probe GNS-OPE2 is at 2216 cm⁻¹, -1 There is a clear signal at that location, but not at 1584cm. -1 (folic acid) and 2126cm -1 When the CTC signal at the (EpCAM antibody) site is detected, it is identified as a platelet present alone. In some embodiments, in step (d), 1584 cm -1 (Folic acid), 2126cm -1 (EpCAM antibody) and 2216cm -1 When a significant SERS signal was observed at all three wavenumbers (CD41 antibody), the CTC was identified as a platelet-adhesive CTC.

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

[0071] In this application, the inventors developed three novel nanoparticles with surface-enhanced Raman scattering (SERS) functionality based on gold nanostars (GNS, or AuNS). These nanoparticles are modified with 4-MPBA (4-mercaptophenylboronic acid, as a Raman reporter molecule), OPE1 (1-thioacetyl-4-[(trimethylsilyl)acetylene]benzene), and OPE2 (4,4'-di(thioacetyl)diphenylacetylene), the latter two of which have strong SERS signals in the Raman silencing region. These nanoparticles are further functionalized with recognition elements to recognize platelet-adhesive CTCs or CTCs. For example, the nanoparticles of this application are conjugated with folic acid (FA, as a targeting ligand) or modified with EpCAM antibody for precise recognition of CTCs, and / or the nanoparticles of this application are modified with CD41 antibody for platelet recognition.

[0072] The present application designs Raman silent zone reporter molecule OPE1 or OPE2 molecule, and is combined with fingerprint zone reporter molecule 4-MPBA to synthesize novel SERS probe combination, the probe combination described in the present application further comprises EpCAM antibody, CD41 antibody or folate (FA) as a recognition element, thereby creatively realizing multiple labeling imaging of platelet-adherent CTC. The signal of the Raman probe located in the Raman silent zone in the technical solution of the present application is not disturbed by the biological background signal, and can well meet the increasing precision requirement of medical imaging for precision medicine. The technical solution of the present application can improve the technical defects of the existing immunofluorescence imaging in visualizing platelet-adherent CTC, overcome the problems of 'non-specific staining, limited multi-labeling capability, signal quenching and fluorescence quenching, light quenching and high detection cost' existing in immunofluorescence imaging by means of SERS imaging technology, and thus provide a brand-new imaging visualization technology for detecting platelet-adherent CTC.

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

[0074] ①High sensitivity: the SERS probe combination and the corresponding immunolabeling imaging method of the present application have single-cell level sensitivity, the SERS effect can significantly enhance the Raman signal of the probe, and when imaging in the Raman silent zone, the target object with extremely low concentration can also be detected, thereby improving the sensitivity of detection.

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

[0076] ③Low background noise: the SERS probe in the Raman silent zone has low background noise, since the biological sample has no significant Raman response in the Raman silent zone, the signal of the SERS probe will not be disturbed by the spontaneous Raman scattering of biological molecules, thereby reducing the background noise and enhancing the detectability of the signal, and realizing the highly selective recognition of platelet-adherent CTC.

[0077] ④Real-time detection: the SERS immunolabeling imaging method has fast analysis speed and real-time detection capability, can complete sample analysis in a short time, and can realize real-time monitoring of molecular changes.

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

[0079] ⑥Signal stability: the SERS immunolabeling imaging method has stable signal, and does not have the defects of photobleaching and photobleaching.

[0080] ⑦Avoid false positive or false negative results: SERS immunolabeling imaging method signal is stable, avoid the false positive or false negative results that the marker may bring.

[0081] ⑧Multiplexing capability: SERS immunolabeling imaging probe in Raman silent zone has multiple imaging capability, in Raman silent zone, different probe design with different Raman characteristic signal can be used, so that multiple imaging and multiple marker detection is possible, multiple target molecules or cell processes can be identified and tracked simultaneously.

[0082] ⑨Good biocompatibility: SERS probe usually used in Raman silent zone is based on functionalized design of noble metal nanomaterials (such as gold or silver), which can exhibit good compatibility in biological system, suitable for biomedical imaging applications in vivo or in vitro.

[0083] Embodiment

[0084] The technical solutions of the present application will be described in more detail below in conjunction with specific examples. The following examples are only examples, and do not constitute any limitation or restriction on the technical solutions of the present application. The specific materials, steps, conditions, numerical values or numerical value ranges and other technical parameters in the following examples are only examples, and are not exhaustive or limited.

[0085] In addition to the specific methods, devices and materials used in the examples, according to the master of the prior art of the person skilled in the art and the description of the present application, any method, device and material of the prior art similar or equivalent to the method, device and material described in the examples of the present application can also be used to realize the present application.

[0086] Table 1. Reagents / devices used

[0087]

[0088]

[0089] Example 1. Synthesis and characterization of two Raman-silent 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 mixture of concentrated hydrochloric acid (88 mL) and water (280 mL) and the mixture was cooled to 0 °C in an ice bath. Subsequently, sodium nitrite (11 g) solution (42 mL) was added dropwise to the ice-cold solution over 2 h and stirring was continued for 15 min after completion. Next, diethyl ether was added to the mixture to generate a precipitate of diazonium tetrafluoroborate. The precipitate was filtered and washed with ice water, methanol and ether successively, and the precipitate was dried under vacuum and used directly for the next reaction without further purification. Potassium thioacetate (11.93 g) was dissolved in dimethyl sulfoxide (300 mL) and then the diazonium tetrafluoroborate DMSO solution (10.5 g in 100 mL) was added dropwise over 0.5 h and stirred at room temperature for 1.5 h. After completion of the reaction, the mixture was poured into saturated brine and then the organic phase was extracted with ether and washed with sodium hydroxide solution and saturated brine successively. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated. The residue was further purified by silica gel column chromatography to give the product, 1-iodo-4-thioacetylbenzene as a light yellow solid. A mixture of 1-iodo-4-thioacetylbenzene (4.44 g), tetrahydrofuran (20.0 mL), trimethylsilyl acetylene (2.35 g), dichlorobis(triphenylphosphine)palladium(II) (0.56 g), copper iodide (0.16 g) and N,N-diisopropylethylamine (3.32 g) was stirred under argon atmosphere for 24 h. 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 give the crude product. The crude product was purified by silica gel column chromatography to give 1-thioacetyl-4-[(trimethylsilyl)ethynyl]benzene as a light white solid.

[0091] 1 -thioacetyl-4-[(trimethylsilyl)ethynyl]benzene characterization results:1H NMR (300 MHz, CDC13) 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, CDC13) 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: 248.42, found: 248.2. In a degassed tetrahydrofuran (THF, 10 mL) solution, 1 -thioacetyl-4-[(trimethylsilyl)ethynyl]benzene (2.5 mg) and ammonium hydroxide (10 μί) were added dropwise. The solution was stirred at room temperature under nitrogen atmosphere for 30 min. The resulting OPE1 solution was used without further purification.

[0092] (2) Synthesis and characterization of OPE2: 1-Thioacetyl-4-[(trimethylsilyl)ethynyl] benzene (1.7 g) was dissolved in tetrahydrofuran (20 mL) at 0 °C, followed by the addition of glacial acetic acid (0.1 g) and acetic anhydride (0.1 g) sequentially, and then a solution of tetrabutylammonium fluoride (28.8 g) in tetrahydrofuran (10 mL) was added dropwise under argon atmosphere. The solution was warmed to room temperature and reacted for 1 h. 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 in vacuo. The residue was further purified by column chromatography to give the target product 4-ethynyl-l-thioacetylbenzene as a yellow to orange solid. N,N-diisopropylethylamine (0.62 g) was added to a solution of 1-iodo-4-thioacetylbenzene (0.53 g) and 4-ethynyl-l-thioacetylbenzene (0.42 g) in tetrahydrofuran (THF, 10 mL) under argon atmosphere. After stirring for 5 min at 23 °C, bis(triphenylphosphine)palladium dichloride (Pd(PPh3)2Cl2, 81 mg) and copper iodide (CuI, 68 mg) were added, and tetrahydrofuran (10 mL) was added. The reaction mixture was stirred at room temperature until complete conversion was detected by TLC (18 h). Dichloromethane (CH2Cl2) and water were added, and the aqueous phase was separated. The aqueous phase was extracted with dichloromethane, and the organic phases were combined, washed with water, and then dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated. The residue was purified by column chromatography to give the product 4,4'-bis(thioacetyl)stilbene as a light yellow solid.

[0093] 4,4'-bis(thioacetyl)stilbene characterization results: 1H NMR (300 MHz, CDC13): 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, CDC13): δ = 30.20, 90.14, 124.06, 128.31, 132.14, 134.15, 193.31; GCMS calculated: 326.43, found: 326.2. In a degassed tetrahydrofuran (THF, 10 mL) solution, 4,4'-bis(thioacetyl)stilbene (2.5 mg) and ammonium hydroxide (10 μL) were added dropwise. The solution was stirred at room temperature under nitrogen atmosphere for 30 min. The resulting OPE2 solution was used without further purification.

[0094] Example 2. Synthesis of gold nanostars

[0095] (1) Synthesis of gold nanostars (GNS): First, 15 mL of 1% sodium citrate solution was added to 100 mL of boiling 1 mM HAuCl4solution under vigorous stirring and cooled after 15 min of continued boiling and filtered through a 0.22 pm cellulose nitrate membrane. The filtered seed solution was stored at 4 °C for long-term use. In a 20 mL glass vial, 100 pL of the above sodium citrate-stabilized seed solution (particle size: 12 ± 0.7 nm; A 520 : 2.81) was added to 10 mL of HAuCl4solution (0.25 mM, containing 10 pL of 1 M HC1) at room temperature with stirring at 700 rpm. Subsequently, 100 pL of AgNO3solution (final concentration 0.5 mM) and 50 pL of ascorbic acid (100 mM) were quickly added. After 30 s of stirring, the solution color rapidly changed from light red to blue or green-black. Subsequently, centrifugation at 3000-5000 g for 15 min was performed to terminate the nucleation process of the nanoparticles. The centrifuged product was resuspended in deionized water and filtered through a 0.22 pm cellulose nitrate membrane and stored at 4 °C for long-term use.

[0096] (2) Physicochemical property characterization of gold nanostars (GNS): The morphology and structure of gold nanostars were characterized by transmission electron microscopy (TEM), and the particle size statistics were analyzed by ImageJ software. The results showed that the diameter of the synthesized gold nanostars was about 50 nm. In addition, the extinction spectrum of gold nanostars was determined using a UV-visible spectrophotometer, and the results showed that the absorption peak was located at about 719 nm.

[0097] Example 3. Preparation of three SERS imaging probes and characterization of their physicochemical properties

[0098] (1) Preparation of SERS imaging probes and gold nanostar modified with targeting molecules: Take 10 mL of gold nanostar sol in three portions, and add 10 μL of 1 mM 4-MPBA, OPE1, and OPE2 tetrahydrofuran solution to each portion, respectively, and let stand for 3 h of reaction. After the reaction is completed, continue to add 1 mL of 0.1% polyallylamine hydrochloride (PAH) aqueous solution, and react for 12 h. Then add 10 μL of 10 mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) hydrochloric acid aqueous solution and 20 μL of 1 mM N-hydroxysuccinimide (NHS) aqueous solution, and let stand for 1 h of carboxyl activation. Then add 20 μL of 1 mM folic acid (FA), EpCAM antibody, and CD41 antibody solution to the three probes, respectively, and let stand for 16 h of reaction. Then use Amicon Ultra-15 centrifugal filter units (Millipore, MWCO 3.0 kDa) to ultrafilter the obtained GNS-MPBA, GNS-OPE1, and GNS-OPE2 conjugated with targeting molecules, and disperse the purified nanoparticles in 2.0 mL of Milli-Q water for recognition and quantitative detection of platelet-adherent CTC. Among them, the SERS probes modified with FA and Epcam antibody have CTC cell membrane targeting ability, and the SERS probes modified with CD41 antibody have platelet targeting ability.

[0099] (2) Characterization of the physicochemical properties of SERS imaging probes and gold nanostars modified with targeting molecules: The extinction spectra of the three modified gold nanostars were determined using a UV-visible spectrophotometer, and the results showed that the absorption peak was located at about 725 nm, which was about 6 nm red-shifted compared with the absorption peak of gold nanostar sol alone, indicating the successful modification of probes and targeting molecules Figure 2 C). Further, the successful modification of probes and targeting molecules Figure 3 B, Figure 4 B, Figure 5 B) was illustrated by the peak situation of Raman spectrum. And the peak situation of Raman spectrum when the three probes coexist is shown in Figure 6 B, which illustrates that the presence of three target molecules can be co-localized.

[0100] Example 4. Construction of CTC imaging chip

[0101] As Figure 7As shown, the slides were cleaned with ethanol and ultrapure water, then immersed in a poly-L-lysine aqueous solution (10 μg / ml) for 10 min, rinsed with ultrapure water, and dried with nitrogen gas. Holes (3 mm in diameter and 1 mm in height) were pre-drilled in the PDMS membrane and loaded onto the poly-L-lysine-modified slides. Due to the electrostatic interaction between PDMS and the poly-L-lysine-modified slides, they bonded firmly, forming micro-reaction pores. Target cells were added to the pores; similarly, the target cells and poly-L-lysine quickly adsorbed onto the slide due to electrostatic interaction, preparing for subsequent SERS imaging.

[0102] Example 5. Specific recognition of CTCs or platelet-adherent CTCs in whole blood

[0103] The specific identification procedure for CTCs or platelet-adhesive CTCs in whole blood is as follows: Figure 8 As shown.

[0104] Isolation of target cells: ① Place the whole blood sample treated with anticoagulant into a centrifuge and centrifuge at 150g for 12 minutes at low speed. This step will separate red blood cells and white blood cells into the lower layer, while the upper layer is platelet-rich plasma. Remove the upper layer; ② Add red blood cell lysis buffer to remove red blood cells; ③ Add CD45 modified magnetic beads to the cell fluid after red blood cell lysis. The magnetic field captures white blood cells; ④ Add the remaining cells to poly-L-lysine modified slides and chip carriers for later use.

[0105] SERS measurement of target cells: ① Remaining cells were added to a poly-L-lysine-modified slide and a microarray carrier, and incubated at 37°C for 2 hours. ② The synthesized SERS probe was added to the microarray and incubated at 37°C for 30 minutes to complete the SERS labeling of the target cells. Then, SERS scanning imaging of the entire well was performed (638nm laser, step size 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, target cells were picked up using a micromanipulator for subsequent related studies.

[0106] Practical examples of SERS scanning measurements, such as Figure 9 As shown.

[0107] Figure 9 A shows the location at 1584cm when only CTC is present. -1 2126cm -1 and 2216cm -1 SERS scan at wavenumber. It can be seen that when only CTC is present, 1584 cm⁻¹... -1 and 2126cm -1The SERS peaks of the GNS-OPE2 labeled with CD41 are obvious at 1584 cm-1, 2126 cm-1 and 2216 cm-1, while the SERS peaks of the GNS-OPE2 labeled with CD41 are not obvious at 2216 cm-1, which indicates that the CTC is not a platelet-adhesion type CTC.

[0108] Figure 9 B shows the SERS scanning graphs located at 1584 cm-1, 2126 cm-1 and 2216 cm-1 respectively when only platelets exist. It can be seen that the SERS peaks of the GNS-OPE2 labeled with CD41 are obvious at 2216 cm-1 when only platelets exist, while the SERS peaks of the GNS-OPE2 labeled with CD41 are not obvious at 2216 cm-1, which indicates that only platelets exist alone. -1 -1 -1

[0109] Figure 9 C shows the SERS scanning graphs located at 1584 cm-1, 2126 cm-1 and 2216 cm-1 respectively when platelet-adhesion type CTC exists. It can be seen that the SERS peaks of the GNS-OPE2 labeled with CD41 are obvious at 1584 cm-1, 2126 cm-1 and 2216 cm-1 when platelet-adhesion type CTC exists, which indicates that the CTC is a platelet-adhesion type CTC. -1 -1 -1

[0110] The current method for identifying CTC or platelet-adhesion type CTC is a multi-color immunofluorescence imaging method. In order to compare the existing method with the method of the present application, the CTC or platelet-adhesion type CTC in whole blood is specifically recognized and separated by the multi-color immunofluorescence method, and compared with the method of the present application.

[0111] ​​​​​​Specific recognition of CTC or platelet-adherent CTC by multicolor immunofluorescence method: ①The target cells obtained by the same separation operation process as described above; ②The cells were fixed with 4% paraformaldehyde for 10 minutes to maintain the cell structure and antigen integrity; ③The cells were permeabilized with 0.1% Triton X-100 PBS solution for 10 minutes to increase the permeability of the cell membrane, so that the antibody can enter; ④5% BSA (bovine serum albumin) was used to block non-specific binding sites to reduce background signal; ⑤EpCAM antibody labeled with phycoerythrin (PE), Alexa Fluor 555 labeled Pan-CK antibody (detecting CTC markers), DAPI (detecting cell nucleus) and Alexa Fluor 647 labeled CD41 antibody (detecting platelet markers) (0.1 mg / mL Alexa Fluor 555 labeled Pan-CK, 0.1 mg / mL phycoerythrin (PE) labeled EpCAM primary antibody mixture, Alexa Fluor 647 labeled CD41 primary antibody) were incubated at 4° overnight, followed by washing the cells with 1x PBS for 3 times, 5 minutes each time, to remove unbound primary antibody; ⑥The cell nucleus was stained with 8.4 μM cell nucleus staining solution DAPI (4', 6-diamidino-2-phenylindole) for 10 minutes, and the chip was washed with 1x PBS; ⑦The target cells were identified as platelet-adherent CTC (identification standard: CTC was identified by EpCAM, Pan-CK and DAPI staining, platelet was identified by CD41 staining, only platelet-adherent CTC adhering to the cell membrane of CTC was captured) by inverted fluorescence microscope imaging, and transferred to PBS buffer for secondary verification by confocal fluorescence microscope, and the confocal fluorescence microscope imaging diagram is shown in Figure 10 The specific parameter conditions of the prior art and the method of the present application are compared from the detection limit (LOD), false positive rate (%), false negative rate (%), detection time (min), repeatability (RSD, %) and cost (yuan / sample), and it is proved that the technical scheme of the present application shows significant advantages in these aspects.

[0112] Table 2. Comparison table of the present application and multicolor immunofluorescence imaging method

[0113]

[0114]

[0115] The above merely is a specific application example of the present application, and does not constitute any limitation to the protection scope of the present application. Based on the above description, other different forms of changes or variations can also be made by those skilled in the art in the field. Here, it is not necessary and also impossible to list all the embodiments. Any similar technical solution formed by equivalent transformation or equivalent replacement falls within the protection scope of the present application.

Claims

1. A surface-enhanced Raman spectroscopy (SERS) probe assembly, the probe assembly comprising: A first probe, comprising nanoparticles, a Raman fingerprint region reporter molecule, and a first recognition element, comprising a reagent that specifically recognizes circulating tumor cells (CTCs); The second probe includes nanoparticles, a Raman silencing region reporter molecule, and a second recognition element, the second recognition element including a reagent that specifically recognizes platelets; and The third probe comprises nanoparticles, a Raman silencing region reporter molecule, and a third recognition element, the third recognition element comprising a reagent that specifically recognizes circulating tumor cells (CTCs) differently from the first recognition element; The Raman fingerprint region reporting molecule includes 4-mercaptophenylboronic acid; The Raman silencing region reporter molecules include 1-thioacetyl-4-[(trimethylsilyl)acetylene]benzene (OPE1) and 4,4'-bis(thioacetyl)diphenylacetylene (OPE2).

2. The SERS probe assembly as described in claim 1, wherein, The reagents that specifically recognize CTCs include folic acid and / or epithelial marker antibodies; the reagents that specifically recognize platelets include platelet membrane glycoprotein antibodies.

3. The SERS probe assembly as described in claim 2, wherein, The epithelial marker antibodies include EpCAM antibodies or cytokeratin antibodies; and / or the platelet membrane glycoprotein antibodies include CD41, CD42a, CD42b, or CD61 antibodies.

4. The SERS probe assembly as described in claim 3, wherein, The cytokeratins include CK8, CK18, or CK19.

5. The SERS probe assembly as described in claim 1, wherein, The nanoparticles include metallic nanoparticles or non-metallic nanoparticles.

6. The SERS probe assembly as described in claim 5, wherein, The metal nanoparticles include gold nanoparticles, silver nanoparticles, or gold-silver composite nanoparticles. The non-metallic nanoparticles include graphene; and / or The nanoparticles include nanopolyhedra and / or nanocrystals.

7. The SERS probe assembly as described in claim 5, wherein, The nanoparticles include nanostars, nanospheres, nanorods, nanoshells, nanoparticle clusters, nanowires, nanocubes, and nanocones.

8. The SERS probe assembly of claim 1, comprising the following probes: The first probe comprises gold nanoparticles, 4-mercaptophenylboronic acid, and folic acid; The second probe comprises gold nanoparticles, 4,4'-bis(thioacetyl)diphenylacetylene, and CD41 antibody; and / or The third probe comprises gold nanoparticles, 1-thioacetyl-4-[(trimethylsilyl)acetylene]benzene, and EpCAM antibody.

9. A reagent kit comprising: The SERS probe combination as described in any one of claims 1 to 8; and Micropore array chip for SERS imaging.

10. Use of the SERS probe combination as described in any one of claims 1 to 8 in the preparation of a kit for identifying and / or quantifying platelet-adhesive CTCs.

Images