A stimuli-responsive magnetic SERS composite nanoprobe, a preparation method and application thereof

Abstract

The application belongs to the technical field of medicines, and relates to a stimulus-responsive magnetic SERS composite nano probe and a preparation method and application thereof. The stimulus-responsive magnetic SERS composite nano probe comprises noble metal nano particles, magnetic nano particles, Raman signal molecules, ligand molecules and organic building units. The noble metal nano particles are sequentially connected with the Raman signal molecules and the ligand molecules on the surface, the magnetic nano particles are connected with the ligand molecules on the surface, and the organic building units are combined with the ligand molecules to self-assemble the noble metal nano particles and the magnetic nano particles together to form a self-assembled nano structure. The stimulus-responsive magnetic SERS composite nano probe provided by the application can efficiently separate and highly sensitively detect tumor cells, and the self-assembled structure can be disassembled under certain conditions, so that the detection sensitivity is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and relates to a stimulus-responsive magnetic SERS composite nanoprobe as well as a preparation method and application thereof. BACKGROUND

[0002] Malignant tumor is an important disease seriously endangering human health all over the world. Circulating tumor cells (CTCs) are tumor cells released into the peripheral blood circulation system from a solid tumor or a metastatic lesion spontaneously or due to a diagnosis and treatment operation, and are a necessary condition for causing tumor metastasis. The monitoring of CTCs has important significance for early diagnosis of cancer, tumor metastasis, prognosis and individualized treatment. However, the number of CTCs in blood is extremely small (only 0-100 CTCs in 1 mL of blood), and how to separate and detect CTCs from blood is the key to the problem. At present, there are many separation and detection methods. The separation methods are mainly based on the physical properties, immunobiological methods and microfluidic technology of CTCs, including microporous filter membranes, gradient density centrifugation, electrostatic electric fields, immunomagnetic beads and lateral fluidic separation methods. The detection techniques mainly include flow cytometry, fluorescence spectroscopy detection technology, PCR technology and electrochemical technology. The Veridex system of Johnson & Johnson is a separation and detection system based on immunomagnetic beads and flow cytometry technology, and has been certified by the US FDA. However, the system has low sensitivity, high missed detection rate, high cost and high detection cost, and has been discontinued at the end of 2015. Therefore, it is necessary to develop a new detection technology with high detection sensitivity and low cost.

[0003] Surface-enhanced Raman scattering (SERS) technology has attracted much attention due to its ultra-high detection sensitivity. SERS is a phenomenon of significant enhancement of Raman signals based on the local plasmon resonance of noble metal or metal compound nanostructures, which can enhance 10 6 -10 10 ​SERS spectroscopy technology has the advantages of good selectivity, high sensitivity, no photo-bleaching, anti-interference, rapid and non-destructive, and is widely used in environmental analysis and biomedical fields. Similarly, separation and enrichment technology is crucial in CTC detection, not only because it can improve the sensitivity of detection, but also because it can be used for traceability analysis and drug screening, which is beneficial to the diagnosis of disease location and medication guidance. Among the many separation methods, immunomagnetic beads are widely used due to their rapid and non-destructive separation. For example, the magnetic SERS nanoparticles developed by Xue Ting et al. can rapidly separate and detect CTCs, with a detection limit of 1 cells / mL. Therefore, the combination of immunomagnetic separation and SERS technology can achieve rapid separation and high-sensitivity detection of CTCs. However, the magnetic SERS nanomaterials reported so far are mostly combined by crystal growth method or core-shell structure, which has the disadvantages of complex and tedious preparation method, poor stability, significant reduction of magnetic or SERS signal, and single detection mode. SUMMARY

[0004] The present application aims at the deficiencies in the prior art, and provides a stimulus-responsive magnetic SERS composite nanoprobe, a preparation method and application thereof.

[0005] One object of the present application is to provide a stimulus-responsive magnetic SERS composite nanoprobe, which comprises noble metal nanoparticles, magnetic nanoparticles, Raman signal molecules, ligand molecules and organic building units.

[0006] As a preferred embodiment, the surface of the noble metal nanoparticles is sequentially connected with the Raman signal molecules and the ligand molecules, the surface of the magnetic nanoparticles is connected with the ligand molecules, and the organic building units are self-assembled together with the noble metal nanoparticles and the magnetic nanoparticles by binding with the ligand molecules to form a self-assembled nanostructure.

[0007] As a preferred embodiment, the noble metal nanoparticles are one or more of gold, silver and platinum group metals.

[0008] As a preferred embodiment, the particle size of the noble metal nanoparticles is <1 μm, and further preferably 1-500 nm. Optionally, the particle size of the noble metal nanoparticles is any one of 1, 10, 20, 30, 40, 50, 80, 100, 200, 500 nm or a range value between any two of them.

[0009] As a preferred embodiment, the morphology of the noble metal nanoparticles includes but is not limited to one or more of spherical, star-shaped, flaky, cubic, triangular pyramidal, rod-shaped and the like.

[0010] As preferred, the magnetic nanoparticles are one or more of magnetic metal elemental nanoparticles, magnetic metal oxide nanoparticles; the magnetic metal element includes but is not limited to one or more of Fe, Zn, Co, Ni, Cr, Mn, and the magnetic metal oxide includes but is not limited to one or more of oxides of one or more of Fe, Co, Ni, Cr, Mn.

[0011] As preferred, the particle size of the magnetic nanoparticles is <1 μm, and further preferred is 1-500 nm. Optionally, the particle size of the magnetic nanoparticles is any one of 1, 10, 20, 30, 40, 50, 80, 100, 200, 500 nm or a range between any two of them.

[0012] As preferred, the morphology of the magnetic nanoparticles includes but is not limited to one or more of spherical, disc-shaped, flaky, cubic, triangular, rod-shaped, flower-shaped, etc.

[0013] As preferred, the Raman signal molecule is an organic molecule with conjugated vibration in Raman spectrum, including but not limited to 4-mercaptobenzoic acid, mercaptopyridine, 4-mercaptobenzene, mercaptonaphthalene, p-fluorothiophenol, rhodamine, crystal violet, Nile blue, etc.

[0014] As preferred, the organic building unit is one or more of benzene ring carboxylic acid type bimercuric organic molecule, anthracene ring carboxylic acid type bimercuric organic molecule, naphthalene ring carboxylic acid type bimercuric organic molecule. For example, 3,5-dimercaptobenzoic acid (as shown below B1), 2,5-dimercapto-p-terephthalic acid (as shown below B2), 3,5-bis(mercaptomethyl)benzoic acid (as shown below B3), 3,7-dimercapto-2-naphthoic acid (as shown below B4), 6,6'-dimercapto-3,3'-methylene diphenic acid (as shown below B5), etc.

[0015]

[0016] As preferred, the molecular weight of the organic building unit is 60-10000 Da.

[0017] As preferred, the ligand molecule is one or more of quaternary ammonium salt type molecule, pyridine salt type molecule, amino type molecule, and the ligand molecule is connected to the surface of the nanoparticle, so that the surface is positively charged.

[0018] As preferred, the quaternary ammonium salt type molecule is alkyl quaternary ammonium salt, and the pyridine salt type molecule is alkyl pyridine salt.

[0019] As preferred, the alkyl quaternary ammonium salt has the structural formula as shown in the following formula I or formula II:

[0020]

[0021] Wherein, X is halogen element, n is an integer of 1-30.

[0022] As preferred, X is one of Cl, Br, I.

[0023] As preferred, the alkyl pyridine salt has the following structural formula of formula III or formula IV:

[0024]

[0025] Wherein, X is halogen element, n is an integer of 1-30.

[0026] As preferred, X is one of Cl, Br, I.

[0027] As preferred, the magnetic SERS composite nano probe further comprises a target molecule.

[0028] As preferred, the target molecule is connected to the surface of the organic building unit.

[0029] As preferred, the target molecule is an antibody or a ligand capable of specifically binding to a cancer cell surface antigen or receptor, and the cancer cell can be breast cancer, lung cancer, liver cancer, esophageal cancer, etc.; as preferred, the target molecule is a polypeptide substance or a non-polypeptide substance, and the polypeptide substance can be listed as a monoclonal antibody (such as EPCAM antibody, CD44 antibody), and the non-polypeptide substance can be listed as folate, galactosamine.

[0030] As preferred, the magnetic SERS nano probe has a particle size of 5-10000nm.

[0031] As preferred, the mass ratio of the noble metal nanoparticles, the magnetic nanoparticles, the Raman signal molecules, the ligand molecules, the organic building units and the target molecules is 1-1200:1-1200:1-2000:0.01-300:0.01-500:0.01-300.

[0032] Another object of the present application is to provide a preparation method of the above-mentioned stimulus-responsive magnetic SERS composite nano probe, comprising the following steps:

[0033] (1) adding a Raman signal molecule solution to a noble metal nanoparticle solution, stirring and reacting, and then adding a ligand molecule solution to continue stirring and reacting;

[0034] (2) stirring and reacting a ligand molecule solution with a magnetic nanoparticle solution;

[0035] (3) adding a mixture comprising the product of step (1) and the product of step (2) to an organic building unit solution to stir and react, to obtain a self-assembled nano structure;

[0036] (4) After the self-assembled nanostructure or the target molecule solution is activated by the catalyst, the activated or non-activated self-assembled nanostructure and the activated or non-activated target molecule solution are mixed and stirred to react, so as to obtain a stimulus-responsive magnetic SERS composite nano probe.

[0037] In step (1), the Raman signal molecule solution is formed by dissolving the Raman signal molecule in a solvent, and any solvent capable of dissolving the Raman signal molecule can be used, such as water, ethanol, etc., and the concentration of the Raman signal molecule solution is not limited; the noble metal nanoparticle solution is formed by dispersing the noble metal nanoparticles in water, and the concentration of the noble metal nanoparticle solution is not limited; and the ligand molecule solution is formed by dissolving the ligand molecule in a solvent, and any solvent capable of dissolving the ligand molecule can be used, such as water, ethanol, etc., and the concentration of the ligand molecule solution is not limited.

[0038] Preferably, in step (1), the temperature of the stirring reaction and the continued stirring reaction is 5-40°C, and the stirring reaction and the continued stirring reaction are preferably carried out at room temperature, the reaction time of the stirring reaction is 5-30 min, and the reaction time of the continued stirring reaction is 1-20 h, and the reaction time of the continued stirring reaction is further preferably 1-10 h.

[0039] The preparation method of the noble metal nanoparticles can be listed as follows:

[0040] a) Gold nanosphere preparation method: 1) Trisodium citrate reduction preparation of gold nanospheres, heating the aqueous solution of chloroauric acid to boiling, then adding the aqueous solution of trisodium citrate, and after a certain reaction time, gold nanospheres are prepared; or mixing the aqueous solutions of chloroauric acid and trisodium citrate, heating to boiling, and after a certain reaction time, gold nanospheres are prepared. 2) Sodium borohydride reduction preparation of gold nanospheres, adding the aqueous solution of sodium borohydride to the aqueous solution of chloroauric acid, and after a certain reaction time, gold nanospheres are prepared. 3) Citric acid reduction preparation of gold nanospheres, adding the aqueous solution of citric acid to the aqueous solution of chloroauric acid, and after a certain reaction time, gold nanospheres are prepared. 4) Sodium oxalate reduction preparation of gold nanospheres, adding the aqueous solution of sodium oxalate to the aqueous solution of chloroauric acid, and after a certain reaction time, gold nanospheres are prepared. 5) Gallic acid reduction preparation of gold nanospheres, adding the aqueous solution of gallic acid to the aqueous solution of chloroauric acid, and after a certain reaction time, gold nanospheres are prepared. 6) Ascorbic acid reduction preparation of gold nanospheres, adding the aqueous solution of ascorbic acid to the aqueous solution of chloroauric acid, and after a certain reaction time, gold nanospheres are prepared. 7) Hydrazine hydrate reduction preparation of gold nanospheres, adding the aqueous solution of hydrazine hydrate to the aqueous solution of chloroauric acid, and after a certain reaction time, gold nanospheres are prepared. 8) Sodium gluconate reduction preparation of silver nanospheres, adding the aqueous solution of sodium gluconate to the aqueous solution of chloroauric acid, and after a certain reaction time, gold nanospheres are prepared. 9) Dextran reduction preparation of silver nanospheres, adding the aqueous solution of dextran to the aqueous solution of chloroauric acid, and after a certain reaction time, gold nanospheres are prepared.

[0041] b) Preparation method of gold nanorods: gold nanorods are prepared by gold seed growth method, 1) gold seed preparation: gold seeds are prepared by reducing chloroauric acid using trisodium citrate or sodium borohydride; 2) growth solution preparation: hexadecyl trimethyl ammonium bromide (CTAB) is mixed with silver nitrate, then chloroauric acid is added, and ascorbic acid is added after uniform mixing; 3) the gold seeds in 1) are added to the growth solution in 2), and gold nanorods are prepared after reacting for a certain period of time.

[0042] c) Preparation method of gold nanostars: gold nanostars are prepared by gold seed growth method, 1) gold seed preparation: gold seeds are prepared by reducing chloroauric acid using trisodium citrate or sodium borohydride; 2) growth solution preparation: hexadecyl trimethyl ammonium bromide is mixed with silver nitrate, then chloroauric acid is added, and ascorbic acid is added after uniform mixing; 3) the gold seeds in 1) are added to the growth solution in 2), and gold nanostars are prepared after reacting for a certain period of time.

[0043] d) Preparation method of silver nanospheres: 1) silver nanospheres are prepared by sodium borohydride reduction, an aqueous solution of sodium borohydride is added to an aqueous solution of silver nitrate, and silver nanospheres are prepared after reacting for a certain period of time. 2) Silver nanospheres are prepared by citric acid reduction, an aqueous solution of citric acid is added to an aqueous solution of silver nitrate, and silver nanospheres are prepared after reacting for a certain period of time. 3) Silver nanospheres are prepared by trisodium citrate reduction, an aqueous solution of trisodium citrate is added to an aqueous solution of silver nitrate, and silver nanospheres are prepared after reacting for a certain period of time. 4) Silver nanospheres are prepared by ascorbic acid reduction, an aqueous solution of ascorbic acid is added to an aqueous solution of silver nitrate, and silver nanospheres are prepared after reacting for a certain period of time. 5) Silver nanospheres are prepared by hydrazine hydrate reduction, an aqueous solution of hydrazine hydrate is added to an aqueous solution of silver nitrate, and silver nanospheres are prepared after reacting for a certain period of time.

[0044] e) Preparation method of silver nanotriangular flake: an aqueous solution of trisodium citrate, an aqueous solution of polyvinylpyrrolidone, an aqueous solution of silver nitrate, and hydrogen peroxide are uniformly mixed, then an aqueous solution of sodium borohydride is added, and the mixture is kept away from light after reacting for a certain period of time, and silver nanotriangular flake is prepared after keeping away from light for a period of time.

[0045] f) Preparation method of gold@silver nanospheres: gold@silver nanospheres are prepared by gold seed growth method, 1) gold seed preparation: gold seeds are prepared by reducing chloroauric acid using trisodium citrate or sodium borohydride; 2) growth solution preparation: hexadecyl trimethyl ammonium bromide (CTAB) is uniformly mixed with silver nitrate, then ascorbic acid is added; 3) the gold seeds in 1) are added to the growth solution in 2), and gold@silver nanospheres are prepared after reacting for a certain period of time.

[0046] g) Preparation of silver@gold nanospheres: silver@gold nanospheres were prepared by gold seed growth method, 1) gold seed preparation: silver seeds were prepared by reduction of silver nitrate using trisodium citrate or sodium borohydride; 2) growth solution preparation, mixing cetyltrimethylammonium bromide (CTAB) with silver nitrate, then adding aqueous chloroauric acid solution, and adding ascorbic acid after mixing uniformly; 3) adding the silver seeds of 1) to the growth solution of 2), and after reacting for a certain time, silver@gold nanospheres were prepared.

[0047] In step (2), the ligand molecule solution is prepared in step (1) by dispersing magnetic nanoparticles in water, and the concentration of the magnetic nanoparticle solution is not limited.

[0048] Preferably, in step (2), the reaction is stirred at a temperature of 5-40°C, preferably at room temperature, and the reaction time is 0.1-20h, further preferably 1-10h.

[0049] Methods for preparing magnetic nanoparticles can be listed as follows:

[0050] a) Non-aqueous phase method: iron organometallic compounds or complexes (iron pentacarbonyl, iron acetylacetone, iron oleate, etc.) are heated to above 250°C in the presence of high-boiling solvents and ligands (such as oleic acid and oleylamine) and reducing agents. The final nanoparticle size is determined by the temperature and the length of heating time. This method can closely control the size distribution of nanoparticles, and also obtain nanoparticles with high crystallinity. This method has been proven to be very versatile, and has been extended to the preparation of different properties of ferrite nanoparticles (MFe2O4, M = Mn, Co, Ni, Zn) and other magnetic materials such as FePt. The main disadvantage of thermal decomposition is that the obtained nanoparticles are usually only soluble in non-polar solvents, so functional groups must be introduced by intermediate ligands before the nucleation and growth of the nanoparticles are stable.

[0051] b) aqueous phase method: 1) microemulsion method: two immiscible solvents form a uniform microemulsion under the action of a surfactant, nucleation and growth of the nanoparticles are avoided, and agglomeration between the nanoparticles is avoided, thereby obtaining the nanoparticles. 2) sol-gel method: the sol-gel method is a method for preparing metal oxides by a wet process. In the sol-gel method, iron chloride or iron nitrate is used as a raw material, and is dissolved in water or alcohol at a certain temperature, and a certain amount of base (or ammonia) and a surfactant for modification are added. The reaction solution is gelled by adjusting the pH of the solution, and iron oxide nanoparticles are obtained by drying, calcining and other steps. 3) coprecipitation method: the coprecipitation method refers to a process in which two or more cations are simultaneously precipitated in a multi-component solution in the presence of a precipitant. Magnetic nanoparticles are prepared by this method based on the principle of solubility product, and are based on a precipitation reaction. In a solution containing a specific proportion of cations, nanoparticles are formed after the addition of a suitable precipitant. The advantages of this method are simple reaction principle, low cost of equipment and raw materials, and suitability for mass production. However, the disadvantages are that it is difficult to obtain high-quality monodisperse colloids, and the saturation magnetization is usually low. For example, iron oxide (Fe3O4 or γ-Fe2O3) is prepared by the coprecipitation method in an inert gas protective atmosphere. Nanoparticles are obtained by adding a precipitant to a solution of Fe 2+ / Fe 3+ salt in a certain proportion at room temperature or a provided temperature. 4) hydrothermal and solvothermal methods: the hydrothermal reaction is carried out in an autoclave. Under high temperature and pressure, the reactants are reacted in a closed system with water as the solvent, and the desired product is finally obtained. 5) chemical reduction method: the metal reduction method is used, and the raw materials can be metal chlorides, nitrates or acetylacetone salts. The reducing agent is selected from super lithium or sodium borohydride, and some organic reducing agents such as polyols, hydrazine hydrate and dihydrogen gas. The chemical reduction method can be divided into aqueous solution method and organic solution method according to the different reaction systems.

[0052] In step (3), the organic building block solution is formed by dissolving the organic building block in a solvent, and any solvent that can dissolve the organic building block can be used, such as PBS buffer, water, ethanol, etc. The concentration of the organic building block solution is not limited. As a preferred embodiment, the pH of the organic building block solution is 7.2 to 7.8. As a preferred embodiment, the organic building block solution is formed by dissolving the organic building block in PBS buffer, and the pH of the PBS buffer is 7.2 to 7.8.

[0053] As a preferred embodiment, in step (3), the temperature for stirring the reaction is 5 to 40°C, and the reaction is preferably carried out at room temperature. The reaction time is 5 to 80h, and the reaction time is further preferably 10 to 70h.

[0054] In step (3), the "SH" in the inorganic building unit forms a dynamic covalent bond of "S-S" through oxidation of oxygen in air and water, forming a dynamic combinatorial chemistry library, and forming a dynamic cyclic molecule through dynamic exchange of "S-S". Under the driving force of non-covalent interactions (including host-guest interactions, electrostatic interactions, hydrogen bonding interactions, π-π conjugation interactions, hydrophobic interactions, dipole interactions, van der Waals interactions, and other non-covalent interactions), the noble metal nanoparticles and magnetic nanoparticles are self-assembled together to form a self-assembled nanostructure. Because the dynamic combinatorial chemistry is a thermodynamically controlled system, that is, when a certain dynamic combinatorial library reaches thermodynamic equilibrium, the components in the system are in a thermodynamic state of minimum energy and are more stable. Therefore, the composite nanomaterials prepared by the self-assembly method based on dynamic combinatorial chemistry are more stable. Under certain conditions or substance triggers, the thermodynamic equilibrium is broken, the dynamic cyclic molecule structure is destroyed, and the self-assembled nanostructure is disassembled, realizing the stimulus-responsive disassembly.

[0055] In step (4), the target molecule solution is formed by dissolving the target molecule in a solvent, and any solvent that can dissolve the target molecule can be used, such as water, ethanol, etc., and the concentration of the target molecule solution is not limited.

[0056] In step (4), the catalyst activates the self-assembled nanostructure or the target molecule solution, depending on: when the self-assembled nanostructure material contains a carboxyl group, a target molecule containing an amino group can be selected, then the carboxyl group on the surface of the self-assembled nanostructure is activated by a catalyst, and then the target molecule solution containing an amino group is added dropwise to make the activated carboxyl group covalently react with the amino group of the target molecule to form a stable amide bond; when the self-assembled nanostructure material contains an amino group, a target molecule containing a carboxyl group can be selected, and then the carboxyl group of the target molecule is activated by the above method and grafted onto the surface of the self-assembled nanostructure material.

[0057] As a preferred embodiment, the catalyst in step (4) is one or more of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), a compound containing an azide group, and dopamine.

[0058] As a preferred embodiment, the activation in step (4) is carried out in the dark, and the activation time is 1-20 h.

[0059] As a preferred embodiment, in step (4), the stirring reaction temperature is 5-40°C, preferably carried out at room temperature, and the reaction time is 5-40 h, further preferably 8-30 h.

[0060] The stirring speed in steps (1)-(4) is not limited, which can be 100-1000 rpm.

[0061] Another object of the present application is to provide the application of the magnetic SERS composite nano-probe in in-vitro detection.

[0062] As preferred, the application comprises the following steps: adding the magnetic SERS composite nano-probe into the system to be detected, combining the magnetic SERS composite nano-probe with the target in the system to be detected, separating through magnetic separation, and detecting through Raman spectrum to determine the concentration of the target in the system to be detected.

[0063] The mononuclear cells in the blood of healthy human body do not express or express a small amount of specific antigens or receptors on the surface, and cannot adsorb or uptake the magnetic SERS composite nano-probe, so the magnetic SERS composite nano-probe cannot be separated and enriched in the magnetic field, and the SERS signal cannot be detected; the blood of cancer patients contains cancer cells, and the cancer cells highly express specific antigens or receptors, so the tumor cells can adsorb or uptake the magnetic SERS composite nano-probe, the tumor cells can be separated and enriched in the magnetic field, and the SERS signal can be detected, so as to achieve the purpose of high-sensitivity detection of cancer cells.

[0064] In addition, in order to avoid the interference of mononuclear cells (such as white blood cells) in the body of a cancer patient, the SERS signal of the probe-labeled cancer cells is tested at different time points, and under the triggering of GSH in the cancer cells, the magnetic SERS composite nano-probe is disassembled, and the SERS signal is obviously reduced, while the magnetic SERS composite nano-probe in the white blood cells cannot be disassembled, and the SRES signal does not change, so that the ratio of the SERS signals at different time points is used to further improve the sensitivity of CTC detection.

[0065] As preferred, the system to be detected includes but is not limited to blood, culture solution, peritoneal fluid, urine and the like.

[0066] Compared with the prior art, the present application has the following beneficial effects:

[0067] 1. The stimulus-responsive magnetic SERS composite nano-probe of the present application comprises noble metal nanoparticles, magnetic nanoparticles, Raman signal molecules, ligand molecules and organic building units; the magnetic nanoparticles can complete the enrichment and separation of tumor cells in a magnetic field, the noble metal nanoparticles have a significant enhancement effect on the Raman signal, and the detection and counting of tumor cells are completed through Raman spectrum testing; the noble metal nanoparticles are sequentially connected with the Raman signal molecules and the ligand molecules on the surface, the magnetic nanoparticles are connected with the ligand molecules on the surface, and the organic building units are self-assembled together with the noble metal nanoparticles and the magnetic nanoparticles by combining with the ligand molecules to form a self-assembled nanostructure;

[0068] 2、The organic building unit benzene ring carboxylic acid type bimercapto organic molecule, anthracene ring carboxylic acid type bimercapto organic molecule, naphthalene ring carboxylic acid type bimercapto organic molecule of the application, one or more of the organic building unit, the "-SH" in the organic building unit can form a dynamic covalent bond of "S-S" through the oxidation of oxygen in air and water, a dynamic cyclic molecule is formed through the dynamic exchange of "S-S", under the driving of non-covalent force, the ligand molecule and the dynamic cyclic molecule self-assemble the noble metal nanoparticles and the magnetic nanoparticles together to form a self-assembled nanostructure;

[0069] 3、The stimulus-responsive magnetic SERS composite nano-probe of the application further comprises a target molecule coupled on the surface of the self-assembled nanostructure, improves the targeting performance of the self-assembled nanostructure on tumor cells, and improves the specific binding of the nano-probe and the tumor cells;

[0070] 4、The stimulus-responsive magnetic SERS composite nano-probe of the application is triggered under a certain condition or substance, the thermodynamic equilibrium is broken, the dynamic cyclic molecule structure is destroyed, and then the self-assembled nanostructure is disassembled, and the SERS signal is obviously reduced, so that the detection sensitivity of tumor cells is further improved through the ratio of SERS signals at different time points;

[0071] 5、The application is based on noble metal nanoparticles and magnetic nanomaterials, and develops a stimulus-responsive magnetic SERS composite nano-probe with simple synthesis, good stability, good separation effect, high sensitivity and good specificity based on a self-assembly method of dynamic combinatorial chemistry, provides a theoretical and technical basis for the research of a high-performance tumor cell detection method and device, and solves important problems such as cancer diagnosis and prognosis. BRIEF DESCRIPTION OF DRAWINGS

[0072] Figure 1 Fig. 1 is a preparation and disassembly diagram of the magnetic SERS composite nano-probe of the application;

[0073] Figure 2 Fig. 2 is a TEM diagram of the magnetic SERS nano-probe prepared in Example 1;

[0074] Figure 3 Fig. 3 is a TEM comparison diagram of the magnetic SERS nano-probe before and after GSH is added;

[0075] Figure 4 Fig. 4 is a SERS spectrum diagram of the magnetic SERS nano-probe prepared in Example 1 for detecting MCF7 breast cancer cells. DETAILED DESCRIPTION

[0076] The technical solutions of the present application are further described and illustrated below by means of specific examples and drawings. It should be understood that the specific examples described herein are only used to help understand the present application and are not used to limit the present application. The drawings used herein are only used to better illustrate the disclosed content of the present application and do not limit the scope of protection. If not specifically stated, the raw materials used in the examples of the present application are all commonly used raw materials in the art, and the methods used in the examples are all conventional methods in the art.

[0077] Figure 1 For the preparation and disassembly of the magnetic SERS composite nanoprobe of the present application, as shown in the figure, the magnetic nanoparticles are connected with ligand molecules, the noble metal nanoparticles are connected with ligand molecules, the "SH" in the organic building unit is oxidized by air and oxygen in water to form "S-S", the dynamic exchange of "S-S" forms a dynamic macrocyclic molecule, the dynamic macrocyclic molecule is combined with the ligand molecules modified on the surface of the noble metal nanoparticles and the magnetic nanoparticles, the noble metal nanoparticles and the magnetic nanoparticles are self-assembled together based on the dynamic combinatorial chemistry self-assembly method to form a self-assembled nanostructure, the target molecules are modified on the outer layer of the self-assembled nanostructure, and have specific interaction with the tumor cells to be detected; under the stimulation of glutathione (GSH), the "S-S" in the self-assembled nanostructure is destroyed, the dynamic macrocyclic molecule reverts to the organic building unit, and at the same time the self-assembled nanostructure disassembles, the SERS signal is weakened, and the stimulation-responsive SERS signal conversion is realized.

[0078] Example 1

[0079] (1) Preparation of gold nanospheres (AuNPs) with SERS function

[0080] 10 mL of 5 mM chloroauric acid solution was added to 30 mL of ultrapure water, stirred uniformly, and then 2 mL of 0.1 M sodium borohydride solution was added. The solution turned wine red, and AuNPs were prepared.

[0081] (2) Preparation of gold nanospheres (AuNP-MBA-CTAB) connected with Raman signal molecules and ligand molecules

[0082] 4-mercaptobenzoic acid (4-MBA) was dissolved in an ethanol solvent to prepare a 4-mercaptobenzoic acid ethanol solution with a concentration of 50 mM. 3 mL of gold nanospheres prepared in step (1) were taken and added to 9 mL of ultrapure water, mixed uniformly, and then 30.0 μL of 4-mercaptobenzoic acid ethanol solution was added. The reaction was stirred at room temperature at 300 rpm. After 10 min of reaction, AuNP-MBA material was prepared.

[0083] Take 10 mL AuNP-MBA material, under stirring state, add 10.0 μL 0.1M ligand molecule solution (hexadecyl trimethyl ammonium bromide, CTAB), under room temperature, 300 rpm stirring reaction, after 8h reaction, AuNP-MBA-CTAB material is prepared.

[0084] (3) Preparation of magnetic nanoparticles (MNPs)

[0085] Put 2 mmol ferrous ammonium sulfate hexahydrate into 20 mL ultrapure water to prepare ferrous ammonium sulfate solution. Put 1 g sodium hydroxide into 10 mL mixed solution of oleic acid and 10 mL ethanol, stir until completely dissolved, then add 20 mL ferrous ammonium sulfate solution, after the color of the mixed solution changes to brown red, transfer it to 50 mL reaction kettle, heat at 230℃ for 8h. After the reaction kettle is cooled, take it out, ethanol centrifugal washing three times, and disperse in 20 mL cyclohexane to prepare the required oil-soluble magnetic nanoparticles. Then add 2 g citric acid and 20 mL oil-soluble magnetic nanoparticles into 30 mL mixed solution of chloroform / DMF (v / v: 1 / 1), stir for 12h, ethanol centrifugal washing three times, and disperse in 20 mL water to prepare the required aqueous magnetic nanoparticles (MNPs).

[0086] (4) Preparation of magnetic nanoparticles connected with ligand molecules (MNP-CTAB)

[0087] Take 1 mL aqueous MNPs material prepared in step (3), add 9 mL water, and under stirring state, add 10.0 μL 0.1M ligand molecule (hexadecyl trimethyl ammonium bromide, CTAB), under room temperature, 300 rpm stirring reaction, after 8h reaction, MNP-CTAB material is prepared.

[0088] (5) Preparation of self-assembled magnetic SERS nanostructure

[0089] Take 0.5 mL AuNP-MBA-CTAB and 0.5 mL MNP-CTAB, add 0.4 mL pH 7.4 50mM PBS buffer solution, then add 0.3 mL 5mM building block solution (3,5-dimercaptobenzoic acid dissolved in pH 7.4 50mM PBS buffer solution to form) and 0.3 mL water, under room temperature, 300 rpm stirring reaction for 48h, centrifugal and disperse in water to prepare self-assembled magnetic SERS nanostructure.

[0090] (6) Antibody coupling of self-assembled magnetic SERS nanostructure

[0091] Take the self-assembled magnetic SERS nanoparticles prepared in step (5), EDC, and NHS (mass ratio 1000:20:15), dissolve them in 10 mM phosphate buffer (pH 7.4), react in the dark for 8 h, and centrifuge. Add 1 mg of cRGD cyclic peptide to 2 mL of activated self-assembled magnetic SERS nanoparticles, stir at 300 rpm for 16 h at room temperature, and centrifuge using an ultrafiltration centrifuge tube (MWCO 3.0 kDa) to obtain magnetic SERS nanoprobes with cRGD target molecules coupled to their surface.

[0092] Figure 2 The image shows a TEM image of the magnetic SERS nanoprobe prepared in Example 1. The image shows that the noble metal nanoparticles and magnetic nanoparticles self-assemble into an aggregated state. The elemental distribution map further confirms the formation of the self-assembled structure.

[0093] GSH was added to the magnetic SERS nanoprobe prepared in Example 1. TEM images of the magnetic SERS nanoprobe before and after GSH addition are shown below. Figure 3 ,from Figure 3 It can be seen that the self-assembled structure of the magnetic SERS nanoprobes undergoes deassembly under the stimulation of GSH, and the noble metal nanoparticles and magnetic nanoparticles are redispersed into single particles.

[0094] The magnetic SERS nanoprobe prepared in Example 1 was co-incubated with MCF7 breast cancer cells. After magnetic separation, SERS spectroscopy was performed, and the SERS spectrum is shown below. Figure 4 As shown, 1076cm -1 The peak at the shift point is the Raman characteristic peak of the Raman signal molecule 4-MBA. After cancer cells were incubated with the magnetic SERS nanoprobe, Raman testing was performed on them, and the characteristic peak of the Raman signal molecule appeared, indicating that the magnetic SERS nanoprobe successfully labeled the cancer cells and performed highly sensitive SERS detection.

[0095] Example 2

[0096] (1) Preparation of SERS-enabled gold nanorods (AuNRPs)

[0097] The first step is the preparation of the gold seed. Mix 7.5 mL of CTAB solution and 0.5 mL of 5 mM chloroauric acid solution evenly, and then quickly add 0.6 mL of 0.01 M sodium borohydride. Once the solution turns brown, the gold seed can be obtained.

[0098] Second step, growth solution preparation, 100 mL 0.1M CTAB solution and 0.1 mL 20 mM silver nitrate solution are mixed, 12 mL 5 mM chloroauric acid solution is added and stirred uniformly, then 0.48 mL ascorbic acid aqueous solution is added, after the solution becomes colorless, 0.2 mL gold seed is added, and the reaction is carried out in the dark, until the color becomes dark blue, the gold nanorod with SERS function (AuNRPs) can be prepared.

[0099] The Raman signal molecules and ligand molecules are connected on the surface of the AuNRPs, and the subsequent steps are the same as those in Example 1, so that the stimulus-responsive magnetic SERS composite nanoprobe can be prepared.

[0100] Example 3

[0101] (1) Preparation of gold nanostar (AuNS) with SERS function

[0102] First step, preparation of gold seed, 3 mL 5 mM chloroauric acid solution is added to 45 mL ultrapure water, heated to boiling, then 2.5 mL 1% trisodium citrate solution is quickly added, the reaction is stopped when the color becomes purple, and the liquid is naturally cooled to room temperature, and the color of the liquid becomes wine red, and the gold seed can be prepared; second step, preparation of growth solution, 1.0 mL 0.1M CTAB, 2.75 mL 5 mM chloroauric acid solution is added to 45 mL ultrapure water, then 50 μL 50 mM silver nitrate solution is added, and then 0.565 mL 10 mg / mL ascorbic acid solution is added, when the color of the liquid becomes colorless, 50 μL gold seed is added, and when the color becomes dark blue, the gold nanostar (AuNS) with SERS function can be prepared.

[0103] The Raman signal molecules and ligand molecules are connected on the surface of the AuNS, and the subsequent steps are the same as those in Example 1, so that the stimulus-responsive magnetic SERS composite nanoprobe can be prepared.

[0104] Example 4

[0105] (1) Preparation of silver nanosphere (AgNPs) with SERS function

[0106] 1 mL 20 mM silver nitrate is added to 97 mL ultrapure water, then 1 mL of protective agent (30 mM trisodium citrate solution) is added, then 1 mL of 0.1M sodium borohydride solution is added, and after the reaction for 2 h, the silver nanosphere (AgNPs) with yellow color can be prepared.

[0107] The Raman signal molecules and ligand molecules are connected on the surface of the AgNPs, and the subsequent steps are the same as those in Example 1, so that the stimulus-responsive magnetic SERS composite nanoprobe can be prepared.

[0108] Example 5

[0109] (1) Preparation of silver nano-triangular plate (AgNRPs) with SERS function

[0110] Into 99.5 mL of ultrapure water, 6 mL of 30 mM trisodium citrate solution and 6 mL of 0.7 mM polyvinylpyrrolidone solution were added and mixed under stirring, and then 0.5 mL of 20 mM silver nitrate solution and 0.24 mL of 30% hydrogen peroxide were added thereto and stirred uniformly. 1 mL of 0.1 M sodium borohydride solution was quickly added, and after reaction for a certain time in the dark, silver nano-triangular plate (AgNRPs) with SERS function was prepared when the color turned blue.

[0111] The Raman signal molecules and ligand molecules were connected to the surface of AgNRPs, and the subsequent steps were the same as those in Example 1, so that the stimulus-responsive magnetic SERS composite nanoprobe was prepared.

[0112] Example 6

[0113] In steps (2) and (4) of Example 1, the ligand molecules were replaced by cetylpyridinium bromide. The specific preparation method is briefly described as follows: 10 mL of AuNP-MBA material prepared in step (2) of Example 1 was taken, 5.0 μL of 0.1 M ligand molecules (cetylpyridinium bromide, CTPB) was added under stirring, and the reaction was carried out under stirring at room temperature and at 300 rpm. After 8 h of reaction, AuNP-MBA-CTPB material was prepared. In step (4), 1 mL of aqueous MNP material prepared in step (3) of Example 1 was taken, added into 9 mL of water, and 5.0 μL of 0.1 M ligand molecules (cetylpyridinium bromide, CTPB) was added under stirring. The reaction was carried out under stirring at room temperature and at 300 rpm. After 8 h of reaction, MNP-CTPB material was prepared. The other steps were the same as those in Example 1, so that the stimulus-responsive magnetic SERS composite nanoprobe was prepared.

[0114] Example 7

[0115] In steps (2) and (4) of Example 1, the ligand molecules were replaced by dodecylpyridinium bromide. The specific preparation method is briefly described as follows: 10 mL of AuNP-MBA material prepared in step (2) of Example 1 was taken, 5.0 μL of 0.1 M ligand molecules (dodecylpyridinium bromide, DPB) was added under stirring, and the reaction was carried out under stirring at room temperature and at 300 rpm. After 8 h of reaction, AuNP-MBA-DPB material was prepared. In step (4), 1 mL of aqueous MNP material prepared in step (3) of Example 1 was taken, added into 9 mL of water, and 5.0 μL of 0.1 M ligand molecules (dodecylpyridinium bromide, DPB) was added under stirring. The reaction was carried out under stirring at room temperature and at 300 rpm. After 8 h of reaction, MNP-DPB material was prepared. The other steps were the same as those in Example 1, so that the stimulus-responsive magnetic SERS composite nanoprobe was prepared.

[0116] Example 8

[0117] The ligand molecules in steps (2) and (4) of Example 1 are replaced by tetradecylpyridinium bromide. The specific preparation method is briefly described as follows: take 10 mL of AuNP-MBA material prepared in step (2) of Example 1, add 5.0 μL of 0.1M ligand molecules (tetradecylpyridinium bromide, TPB) under stirring, and react at room temperature under 300 rpm stirring. After 8h of reaction, AuNP-MBA-TPB material is prepared. Step (4): take 1 mL of water-soluble MNPs material prepared in step (3) of Example 1, add 9 mL of water, and add 5.0 μL of 0.1M ligand molecules (tetradecylpyridinium bromide, TPB) under stirring. React at room temperature under 300 rpm stirring. After 8h of reaction, MNP-TPB material is prepared. Other steps are the same as those in Example 1, and a stimulus-responsive magnetic SERS composite nanoprobe is prepared.

[0118] Example 9

[0119] The ligand molecules in steps (2) and (4) of Example 1 are replaced by tetradecyltrimethylammonium bromide (TTAB). The specific preparation method is briefly described as follows: take 10 mL of AuNP-MBA material prepared in step (2) of Example 1, add 5.0 μL of 0.1M ligand molecules (TTAB) under stirring, and react at room temperature under 300 rpm stirring. After 8h of reaction, AuNP-MBA-TTAB material is prepared. Step (4): take 1 mL of water-soluble MNPs material prepared in step (3) of Example 1, add 9 mL of water, and add 5.0 μL of 0.1M ligand molecules (TTAB) under stirring. React at room temperature under 300 rpm stirring. After 8h of reaction, MNP-TTAB material is prepared. Other steps are the same as those in Example 1, and a stimulus-responsive magnetic SERS composite nanoprobe is prepared.

[0120] Example 10

[0121] The ligand molecules in steps (2) and (4) of Example 1 are replaced by dodecyl trimethyl ammonium bromide (DTAB). The specific preparation method is briefly described as follows: 10 mL of AuNP-MBA material prepared in step (2) of Example 1 is taken, 5.0 μL of 0.1M ligand molecules (DTAB) is added under stirring, and the reaction is stirred at 300 rpm at room temperature. After 8 h of reaction, the AuNP-MBA-DTAB material is prepared. Step (4): 1 mL of water-soluble MNPs material prepared in step (3) of Example 1 is taken, added into 9 mL of water, and 5.0 μL of 0.1M ligand molecules (DTAB) is added under stirring. The reaction is stirred at 300 rpm at room temperature. After 8 h of reaction, the MNP-DTAB material is prepared. Other steps are the same as those in Example 1, and the stimulus-responsive magnetic SERS composite nanoprobe is prepared.

[0122] Example 11

[0123] The organic building unit in step (5) of Example 1 is replaced by 2,5-dimercaptoterephthalic acid. The specific preparation method is briefly described as follows: 0.5 mL of AuNP-MBA-CTAB and 0.5 mL of MNP-CTAB are added into 0.4 mL of 50 mM PBS buffer solution at pH 7.4, 0.3 mL of 5 mM organic building unit solution (2,5-dimercaptoterephthalic acid is dissolved in 50 mM PBS buffer solution at pH 7.4 to form) and 0.3 mL of water are added, and the reaction is stirred for 48 h. After centrifugation and dispersion in water, the self-assembled magnetic SERS nanostructure is prepared. Other steps are the same as those in Example 1, and the stimulus-responsive magnetic SERS composite nanoprobe is prepared.

[0124] Example 12

[0125] The organic building unit in step (5) of Example 1 is replaced by 3,5-bis(mercaptomethyl)benzoic acid. The specific preparation method is briefly described as follows: 0.5 mL of AuNP-MBA-CTAB and 0.5 mL of MNP-CTAB are added into 0.4 mL of 50 mM PBS buffer solution at pH 7.4, 0.3 mL of 5 mM organic building unit solution (3,5-bis(mercaptomethyl)benzoic acid is dissolved in 50 mM PBS buffer solution at pH 7.4 to form) and 0.3 mL of water are added, and the reaction is stirred for 48 h. After centrifugation and dispersion in water, the self-assembled magnetic SERS nanostructure is prepared. Other steps are the same as those in Example 1, and the stimulus-responsive magnetic SERS composite nanoprobe is prepared.

[0126] Example 13

[0127] The organic building unit in step (5) of Example 1 is replaced by 3,7-dimercapto-2-naphthoic acid, and the preparation method is briefly described as follows: 0.5 mL of AuNP-MBA-CTAB and 0.5 mL of MNP-CTAB are added into 0.4 mL of 50 mM PBS buffer solution at pH 7.4, and then 0.3 mL of 5 mM organic building unit solution (3,7-dimercapto-2-naphthoic acid is dissolved in 50 mM PBS buffer solution at pH 7.4 to form) and 0.3 mL of water are added, and the reaction is stirred for 48 h, and then centrifuged and dispersed in water to obtain self-assembled magnetic SERS nanostructures. Other steps are the same as those in Example 1, that is, a stimulus-responsive magnetic SERS composite nanoprobe can be prepared.

[0128] Example 14

[0129] The organic building unit in step (5) of Example 1 is replaced by 6,6'-dimercapto-3,3'-methylene dibenzoic acid, and the preparation method is briefly described as follows: 0.5 mL of AuNP-MBA-CTAB and 0.5 mL of MNP-CTAB are added into 0.4 mL of 50 mM PBS buffer solution at pH 7.4, and then 0.3 mL of 5 mM organic building unit solution (6,6'-dimercapto-3,3'-methylene dibenzoic acid is dissolved in 50 mM PBS buffer solution at pH 7.4 to form) and 0.3 mL of water are added, and the reaction is stirred for 48 h, and then centrifuged and dispersed in water to obtain self-assembled magnetic SERS nanostructures. Other steps are the same as those in Example 1, that is, a stimulus-responsive magnetic SERS composite nanoprobe can be prepared.

[0130] Aspects, embodiments, features, and the like of the present application are to be considered illustrative only and not restrictive in all respects. The scope of the present application is defined only by the claims. Other embodiments, modifications, and uses can be apparent to those skilled in the art, without departing from the spirit and scope of the claimed application.

[0131] In the preparation method of the present application, the order of the steps is not limited to the order listed, and for those skilled in the art, the order of the steps can be changed without creative labor, which is within the protection scope of the present application. In addition, two or more steps or actions can be performed simultaneously.

[0132] It should be noted that the embodiments described herein are merely illustrative of the present application and should not be construed as limiting the scope of the present application. Those skilled in the art can make various modifications or additions to the embodiments described herein or adopt similar ways to replace them without departing from the spirit of the present application. It is not necessary or possible to describe all the embodiments herein. Any obvious changes or variations derived from the spirit of the present application are still within the scope of the present application, and any additional limitations are contrary to the spirit of the present application.

Claims

1. A stimuli-responsive magnetic SERS composite nanoprobe, characterized in that, The noble metal nanoparticles, magnetic nanoparticles, Raman signal molecules, ligand molecules, organic building units and target molecules are included. The noble metal nanoparticles are sequentially connected with Raman signal molecules and ligand molecules on the surface, the magnetic nanoparticles are connected with ligand molecules on the surface, the organic building units are combined with the ligand molecules to self-assemble the noble metal nanoparticles and the magnetic nanoparticles together to form a self-assembled nanostructure; after the self-assembled nanostructure is activated by a catalyst, the activated self-assembled nanostructure and a non-activated target molecule solution are mixed and stirred to obtain a stimulus-responsive magnetic SERS composite nanoprobe. The organic building unit is 3,5-dimercaptobenzoic acid, 2,5-dimercaptoterephthalic acid, 3,5-bis(mercaptomethyl)benzoic acid, 3,7-dimercapto-2-naphthoic acid or 6,6'-dimercapto-3,3'-methylene dibenzoic acid. The ligand molecule is one or more of quaternary ammonium salt molecules and pyridine salt molecules, The quaternary ammonium salt molecule is an alkyl quaternary ammonium salt, and has a structural formula as shown in the following formula I: Formula I In the formula, X is a halogen element, and n is an integer of 1-30. The pyridine salt molecule is an alkyl pyridine salt, and has a structural formula as shown in the following formula III: Formula III In the formula, X is a halogen element, and n is an integer of 1-30.

2. The stimuli-responsive magnetic SERS composite nanoprobe of claim 1, wherein, The noble metal nanoparticles are one or more of gold, silver and platinum group metals; the particle size of the noble metal nanoparticles is less than 1 μm. 3.The stimuli-responsive magnetic SERS composite nanoprobe of claim 1, wherein, The magnetic nanoparticles are one or more of magnetic metal element nanoparticles and magnetic metal oxide nanoparticles; the magnetic metal element includes one or more of Fe, Zn, Co, Ni, Cr and Mn; the magnetic metal oxide includes one or more of oxides of one or more of Fe, Co, Ni, Cr and Mn; The particle size of the magnetic nanoparticles is less than 1 μm.

4. The stimuli-responsive magnetic SERS composite nanoprobe of claim 1, wherein, The Raman signal molecule is an organic substance having conjugate vibration in Raman spectrum.

5. The stimuli-responsive magnetic SERS composite nanoprobe of claim 1, wherein, The ligand molecule is cetyltrimethylammonium bromide, cetylpyridinium bromide, dodecylpyridinium bromide, tetradecylpyridinium bromide, tetradecyltrimethylammonium bromide or dodecyltrimethylammonium bromide.

6. The method for preparing the stimulus-responsive magnetic SERS composite nanoprobe as described in claim 1, characterized in that, The method comprises the following steps: (1) adding a Raman signal molecule solution into a noble metal nanoparticle solution, stirring and reacting, and then adding a ligand molecule solution to continue stirring and reacting; (2) stirring and reacting a ligand molecule solution and a magnetic nanoparticle solution; (3) adding a mixture of the product of step (1) and the product of step (2) into an organic building unit solution to stir and react, to obtain a self-assembled nanostructure; (4) after the self-assembled nanostructure is activated by a catalyst, mixing and stirring the activated self-assembled nanostructure and a non-activated target molecule solution to obtain a stimulus-responsive magnetic SERS composite nanoprobe.

7. Application of the stimulus-responsive magnetic SERS composite nanoprobe of claim 1 in in-vitro detection, which is for non-disease diagnosis.

8. Use according to claim 7, characterized in that, The application comprises the following steps: adding the above-mentioned stimulus-responsive magnetic SERS composite nanoprobe into a to-be-detected system, combining the above-mentioned magnetic SERS composite nanoprobe with a target object in the to-be-detected system, performing magnetic separation, detecting through a Raman spectrum, and determining the concentration of the target object in the to-be-detected system.

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