CN116223475A - Raman nanoparticle lamellar treatment method and application - Google Patents
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Abstract
The invention discloses a Raman nanoparticle lamellar treatment method and application. By forming the self-assembled Raman nanoparticle thin layers which are uniformly distributed, the aggregation of Raman nanoparticles is avoided, so that the same signal can be obtained from any position of the Raman nanoparticle thin layers, and the stability of Raman signals is ensured. The Raman nano particle thin layer is further enriched and transferred onto the solid-phase substrate, so that the concentration of the Raman nano particles in unit area is improved, and further the detection signal intensity and the detection sensitivity are improved. Through the technical routes of specific combination, dissociation and magnetic bar separation of sandwich complexes, the quantitative relation between the biomarker and the Raman nano particles is ensured to be in linear correlation, and the problems of nonspecific residue and false positive of the Raman nano particles in the prior art are avoided. The invention is simple and easy to implement, has outstanding sensitivity, accuracy, reproducibility and specificity, and has great application potential in the field of biological detection.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a Raman nanoparticle lamellar treatment method and application.
Background
The presence or change (e.g., concentration, in vivo distribution, etc.) of a particular active substance within a living being is highly correlated to the important changes that are occurring and will occur to the living being. Ultrasensitive detection of specific substances (biomarkers) in the living body, such as proteins, nucleic acids, cells, etc., is of great importance for diagnosing individual diseases or potential diseases. For example, achieving early diagnosis of tumors based on biomarkers can significantly improve disease treatment efficacy; reliable detection and rapid reporting of viruses based on biomarkers can effectively block transmission of infectious diseases. Thus, the development of advanced diagnostic techniques is an important goal of modern medicine.
The raman spectrum is an analysis method for analyzing a scattering spectrum different from the frequency of incident light to obtain information on the vibration and rotation of molecules, and is applied to the study of molecular structures. The Raman spectrum derived from induced dipole moment change can provide fingerprint spectrum information of molecular structure vibration, and the Raman spectrum peak with narrow distribution can be used for simultaneously researching various components, and the detection process is rapid, non-contact and lossless. In addition, the Raman signal of water is extremely weak, and the Raman spectrum technology can be directly used for detecting the water-containing sample, so that the advantages are remarkable in biological sample analysis. However, the molecular intrinsic raman scattering cross section is generally small, only 10 of the infrared and fluorescence processes -6 And 10 -14 Therefore, the sensitivity is low and the wide application is not possible. In 1974, the university of southern Anputon, england Fleischmann et al unexpectedly obtained a high intensity Raman spectrum of a monolayer of pyridine molecules adsorbed on the surface of silver electrodes (after Van Duyne and Cright on, the Raman signal of pyridine molecules on the surface of roughened electrodes was enhanced by about 10 compared to the solution state 6 ). Accordingly, a Surface enhanced effect associated with rough surfaces, surface Enhanced Raman Scattering (SERS), occurs. Recent studies indicate that SERS effects are mainly caused by surface plasmon resonance, and enhancement performance can be evaluated with enhancement factors. Previous studies have demonstrated enhancement factors for SERS as high as 10 15 Single molecule level detection can be achieved.
Compared with the conventional fluorescence and colorimetric technologies, the SERS technology has the characteristics of ultrahigh sensitivity (trace level detection of molecules), molecular fingerprint property, no damage, insusceptibility of SERS markers to self-quenching and photo-bleaching, capability of excitation of various SERS markers under the same excitation wavelength, narrow Raman spectrum peak (much narrower than fluorescence), strong anti-interference capability and the like, so that the SERS technology has great application potential in the field of biological detection. Chinese patent CN114720450a discloses a new coronavirus raman-labeled antigen detection test strip and a preparation method thereof, wherein the method combines a raman probe onto an immune nitrocellulose membrane to realize new coronavirus detection based on raman spectrum signals. Chinese patent CN114544594a discloses a SERS virus detection chip for detecting novel coronaviruses and a method for preparing the same, and the method combines ACE2 biological probe molecules with graphene/periodic nano metal structure SERS detection substrates to form a novel coronavirus biological detection chip, so as to realize high-efficiency, rapid and high-sensitivity detection of novel coronaviruses.
Although some SERS biological detection studies are reported at home and abroad, polymerase Chain Reaction (PCR) based nucleic acid detection is still a gold standard for clinical detection of biomarkers such as new coronaviruses. Because the technology meeting the clinical detection requirement should be mature, each detection can be ensured to be timely and accurate. However, the related research of the existing SERS detection is mainly focused on the design of the nano-substrate structure, and the improvement of the enhancement factors is simply pursued. The problems of difficult signal stabilization, poor reproducibility, non-specific retention interference and the like in detection application seriously prevent the SERS technology from going to clinical application.
Compared with PCR technology, SERS does not need amplification process, and detection time can be greatly reduced. The invention discloses a clinical SERS detection method with strong signals, good reproducibility and no non-specific interference, which promotes the technology to realize clinical application and has important academic significance and industrial value.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide a method for detecting biomarkers based on surface-enhanced raman spectroscopy, which aims to solve the problems existing in the prior art.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
The first aspect of the invention provides a raman nanoparticle thinning treatment method, comprising the following steps:
1) Dissolving Raman nano particles in an aqueous phase, wherein the Raman nano particles are provided with Raman signal molecules, and can generate Raman spectrum signals under laser irradiation;
2) Adding an oily solvent and an accelerator, forming a self-assembled Raman nanoparticle thin layer on an oil-water interface, enriching the Raman nanoparticle thin layer, and transferring the Raman nanoparticle thin layer to a solid-phase substrate.
According to the method, the oil phase solvent and the accelerator are arranged, so that hydrophilic Raman nanoparticles in the water phase are converted from a one-body distribution state to a one-surface distribution state which is uniformly distributed at an oil-water interface, a Raman nanoparticle thin layer is formed by self-assembly above the oil-water interface, the Raman nanoparticle thin layer is further enriched, the concentration of Raman nanoparticles in unit area is improved, and the improvement of Raman spectrum signal intensity is facilitated.
In certain embodiments, the raman nanoparticle comprises a noble metal nano-core, a raman signal molecule layer, a protective shell layer and a specific binding molecule of an object to be detected from inside to outside in sequence; preferably, the raman nanoparticle is bound to an analyte.
Preferably, the particle size of the noble metal nano-core is 20-30 nm.
Preferably, the raw material of the noble metal nano-core is selected from one of gold, silver, ruthenium, rhodium, palladium, osmium, iridium and platinum.
Preferably, the thickness of the Raman signal molecule layer is 0.05-5 nm.
Preferably, the raw material of the raman signal molecular layer is selected from one of p-dimercaptophene, p-toluenesulfonate, 4-acetaminophen, p-aminophenylsulfate, 2-naphthalene thiol and alkynyl sulfide.
Preferably, the thickness of the protective shell layer is 0.1 nm-30 nm.
Preferably, the raw material of the protective shell layer is selected from one of gold, silver, silicon dioxide, polyethylene glycol, polystyrene, polyacrylonitrile, polydopamine and derivatives thereof.
In certain embodiments, the oily solvent has a density greater than that of water.
Preferably, the oily solvent is selected from one of dichloromethane, chloroform and carbon tetrachloride.
In certain embodiments, the promoter is an ionic salt having an opposite charge to the raman nanoparticle.
Preferably, the accelerator is selected from tetrabutylammonium nitrate.
The second aspect of the invention protects the use of a raman nanoparticle thinning treatment method as described above in any one of the following:
The method is used for improving the stability of Raman spectrum signals of the Raman nano particles;
the method is used for improving the Raman spectrum signal intensity of the Raman nano particles;
for biomarker detection based on raman spectroscopic signals.
The third aspect of the invention provides a method for detecting a biomarker based on surface-enhanced raman spectroscopy, comprising the following steps:
1) Combining a sample to be detected, an excessive amount of Raman nano particles coated with a targeting molecule A and an excessive amount of magnetic nano particles coated with a targeting molecule B in a lysate, and separating magnetic substances to obtain a magnetic mixture I, wherein the targeting molecule B and the targeting molecule A can specifically bind with a biomarker in the sample to be detected and are suitable for forming a sandwich complex with the biomarker;
2) Adjusting the pH value of the magnetic mixture I by adopting a reagent A to enable the sandwich compound to be dissociated, separating and removing magnetic substances to obtain a non-magnetic mixture II, wherein the non-magnetic mixture II at least comprises Raman nano particles coated with the targeting molecule A;
3) Adjusting the pH value of the nonmagnetic mixture II by adopting a reagent B, enabling the Raman nano particles coated with the targeting molecule A to have the same charge, adding an oily solvent and an accelerator for mixing, forming a self-assembled Raman nano particle thin layer on an oil-water interface, enriching the Raman nano particle thin layer, and transferring the Raman nano particle thin layer to a solid phase substrate;
4) And (3) carrying out signal acquisition on the Raman nano particle thin layer on the solid-phase substrate by using a Raman spectrometer, and carrying out qualitative analysis or quantitative analysis.
In certain embodiments, in 1), the biomarker is selected from one or more of a protein, a nucleic acid, and an enzyme.
In certain embodiments, 1), the targeting molecule B and targeting molecule a specifically bind to different sites of the biomarker.
In certain embodiments, in 1), the separation is performed using magnetic attraction of a magnetic rod.
In certain embodiments, 1) the raman nanoparticle coated with the targeting molecule a comprises, from inside to outside, a noble metal nano-core, a raman signal molecule layer, a protective shell layer and the targeting molecule a, wherein the raman nanoparticle coated with the targeting molecule a is hydrophilic.
In certain embodiments, in 2), the agent a is an aqueous solution. Preferably, the reagent a is selected from pH buffers.
In certain embodiments, in 2), the pH is from 1 to 10.
In certain embodiments, 3), the reagent B is an aqueous solution. Preferably, the reagent B is selected from pH buffers.
In certain embodiments, in 3), the pH is from 1 to 10.
In certain embodiments, in 3), the promoter is an ionic salt having an opposite charge to the raman nanoparticle.
In certain embodiments, the accelerator is selected from tetrabutylammonium nitrate.
In certain embodiments, in 3), the oily solvent has a density greater than that of water.
In certain embodiments, the oily solvent is selected from one of dichloromethane, chloroform, and carbon tetrachloride.
Preferably, the oily solvent is selected from one of dichloromethane and chloroform.
In certain embodiments, 1) the raman nanoparticle coated with the targeting molecule a comprises, in order from inside to outside, a noble metal nanocore, a raman signal molecule layer, a protective shell layer and the targeting molecule a, the raman nanoparticle being hydrophilic.
Preferably, the particle size of the noble metal nano-core is 20-30 nm.
More preferably, the raw material of the noble metal nano-core is selected from one of gold, silver, ruthenium, rhodium, palladium, osmium, iridium and platinum.
Preferably, the thickness of the Raman signal molecule layer is 0.05-5 nm.
More preferably, the raw material of the raman signal molecular layer is selected from one of p-dimercaptophene, p-tolylthiophenol, 4-acetaminophen thiophenol, p-aminophenylthiophenol, 2-naphthalene thiol, and alkynyl sulfide. Specifically, the alkynyl sulfide is alkynyl polyethylene glycol sulfhydryl.
Preferably, the thickness of the protective shell layer is 0.1 nm-30 nm.
More preferably, the raw material of the protective shell layer is selected from one of gold, silver, silicon dioxide, polyethylene glycol, polystyrene, polyacrylonitrile, polydopamine and derivatives thereof.
Preferably, the targeted molecule a is linked to the protective shell by a chemical bond. More preferably, the targeting molecule a is attached to the protective shell via a thiol group by linking the carboxyl group of the carboxyl polyethylene glycol thiol group (COOH-PEG-SH).
In certain embodiments, in 1), the magnetic nanoparticle coated with targeting molecule B comprises a magnetic core and targeting molecule B in that order from inside to outside.
Preferably, the particle size of the magnetic particles coated with the targeting molecule B is 50 nm-1000 nm.
Preferably, the magnetic core is obtained by coating silica or polystyrene with nano ferroferric oxide particles.
Preferably, the targeting molecule B is linked to the magnetic core by a chemical bond. More preferably, the targeting molecule B is linked to the magnetic core via an amide bond.
In certain embodiments, the biomarker is selected from one or more of a protein, a nucleic acid, and an enzyme.
In certain embodiments, both targeting molecule B and targeting molecule a are capable of specifically binding to the biomarker.
The fourth aspect of the present invention provides a container suitable for enriching a raman nanoparticle thin layer, comprising a tube body, wherein the tube body comprises an upper tube body and a lower tube body, the upper tube body and the lower tube body are connected into an integrated structure, the upper tube body is provided with an opening, and the tube body further comprises a piston body slidably arranged in the lower tube body so as to move up and down along the inner wall of the tube body;
the inner wall of the lower pipe body is provided with a lipophilic layer; along the export direction, the upper tube body includes first portion and second portion, the inner wall of first portion is equipped with the lipophilicity layer, the inner wall of second portion is equipped with the hydrophilicity layer.
In certain embodiments, the hydrophilic layer is formed by washing the inner wall of the tube with a piranha solution. Specifically, the piranha solution is prepared by diluting 100 times of concentrated sulfuric acid and diluting 10 times of 30% hydrogen peroxide in a mass ratio of 5:1.
In certain embodiments, the oleophilic layer is formed by coating the inner wall of the tube with a silane coupling agent. The silane coupling agent is 1H, 2H-perfluoro decyl triethoxysilane.
In some embodiments, the upper tube body is inverted horn-shaped.
In some embodiments, the upper tube is made of glass.
In a fifth aspect, the present invention provides a kit for detecting a biomarker based on surface-enhanced raman spectroscopy, comprising:
The target molecule A and the target molecule B can be specifically combined with the biomarker, and are suitable for forming a sandwich compound with the biomarker, and the binding force between the target molecule B and the biomarker is smaller than that between the target molecule A and the biomarker.
In certain embodiments, the raman nanoparticle coated with the targeting molecule a comprises a noble metal nano-core, a raman signal molecule layer, a protective shell layer and the targeting molecule a from inside to outside in sequence, and the raman nanoparticle coated with the targeting molecule a is hydrophilic.
Preferably, the particle size of the noble metal nano-core is 20-30 nm.
More preferably, the raw material of the noble metal nano-core is selected from one of gold, silver, ruthenium, rhodium, palladium, osmium, iridium and platinum.
Preferably, the thickness of the Raman signal molecule layer is 0.05-5 nm.
More preferably, the raw material of the raman signal molecular layer is selected from one of p-dimercaptophene, p-tolylthiophenol, 4-acetaminophen thiophenol, p-aminophenylthiophenol, 2-naphthalene thiol and alkynyl sulfide. Specifically, the alkynyl sulfide is alkynyl polyethylene glycol sulfhydryl.
Preferably, the thickness of the protective shell layer is 0.1 nm-30 nm.
More preferably, the raw material of the protective shell layer is selected from one of gold, silver, silicon dioxide, polyethylene glycol, polystyrene, polyacrylonitrile, polydopamine and derivatives thereof.
Preferably, the targeting molecule a is linked to the protective shell by a chemical bond. More preferably, the targeting molecule a is attached to the protective shell via a thiol group by linking the carboxyl group of the carboxyl polyethylene glycol thiol group (COOH-PEG-SH).
In certain embodiments, the magnetic nanoparticle coated with targeting molecule B comprises a magnetic core and targeting molecule B in that order from the inside out.
Preferably, the particle size of the magnetic particles coated with the targeting molecule B is 50 nm-1000 nm.
Preferably, the magnetic core is obtained by coating silicon dioxide or polystyrene with nano ferroferric oxide particles.
Preferably, the targeting molecule B is linked to the magnetic core by a chemical bond. More preferably, the targeting molecule B is linked to the magnetic core via an amide bond.
In certain embodiments, the kit further comprises one or more of a dissociation reagent, a particle charge adjustment reagent, an oily solvent, an accelerator, a thin layer enrichment vessel.
Preferably, the dissociating agent is selected from agent a. More preferably, the reagent a is selected from pH buffers.
Preferably, the particulate charge modulating agent is selected from agent B; preferably, the reagent B is selected from pH buffers.
Preferably, the oily solvent is selected from one of dichloromethane, chloroform and carbon tetrachloride.
Preferably, the accelerator is selected from tetrabutylammonium nitrate.
A sixth aspect of the invention provides the use of a kit as described above in the preparation of a test product.
Compared with the prior art, the invention has the following beneficial effects:
1) According to the method for detecting the biomarker based on the surface-enhanced Raman spectrum, through the technical route of specific combination of the biomarker and 2 excessive Raman nanoparticles coated with the targeting molecule A and the magnetic nanoparticles coated with the targeting molecule B, magnetic rod separation and adjustment of the pH value of the aqueous phase solution, the sandwich compound is dissociated, the fact that the Raman nanoparticles are combined with the biomarker only in the presence of the biomarker is ensured, the quantitative relation between the biomarker and the Raman nanoparticles is in linear correlation, and the problem that non-specific residues occur to the Raman nanoparticles in the prior art, and false positives are avoided. Further, by forming the Raman nanoparticle thin layer in a uniformly dispersed state, the intervals among the particles are the same, and aggregation of Raman nanoparticles is avoided, so that detection light irradiates any position of the Raman nanoparticle thin layer to obtain the same signal, and stability of Raman signals is ensured. By further enriching and transferring the Raman nano particle thin layer onto the solid-phase substrate, the concentration of the Raman nano particles in unit area is improved, and then the detection signal intensity and the detection sensitivity are improved. The specific detection of the biomarker is realized by designing specific targeting molecules.
2) The method for detecting the biomarker based on the surface enhanced Raman spectrum is simple and easy to implement, has outstanding sensitivity, accuracy, reproducibility and specificity, and has great application potential in the field of biological detection. The stability and the detection sensitivity of the signal are obviously improved, wherein the lowest detection limit of the biomarker is lower than 0.5fg/mL, and the linear relation between the concentration of the biomarker and the signal intensity is good; the signal curves of the 50 detection points in the detection area are well overlapped. Furthermore, the minimum detection limit of the present application is reduced by 2 orders of magnitude compared to a container without the enriched raman nanoparticle thin layer.
Drawings
Fig. 1 shows a schematic flow chart of a method for detecting biomarkers based on surface enhanced raman spectroscopy according to the invention.
Fig. 2 shows a schematic partial flow diagram of a method of detecting biomarkers based on surface enhanced raman spectroscopy according to the invention.
The labels in fig. 2 are as follows:
1 biomarker
2 Raman nanoparticle coated with targeting molecule A
3 magnetic nanoparticles coated with targeting molecule B
4 magnetic bar
6 oily solvent
7 promoter
8 Raman nanoparticle thin layer
9 thin layer suitable for enriching Raman nano particle
Fig. 3 shows a schematic diagram of raman nanoparticle thin layer formation by enrichment, transfer in the method of detecting biomarkers based on surface enhanced raman spectroscopy according to the invention.
The labels in fig. 3 are as follows:
8 Raman nanoparticle thin layer
931 first part
932 second portion
Fig. 4 shows a schematic structural diagram of a container suitable for enriching a thin layer of raman nanoparticles according to the present invention.
The labels in fig. 4 are as follows:
91 piston body
92 lower pipe body
93 upper pipe body
931 first part
932 second portion
Fig. 5 shows a schematic structural diagram of raman nanoparticle coated with targeting molecule a according to the present invention.
The labels in fig. 5 are as follows:
21 noble metal nanonucleus
22 Raman signal molecules
23 protective housing
24 targeting molecule A
Fig. 6 shows a schematic structural diagram of a magnetic nanoparticle coated with a targeting molecule B according to the present invention.
The labels in fig. 6 are as follows:
31 magnetic core
32 targeting molecule B
Fig. 7 shows a schematic structural diagram of a biomarker of the present invention.
The labels in fig. 7 are as follows:
11 target a
12 target b
Fig. 8 shows a schematic structural diagram of a sandwich complex of raman nanoparticle coated with targeting molecule a, biomarker and magnetic particle coated with targeting molecule B according to the present invention.
The labels in fig. 8 are as follows:
5 biomarkers
Detailed Description
Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described by the following specific examples.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. The test methods in the following examples, in which specific conditions are not noted, are generally conducted under conventional conditions or under conditions recommended by the respective manufacturers.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
As shown in fig. 2, one of the objects of the present invention is to provide a raman nanoparticle thinning treatment method, which comprises the following steps:
1) Dissolving the Raman nano particles 2 in an aqueous phase, wherein the Raman nano particles 2 are provided with Raman signal molecules, and can generate Raman spectrum signals under the irradiation of laser;
2) Adding an oily solvent 6 and an accelerator 7, forming a self-assembled Raman nanoparticle thin layer 8 on an oil-water interface, enriching the Raman nanoparticle thin layer 8, and transferring the Raman nanoparticle thin layer to a solid-phase substrate.
In some embodiments, the raman nanoparticle 2 includes, from inside to outside, a noble metal nano-core 21, a raman signal molecule layer 22, a protective shell layer 23, and an analyte specific binding molecule 24. Preferably, the raman nanoparticle 2 is bound to the analyte 1.
Preferably, the noble metal nano-core 21 has a particle diameter of 20 to 30nm.
Preferably, the raw material of the noble metal nano-core is selected from one of gold, silver, ruthenium, rhodium, palladium, osmium, iridium and platinum.
Preferably, the thickness of the raman signal molecule layer 22 is 0.05 to 5nm.
Preferably, the raw material of the raman signal molecular layer is selected from one of p-dimercaptophene, p-toluenesulfonate, 4-acetaminophen, p-aminophenylsulfate, 2-naphthalene thiol and alkynyl sulfide.
Preferably, the thickness of the protective shell layer 23 is 0.1nm to 30nm.
Preferably, the raw material of the protective shell layer is selected from one of gold, silver, silicon dioxide, polyethylene glycol, polystyrene, polyacrylonitrile, polydopamine and derivatives thereof.
In certain embodiments, the oily solvent 6 has a density greater than that of water.
Preferably, the oily solvent is selected from one of dichloromethane, chloroform and carbon tetrachloride.
In certain embodiments, the promoter 7 is an ionic salt having an opposite charge to the raman nanoparticle.
Preferably, the accelerator is selected from tetrabutylammonium nitrate.
It is a second object of the present invention to provide the use of the raman nanoparticle thin layer treatment method as described above in any one of the following:
the method is used for improving the stability of Raman spectrum signals of the Raman nano particles;
the method is used for improving the Raman spectrum signal intensity of the Raman nano particles;
for biomarker detection based on raman spectroscopic signals.
As shown in the flowcharts of fig. 1, 2 and 3, the third objective of the present invention is to provide a method for detecting a biomarker based on surface-enhanced raman spectroscopy. The method of the invention comprises the following steps:
1) Combining a sample to be detected, an excessive amount of Raman nano particles 2 coated with a targeting molecule A and an excessive amount of magnetic nano particles 3 coated with a targeting molecule B in a lysate, and separating magnetic substances to obtain a magnetic mixture I, wherein the targeting molecule B and the targeting molecule A can specifically bind with a biomarker 1 in the sample to be detected and are suitable for forming a sandwich compound with the biomarker;
2) Adjusting the pH value of the magnetic mixture I by adopting a reagent A to enable the sandwich compound to be dissociated, separating and removing magnetic substances to obtain a non-magnetic mixture II, wherein the non-magnetic mixture II at least comprises Raman nano particles coated with the targeting molecule A; the method comprises the steps of carrying out a first treatment on the surface of the
3) Adjusting the pH value of the nonmagnetic mixture II by adopting a reagent B, enabling the Raman nano particles coated with the targeting molecule A to have the same charge, adding an oily solvent and an accelerator for mixing, forming a self-assembled Raman nano particle thin layer 8 on an oil-water interface, enriching the Raman nano particle thin layer 8, and transferring the Raman nano particle thin layer 8 to a solid phase substrate;
4) And (3) carrying out signal acquisition on the Raman nano particle thin layer on the solid-phase substrate by using a Raman spectrometer, and carrying out qualitative analysis or quantitative analysis.
In the invention, in the step 2), after the pH value is regulated by adopting the reagent A, the sandwich compound is dissociated into a combination body formed by the Raman nano-particles coated with the targeting molecule A and the magnetic nano-particles coated with the biomarker and the targeting molecule B, or the sandwich compound is dissociated into three substances such as the Raman nano-particles coated with the targeting molecule A, the magnetic nano-particles coated with the biomarker and the targeting molecule B.
In the present invention, in the step 2), the pH is 1 to 10.
In the present invention, in step 2), the reagent a is an aqueous solution. Preferably, the reagent a is selected from pH buffers.
In the present invention, in step 3), the pH is 1 to 10.
In the present invention, in step 3), the reagent B is an aqueous phase solution. Preferably, the reagent B is selected from pH buffers.
In the present invention, in step 3), the density of the oily solvent is greater than that of water. Preferably, the oily solvent is selected from one of dichloromethane, chloroform and carbon tetrachloride.
In the present invention, in step 3), the promoter is an ionic salt having an opposite charge to the raman nanoparticle coated with the targeting molecule a. Preferably, the accelerator is selected from tetrabutylammonium nitrate.
In the invention, in the step 1) or the step 2), the separation is carried out by magnetic attraction of a magnetic rod. If the magnetic rod 4 is used for separation, the magnetic rod 4 is sleeved with a magnetic sleeve made of high polymer materials, and the magnetic attraction and the demagnetizing attraction are realized through combination of the magnetic rod and the magnetic sleeve or separation.
In a specific embodiment, in step 1), the magnetic rod 4 is used to promote sufficient contact of the biomarker 1, the raman nanoparticle 2 coated with the targeting molecule a, and the magnetic nanoparticle 3 coated with the targeting molecule B to form a sandwich complex 5 formed by the raman nanoparticle, the biomarker and the magnetic nanoparticle. According to the method, the magnetic rod 4 vibrates up and down around the mixed solution in a reciprocating mode, so that magnetic substances in the mixed solution are driven to continuously move, and the system is promoted to rapidly and fully conduct mass transfer and reaction. The schematic structure of the sandwich complex 5 is shown in fig. 8. The biomarker is located between the raman nanoparticle coated with targeting molecule a and the magnetic nanoparticle coated with targeting molecule B.
The sandwich complex 5 is formed by the following principle: targeting molecule a 24 in raman nanoparticle 2 coated with targeting molecule a specifically binds to target 12 of biomarker 1, and targeting molecule B32 in magnetic nanoparticle 3 coated with targeting molecule B specifically binds to target 11 of biomarker 1.
The method for separating by using the magnetic rod 4 comprises the following steps: the magnetic rod 4 covered with the magnetic sleeve is placed in a reaction vessel to extract the magnetic mixture I by magnetic attraction, then the magnetic mixture I is transferred to a new vessel, and then the magnetic sleeve and the magnetic rod 4 are separated so that the magnetic mixture I is transferred to the new vessel, thereby separating the magnetic mixture I.
In a specific embodiment, in step 2), the reagent a is used to adjust the pH value of the magnetic mixture I, so as to promote the separation of the sandwich compound 5, and the magnetic rod 4 is used to separate and remove the magnetic material in the system, so as to obtain a non-magnetic mixture II.
The method for separating and removing the magnetic substances by using the magnetic rod 4 comprises the following steps: the magnetic rod 4 covered with the magnetic sleeve is adopted to be placed in a system, and then the magnetic substance is transferred to the outside of the container through the magnetic rod 4 covered with the magnetic sleeve. The magnetic sheath and magnetic rod are then separated so that the magnetic substance falls outside the container.
In the invention, in the step 3), the pH value of the nonmagnetic mixture II is regulated by adopting a reagent B, so that the Raman nano particles 2 coated with the targeting molecule A have the same charge (such as negative electricity or positive electricity), an oily solvent and an accelerator are added for mixing, an oil-water interface forms a self-assembled Raman nano particle thin layer after standing, the Raman nano particle thin layer is enriched, and the Raman nano particle thin layer is transferred onto a solid-phase substrate.
As shown in fig. 2 and 3, the raman nanoparticle 2 coated with the targeting molecule a with the same charge is transferred to a container for enriching the oil-water interface particle thin layer, an oily solvent and an accelerator are added for mixing, a self-assembled raman nanoparticle thin layer is formed on the oil-water interface, the raman nanoparticle thin layer is enriched, and the raman nanoparticle thin layer is transferred to a solid phase substrate.
As shown in fig. 4, a container suitable for enriching a thin layer of raman nanoparticles is provided for the fourth object of the present invention. The container comprises a pipe body, wherein the pipe body comprises an upper pipe body 93 and a lower pipe body 92, the upper pipe body 93 and the lower pipe body 92 are connected into an integrated structure, the upper pipe body 93 is provided with an opening, and the pipe body also comprises a piston body 91 which is slidably arranged in the lower pipe body 92 so as to move up and down along the inner wall of the pipe body; the inner wall of the lower pipe body 92 is provided with a lipophilic layer; along the outlet direction, the upper tube 93 includes a first portion 931 and a second portion 932, wherein an inner wall of the first portion 931 is provided with a hydrophilic layer, and an inner wall of the second portion 932 is provided with a hydrophilic layer. Preferably, the upper tube 93 has an inverted horn shape. Preferably, the upper tube 93 is made of glass.
As shown in fig. 2 and 3, a schematic flow chart of forming a self-assembled thin raman nanoparticle layer and enriching the thin raman nanoparticle layer using a container suitable for enriching the thin raman nanoparticle layer is provided. Adding an oily solvent and an accelerator to the container and oscillating, wherein the raman nano particles are selectively accumulated on the lower tube 92 of the container, and a thin layer of the raman nano particles is formed at the oil-water interface, moving the piston body 91 to enable the oil-water mixture to move towards the upper tube 93, and sequentially passing through the first part 931 to finally reach the second part 932, so that the thin layer of the raman nano particles is densely attached on the second part 932 of the container. The detection signal is collected from the second portion 932 by using a raman spectrometer, and qualitative analysis or quantitative analysis is performed.
In the invention, the Raman nano particles 2 with one Raman signal molecule 22 can be specifically combined with one biomarker 1, and the Raman nano particles 2 with different Raman signal molecules 22 can be respectively specifically combined with a plurality of biomarkers 1 to realize one-time detection of a plurality of indexes, thereby improving the detection efficiency or accuracy.
As shown in fig. 7, a schematic structural diagram of the biomarker of the present invention is shown. The biomarker 1 is selected from one or more of a protein, a nucleic acid, and an enzyme. Preferably, the biomarker 1 comprises a target 11 and a target 12. Preferably, the biomarker 1 is selected from proteins. In a particular embodiment, is SARS-CoV-2 spike protein.
The specific targeting molecule A is SARS-CoV-2 spike protein antibody I (Anti-NTD Ab), the specific targeting molecule B is SARS-CoV-2 spike protein antibody II (Anti-RBD Ab), the SARS-CoV-2 spike protein antibody I and the SARS-CoV-2 spike protein antibody II respectively bind to different sites of SARS-CoV-2 spike protein (NTD domain in S1 subunit and RBD domain in S1 subunit).
As shown in fig. 5, the structure of the raman nanoparticle coated with the targeting molecule a according to the present invention is schematically shown. The Raman nanoparticle 2 coated with the targeting molecule A sequentially comprises a noble metal nano-core 21, a Raman signal molecule layer 22, a protection shell layer 23 and the targeting molecule A24 from inside to outside. The particle size of the Raman nanoparticle 2 coated with the targeting molecule A is 20-100 nm, the size is adjustable, and the Raman nanoparticle is hydrophilic.
In the present invention, the noble metal nano-core 21 is a regular spherical nano-structure, and is made of one of gold, silver and platinum group metals (ruthenium, rhodium, palladium, osmium, iridium and platinum). In a specific embodiment, gold is 25nm in diameter.
In the present invention, the raw material of the raman signal molecular layer 22 is selected from one of p-dimercaptophenone, p-tolylthiophenol, 4-acetaminophen thiophenol, p-aminophenylthiophenol, 2-naphthalene thiol, and sulfide containing alkynyl group (e.g., alkynyl polyethylene glycol mercapto group, etc.). The raman signal molecule layer 22 is uniformly arranged between the noble metal nano-core 21 and the protective shell layer 23 by a self-assembled monolayer state from raw materials (raman signal molecules). Since only one layer is arranged on the noble metal nano-core 21, the raman signal molecular layer 22 presents high spatial symmetric distribution and isotropy. In a specific embodiment, the thickness of p-dimercaptobenzene is 0.8nm.
In the invention, the protective shell layer 23 is a regular spherical shell-shaped nano structure, and the thickness of the protective shell layer 23 is 0.1 nm-30 nm. The protective shell layer in the application cannot be too thick or too thin, otherwise the optical signal transmission is affected. The raw materials of the protective shell are selected from one of gold, silver, silicon dioxide and organic matters (such as polyethylene glycol, polystyrene, polyacrylonitrile, polydopamine and derivatives thereof). In a specific embodiment, gold is 16nm thick.
In the present invention, the targeting molecule a 24 is connected to the protective shell 23 through a chemical bond. Preferably, the targeting molecule a is attached to the protective shell 23 via a thiol group by linking the carboxyl group of the carboxyl polyethylene glycol thiol group (COOH-PEG-SH). The connection mode between the targeting molecule a 24 and the protective shell 23 in the present application is not limited to the one provided in the present application, and any connection mode between the targeting molecule a 24 and the protective shell 23 can be realized in the prior art.
As shown in fig. 6, the magnetic nanoparticle coated with the targeting molecule B of the present invention is schematically shown in structure. The magnetic nanoparticle 3 coated with the targeting molecule B sequentially comprises a magnetic inner core 31 and the targeting molecule B32 from inside to outside. The diameter of the magnetic nano particles 3 coated with the targeting molecule B is 50 nm-1000 nm, and the size is adjustable.
In the present invention, the magnetic core 31 is a magnetic particle prepared by coating silica (or polystyrene coated by emulsion polymerization) by chemical coprecipitation (to obtain nano ferroferric oxide particles) to sol-gel method, and one of hydroxyl, carboxyl or amino connecting groups is modified on the surface of the magnetic particle.
In the present invention, the targeting molecule B32 is connected to the magnetic core 31 through a chemical bond. Preferably, the targeting molecule B32 is linked to the magnetic core 31 by an amide bond.
The invention aims to provide a kit for detecting a biomarker based on surface-enhanced Raman spectroscopy, which comprises the following components:
the target molecule A and the target molecule B can be specifically combined with the biomarker, and are suitable for forming a sandwich compound with the biomarker, and the binding force between the target molecule B and the biomarker is smaller than that between the target molecule A and the biomarker.
In the invention, the Raman nanoparticle 2 coated with the targeting molecule A sequentially comprises a noble metal nano-core 21, a Raman signal molecule layer 22, a protection shell layer 23 and the targeting molecule A24 from inside to outside, and the Raman nanoparticle 2 coated with the targeting molecule A is hydrophilic. Preferably, the noble metal nano-core 21 has a particle diameter of 20 to 30nm. More preferably, the raw material of the noble metal nano-core 21 is selected from one of gold, silver, ruthenium, rhodium, palladium, osmium, iridium, and platinum. Preferably, the thickness of the raman signal molecule layer 22 is 0.05 to 5nm. More preferably, the raman signal molecular layer 22 is made of one selected from the group consisting of p-dimercaptophenone, p-tolylthiophenol, 4-acetaminophen thiophenol, p-aminophenylthiophenol, 2-naphthalene thiol, and alkynyl sulfide. Specifically, the alkynyl sulfide is alkynyl polyethylene glycol sulfhydryl. Preferably, the thickness of the protective shell layer 23 is 0.1nm to 30nm. More preferably, the raw material of the protective shell layer 23 is selected from one of gold, silver, silicon dioxide, polyethylene glycol, polystyrene, polyacrylonitrile, polydopamine and derivatives thereof. Preferably, the targeting molecule a 24 is attached to the protective shell by a chemical bond. More preferably, the targeting molecule a is attached to the protective shell via a thiol group by linking the carboxyl group of the carboxyl polyethylene glycol thiol group (COOH-PEG-SH). The connection mode between the targeting molecule a 24 and the protective shell 23 in the present application is not limited to the one provided in the present application, and any connection mode between the targeting molecule a 24 and the protective shell 23 can be realized in the prior art.
In the invention, the magnetic nanoparticle 3 coated with the targeting molecule B sequentially comprises a magnetic inner core 31 and the targeting molecule B32 from inside to outside. Preferably, the particle size of the magnetic core 31 is 50nm to 1000nm, and the size is adjustable. Preferably, the magnetic core 31 is obtained by coating silica or polystyrene with nano ferroferric oxide particles. Preferably, the targeting molecule B32 is linked to the magnetic core 31 by a chemical bond. More preferably, the targeting molecule B32 is linked to the magnetic core 31 via an amide bond. In one embodiment, the magnetic core 31 is a magnetic particle prepared by coating silica (or polystyrene coated by emulsion polymerization) by chemical coprecipitation (to obtain nano ferroferric oxide particles), and modifying the surface thereof with one of hydroxyl, carboxyl or amino groups.
In the invention, the kit further comprises one or more of a dissociation reagent, a particle charge adjustment reagent, an oily solvent, an accelerator and a thin-layer enrichment container. The dissociation reagent is selected from reagent A, so that the Raman nano-particles coated with the targeting molecule A in the sandwich complex are dissociated from the biomarker; preferably, the reagent a is selected from pH buffers. The particle charge regulating reagent is selected from a reagent B, so that the dissociated Raman nano particles coated with the targeting molecule A have the same charge, and the same charge repels each other to ensure that the interval arrangement among the particles is uniform; preferably, the reagent B is selected from pH buffers. The oily solvent is selected from one of dichloromethane, chloroform and carbon tetrachloride, and the promoter is selected from tetrabutylammonium nitrate, so that the Raman nano particles coated with the targeting molecule A in the water phase are positioned on the upper surface of an oil-water interface, and the Raman nano particles are changed from a state of bulk distribution into a state of bulk distribution at the oil-water interface.
A fifth aspect of the invention provides the use of a kit as described above in the preparation of a test product.
Example 1
As shown in fig. 4, a container suitable for enriching raman nano-particle thin layers comprises a pipe body, wherein the pipe body comprises an upper pipe body 93 and a lower pipe body 92, the upper pipe body 93 and the lower pipe body 92 are connected into an integrated structure, the upper pipe body 93 is provided with an opening, and the pipe body also comprises a piston body 91 which is slidably arranged in the lower pipe body 92 so as to move up and down along the inner wall of the lower pipe body 92; the inner wall of the lower pipe body 92 is provided with a lipophilic layer; along the outlet direction, the upper tube 93 includes a first portion 931 and a second portion 932, wherein an inner wall of the first portion 931 is provided with a hydrophilic layer, and an inner wall of the second portion 932 is provided with a hydrophilic layer. According to the method, by arranging the oil phase liquid, the water phase liquid, the hydrophilic Raman nano particles and the accelerator system, the Raman nano particles in the liquid are converted from a state of distribution of one body to a state of distribution of one surface at an oil-water interface, and the first enrichment of the Raman nano particles is realized. Pushing the piston body 91 makes the oil-water mixture in the container move from the lower pipe body 92 to the upper pipe body 93, and the inner wall of the lower pipe body 92 is provided with a lipophilic layer, so that the area behind the raman particulate matter thin layer at the oil-water interface enters the first part 931 of the upper pipe body is gradually narrowed, and the second enrichment of the raman nano particles is realized. And the piston is continuously pushed to move towards the outlet, and after the Raman particle thin layer positioned at the oil-water interface enters the second part 932 of the upper pipe body, the Raman nanoparticle thin layer is compactly attached to the inner wall of the second part 932 of the container, so that the transfer from the liquid phase substrate to the solid phase substrate is realized, and a stable compact particle thin layer to-be-detected state is formed.
In some embodiments, the upper tube 93 is made of glass.
In some embodiments, the lower tube 92 is made of glass.
In some embodiments, the upper tube 93 is inverted horn-shaped. The inverted horn-shaped upper pipe body is beneficial to the concentration of the particle thin layer at the oil-water interface in the process of moving to the upper part due to the continuous reduction of the area.
In certain embodiments, the hydrophilic layer is formed by washing the inner wall of the tube with a piranha solution.
In certain embodiments, the oleophilic layer is formed by coating the inner wall of the tube with a silane coupling agent. The silane coupling agent is 1H, 2H-perfluoro decyl triethoxysilane.
In certain preferred embodiments, the ratio of the diameter of the lower tube 92 to the diameter at the interface of the first portion 9331 and the second portion 392 is (2-20): 1.
Example 2
In this example, a novel coronavirus (SARS-CoV-2 spike protein) was used as a biomarker, and a Raman nanoparticle thin layer was formed by using the container for enriching the oil-water interface particle thin layer of example 1, and the novel coronavirus was detected.
The probe combination based on the surface-enhanced Raman nano particles comprises: a Raman nanoparticle marked by a specific targeting molecule A and a magnetic nanoparticle marked by a specific targeting molecule B, wherein the specific targeting molecule A is a SARS-CoV-2 spike protein antibody I (Anti-NTD Ab), the specific targeting molecule B is a SARS-CoV-2 spike protein antibody II (Anti-RBD Ab), and the SARS-CoV-2 spike protein antibody I and the SARS-CoV-2 spike protein antibody II respectively specifically bind different sites of the SARS-CoV-2 spike protein (NTD domain in S1 subunit and RBD domain in S1 subunit).
The Raman nanoparticle coated with the targeting molecule A sequentially comprises a noble metal nano core, a Raman molecule layer, a protection shell layer and the targeting molecule A from inside to outside, wherein the noble metal is gold, the diameter of the noble metal nano core is 25nm, the raw material of the Raman molecule layer is p-dimercaptobenzene, the thickness of the Raman molecule layer is 0.8nm, the raw material of the protection shell is gold, the thickness of the protection shell is 16nm, and the targeting molecule A is connected with the gold of the protection shell under the EDC+NHS effect through SH-PEG-COOH. (preparation of Raman nanoparticles see Gandra N, singamannei S (2013) bilayed Raman-intense gold nanostructures with hidden tags (BRIGHTs) for high-resolution biological Mater 25 (7): 1022-1027).
The magnetic particles coated with the targeting molecule B comprise a magnetic inner core and the targeting molecule B, wherein the magnetic inner core is carboxyl magnetic beads with the diameter of 300nm, and the targeting molecule B is connected with the magnetic inner core under the action of excessive EDC and NHS.
The preparation method of the magnetic core comprises the following steps: the silica is coated by a chemical coprecipitation method (nano ferroferric oxide particles are obtained) to a sol-gel method. (preparation of magnetic core see CN 1923857)
The detection method of the novel coronavirus comprises the following steps:
1) Sample 1 (nasal swab) and lysate (manufacturer: mixing a PBS buffer solution and triton X-100 serving as main components to release a biomarker (SARS-CoV-2 spike protein), then adding the Raman nanoparticle 2 coated with the targeting molecule A in excess and the magnetic nanoparticle 3 coated with the targeting molecule B in excess to form a mixed solution, and utilizing a magnetic rod 4 to reciprocate up and down around the mixed solution to promote the full contact effect of the biomarker, the Raman nanoparticle and the magnetic nanoparticle to form a sandwich compound consisting of the Raman nanoparticle, the biomarker and the magnetic nanoparticle; separating the magnetic substance with a magnetic rod 4 to obtain a magnetic mixture I comprising a sandwich complex 5 formed by the raman nanoparticle 2 coated with the targeting molecule a, the biomarker 3 and the magnetic nanoparticle 3 coated with the targeting molecule B and unbound magnetic nanoparticle coated with the targeting molecule B and transferring the magnetic mixture I into a new container.
2) And adding the reagent A to adjust the pH value of the magnetic mixture I to 2.8, promoting the complete dissociation of the sandwich compound 5, and then magnetically attracting and removing all magnetic substances by using the magnetic rod 4 to obtain a non-magnetic mixture II, wherein the magnetic substances comprise unbound magnetic nanoparticles coated with the targeting molecule B and dissociated magnetic nanoparticles. Wherein, the composition of the reagent A is: 0.1mol/L Gly-HCl+0.15mol/L NaCl.
3) And adding a reagent B to adjust the pH value of the nonmagnetic mixture II in the step 2) to 6.8, transferring the nonmagnetic mixture II into a container suitable for enriching the Raman nano particle thin layer, adding an oily solvent and an accelerant, sufficiently oscillating, standing, forming a self-assembled Raman nano particle thin layer 2 at an oil-water interface of a lower pipe body 92, pushing the Raman nano particle thin layer 2 to an upper pipe body 93 through a piston body 91, sequentially passing through a lipophilic layer of a first part 931 and a hydrophilic layer of a second part 932, reducing the area of the Raman nano particle thin layer 2, and realizing transfer of the Raman nano particle thin layer 2 from a liquid phase substrate to a solid phase substrate to form a stable compact particle thin layer to be tested state. Wherein, the composition of the reagent B is as follows: 0.2mol/L KH 2 PO 4 +0.15mol/L NaOH, the oily solvent is dichloromethane, and the accelerator is tetrabutylammonium nitrate.
4) The raman spectrometer is used to align the signal acquisition with the second portion 932 of the container for qualitative and quantitative analysis.
Experimental results show that the lowest detection limit of SARS-CoV-2 spike protein is 0.5fg/mL, and the linear relation between the concentration of the biomarker and the signal intensity is in the range of 0.6 fg/mL-1 ng/mL; the signal curves of 50 detection points in the detection area are well overlapped, the signal curves are well overlapped after being repeated 50 times, and the detection has good signal stability. Furthermore, the minimum detection limit of the present application is reduced by 2 orders of magnitude compared to a container without the enriched raman nanoparticle thin layer.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (14)
1. The Raman nanoparticle lamellar treatment method is characterized by comprising the following steps of:
1) Dissolving Raman nano particles in an aqueous phase, wherein the Raman nano particles are provided with Raman signal molecules, and can generate Raman spectrum signals under laser irradiation;
2) Adding an oily solvent and an accelerator, forming a self-assembled Raman nanoparticle thin layer on an oil-water interface, enriching the Raman nanoparticle thin layer, and transferring the Raman nanoparticle thin layer to a solid-phase substrate.
2. The method of claim 1, wherein the raman nanoparticle comprises, from inside to outside, a noble metal nanocore, a raman signal molecule layer, a protective shell layer, and a specific binding molecule of an analyte; preferably, the raman nanoparticle is bound to an analyte;
and/or the oily solvent has a density greater than that of water;
and/or the promoter is an ionic salt having an opposite charge to the raman nanoparticle.
3. The process of claim 2, comprising at least one of the following features:
1) The promoter is selected from tetrabutylammonium nitrate;
2) The oily solvent is selected from one of dichloromethane, chloroform and carbon tetrachloride;
3) The particle size of the noble metal nano-core is 20-30 nm;
4) The thickness of the Raman signal molecule layer is 0.05-5 nm;
5) The thickness of the protective shell layer is 0.1 nm-30 nm;
6) The raw material of the noble metal nano-core is selected from one of gold, silver, ruthenium, rhodium, palladium, osmium, iridium and platinum;
7) The raw material of the Raman signal molecular layer is selected from one of p-dimercaptophene, p-toluenesulfonic acid, 4-acetaminophen thiophenol, p-aminophenylsulfol, 2-naphthalene thiol and alkynyl sulfide;
8) The raw materials of the protective shell layer are selected from one of gold, silver, silicon dioxide, polyethylene glycol, polystyrene, polyacrylonitrile, polydopamine and derivatives thereof.
4. A use of the raman nanoparticle thin layer treatment method according to any one of claims 1 to 3 in any one of the following:
the method is used for improving the stability of Raman spectrum signals of the Raman nano particles;
the method is used for improving the Raman spectrum signal intensity of the Raman nano particles;
for biomarker detection based on raman spectroscopic signals.
5. A method for detecting a biomarker based on surface-enhanced raman spectroscopy, comprising the steps of:
1) Combining a sample to be detected, an excessive amount of Raman nano particles coated with a targeting molecule A and an excessive amount of magnetic nano particles coated with a targeting molecule B in a lysate, and separating magnetic substances to obtain a magnetic mixture I, wherein the targeting molecule B and the targeting molecule A can specifically bind with a biomarker in the sample to be detected and are suitable for forming a sandwich complex with the biomarker;
2) Adjusting the pH value of the magnetic mixture I by adopting a reagent A so that the sandwich compound is dissociated and separated to remove magnetic substances, and obtaining a non-magnetic mixture II, wherein the non-magnetic mixture II at least comprises Raman nano particles coated with the targeting molecule A;
3) Adjusting the pH value of the nonmagnetic mixture II by adopting a reagent B, enabling the Raman nano particles coated with the targeting molecule A to have the same charge, adding an oily solvent and an accelerator for mixing, forming a self-assembled Raman nano particle thin layer on an oil-water interface, enriching the Raman nano particle thin layer, and transferring the Raman nano particle thin layer to a solid phase substrate;
4) And (3) carrying out signal acquisition on the Raman nano particle thin layer on the solid-phase substrate by using a Raman spectrometer, and carrying out qualitative analysis or quantitative analysis.
6. The method of claim 5, comprising at least one of the following features:
1) Wherein the biomarker is selected from one or more of a protein, a nucleic acid, and an enzyme;
1) In the step of separation, magnetic rod magnetic attraction is adopted for separation;
1) Wherein the Raman nanoparticle coated with the targeting molecule A sequentially comprises a noble metal nano core, a Raman signal molecule layer, a protective shell layer and the targeting molecule A from inside to outside, and the Raman nanoparticle coated with the targeting molecule A is hydrophilic;
1) The magnetic nanoparticle coated with the targeting molecule B sequentially comprises a magnetic inner core and the targeting molecule B from inside to outside;
1) Wherein the targeting molecule B and targeting molecule a specifically bind to different sites of the biomarker;
2) Wherein the pH value is 1-10;
2) Wherein the reagent A is an aqueous phase solution; preferably, the reagent a is selected from pH buffers;
2) The separation is carried out by magnetic attraction of a magnetic rod;
3) Wherein the pH value is 1-10;
3) Wherein the oily solvent has a density greater than that of water;
3) Wherein the promoter is an ionic salt having an opposite charge to the raman nanoparticle coated with the targeting molecule a;
3) Wherein the reagent B is an aqueous phase solution; preferably, the reagent B is selected from pH buffers.
7. The container suitable for enriching the Raman nano particle thin layer comprises a tube body, wherein the tube body comprises an upper tube body (93) and a lower tube body (92), the upper tube body (93) and the lower tube body (92) are connected into an integrated structure, and the upper tube body (93) is provided with an opening, and the container is characterized by further comprising a piston body (91) which is slidably arranged in the lower tube body (92) so as to move up and down along the inner wall of the tube body;
the inner wall of the lower pipe body (92) is provided with a lipophilic layer; along the outlet direction, the upper tube body (93) comprises a first part (931) and a second part (932), the inner wall of the first part (931) is provided with a lipophilic layer, and the inner wall of the second part (932) is provided with a hydrophilic layer.
8. The container according to claim 7, wherein the upper tubular body (93) is of inverted horn shape;
and/or the upper tube body (93) is made of glass;
and/or, the hydrophilic layer is formed by cleaning the inner wall of the tube body with a piranha solution;
and/or, the lipophilic layer is formed by coating a silane coupling agent on the inner wall of the pipe body.
9. A kit for detecting a biomarker based on surface enhanced raman spectroscopy, comprising:
a raman nanoparticle coated with a targeting molecule a and a magnetic nanoparticle coated with a targeting molecule B, both of which are capable of specifically binding to the biomarker and are adapted to form a sandwich complex with the biomarker.
10. The kit of claim 9, wherein the raman nanoparticle coated with the targeting molecule a comprises a noble metal nano-core, a raman signal molecule layer, a protective shell layer and the targeting molecule a in sequence from inside to outside, and the raman nanoparticle coated with the targeting molecule a is hydrophilic;
and/or the magnetic nanoparticle coated with the targeting molecule B sequentially comprises a magnetic inner core and the targeting molecule B from inside to outside.
11. The kit of claim 9, comprising at least one of the following features:
1) The particle size of the noble metal nano-core is 20-30 nm;
2) The raw material of the noble metal nano-core is selected from one of gold, silver, ruthenium, rhodium, palladium, osmium, iridium and platinum;
3) The thickness of the Raman signal molecule layer is 0.05-5 nm;
4) The raw material of the Raman signal molecular layer is selected from one of p-dimercaptophene, p-toluenesulfonic acid, 4-acetaminophen thiophenol, p-aminophenylsulfol, 2-naphthalene thiol and alkynyl sulfide;
5) The thickness of the protective shell layer is 0.1 nm-30 nm;
6) The raw materials of the protective shell layer are selected from one of gold, silver, silicon dioxide, polyethylene glycol, polystyrene, polyacrylonitrile, polydopamine and derivatives thereof;
7) The targeting molecule A is connected with the protective shell through a chemical bond; preferably, the targeting molecule a is linked to the protective shell via a thiol group by linking the carboxyl group of a carboxyl polyethylene glycol thiol group (COOH-PEG-SH);
8) The particle size of the magnetic core is 50 nm-1000 nm;
9) The magnetic core is obtained by coating silicon dioxide or polystyrene with nano ferroferric oxide particles;
10 The targeting molecule B is connected with the magnetic core through a chemical bond; preferably, the targeting molecule B is linked to the magnetic core via an amide bond.
12. The kit of claim 9, further comprising one or more of a dissociation reagent, a particulate charge conditioning reagent, an oily solvent, an accelerator, a lamellar enrichment vessel.
13. The kit of claim 12, wherein the dissociating reagent is selected from reagent a; preferably, the reagent a is selected from pH buffers;
and/or the particulate charge modulating agent is selected from agent B; preferably, the reagent B is selected from pH buffers;
and/or the oily solvent is selected from one of dichloromethane, chloroform and carbon tetrachloride;
and/or the accelerator is selected from tetrabutylammonium nitrate.
14. Use of a kit according to any one of claims 10-13 for the preparation of an assay product.
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