CN108645832B - SERS chip and preparation method and application thereof - Google Patents

SERS chip and preparation method and application thereof Download PDF

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CN108645832B
CN108645832B CN201810240142.3A CN201810240142A CN108645832B CN 108645832 B CN108645832 B CN 108645832B CN 201810240142 A CN201810240142 A CN 201810240142A CN 108645832 B CN108645832 B CN 108645832B
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pits
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CN108645832A (en
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孙海龙
郭清华
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Suzhou Nawei Life Technology Co ltd
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Suzhou Infineon Nanotechnology Co Ltd
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Abstract

The invention provides a SERS substrate and a preparation method and application thereof, wherein the SERS chip comprises a SERS substrate and a plurality of nano-structure units arranged on the surface of the SERS substrate, each nano-structure unit comprises a plurality of pits arranged on the surface of the SERS substrate and a nano-particle aggregate arranged in each pit, each nano-structure unit also comprises a functional molecular layer connected to the surface of the nano-particle aggregate, and each functional molecular layer is used for fixing a molecule to be detected to the surface of the nano-particle aggregate. The invention modifies one or more functional molecules on the substrate, so that the chip has different functions, thereby being applicable to different detection fields. The preparation process of the chip is simple, the efficiency is high, the cost is low, the high-performance SERS chip can be produced on a large scale, and the commercialization requirement can be well met. The SERS chip prepared by the invention has the advantages of high repeatability, uniform hot spot, stable property, large-area growth and high sensitivity.

Description

SERS chip and preparation method and application thereof
Technical Field
The invention relates to a Surface-Enhanced Raman Scattering (SERS) technology, in particular to a preparation method of an SERS chip.
Background
The Raman spectroscopy is a scattering spectrum, and the Raman spectroscopy analysis is an analysis method which is used for analyzing a scattering spectrum with different incident light frequencies based on a Raman scattering effect found by indian scientists c.v. Raman (Raman) to obtain information on molecular vibration and rotation, and is applied to molecular structure research. The technology is widely applied to various fields of chemistry, physics, biology, medicine and the like because of the advantages of rapid, simple, repeatable and nondestructive qualitative and quantitative analysis, and also shows unique advantages in the aspects of pure qualitative analysis, high-degree quantitative analysis and molecular structure determination. The SERS enhancing source comprises a noble metal sol and an enhancing substrate.
In the existing SERS research, researchers are all focused on preparing controllable, repeatable and hot-spot concentrated SERS substrates with metal nanostructures. Such as patent numbers: 201610658664.6, the patent names: a method for preparing ordered silver nanosphere array includes evaporating a silver film with thickness of 10nm on surface of ordered aluminium nanometer bowl OAB array template sample, then vacuum annealing OAB template at 500 deg.C for 1h to obtain ordered silver nanometer array structure. Patent numbers: 201610327475.0, patent name: a large-area surface-enhanced Raman scattering substrate and a preparation method thereof are disclosed, wherein a template with a three-dimensional micron structure is prepared, a layer of silver is evaporated to form silver nanoparticles, then a layer of oxide is evaporated, and a layer of silver is evaporated to obtain a large-area SERS substrate. Patent numbers: 201610929950.1, patent name: a preparation method of an SERS substrate with controllable precious metal nanoparticle spacing comprises the steps of cleaning an AAO template by hydrochloric acid, then obtaining precious metal nanoparticle clusters by a physical or chemical method, and filling the whole AAO template holes. And further leading the AAO template on PMMA, carrying out heat treatment to ensure that the noble metal cluster is immersed in the PMMA, cleaning by hydrochloric acid to remove the AAO template, and drying to obtain the SERS substrate with the noble metal nanoparticles regularly distributed. The method has complicated template transfer and hydrochloric acid cleaning operations, is difficult to realize large-area preparation by transferring AAO to other templates, and has high cost. Few functionalized SERS substrates exist in the prior art.
In addition, at present, an immunoassay method is generally adopted to detect biomolecules, and the immunoassay method requires secondary antibodies or tertiary antibodies, so that material consumption is serious, colored substrates or external markers are required for display, consumables are further increased, and cost is high.
Disclosure of Invention
The invention aims to provide a preparation method of a functionalized SERS chip, which can detect specific target molecules or biomolecules according to artificial design, further construct molecular devices or supermolecule devices and expand the application of SERS in the aspects of special molecules and biomedicine.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention provides a SERS chip which comprises a SERS substrate and a plurality of nano-structure units arranged on the surface of the SERS substrate, wherein each nano-structure unit comprises a plurality of pits arranged on the surface of the SERS substrate and a nano-particle aggregate arranged in each pit, and the nano-structure unit also comprises a functional molecular layer connected to the surface of the nano-particle aggregate, and the functional molecular layer is used for fixing a molecule to be detected to the surface of the nano-particle aggregate.
Preferably, the functional molecule layer is composed of one or more functional molecules, and the functional molecules include at least a first functional group and a second functional group, wherein the first functional group is used for connecting the nanoparticle aggregate, and the second functional group is used for connecting the molecule to be detected.
Further preferably, the first functional group includes-SH, ═ S, -NH2At least one of NH and ≡ N; the second functional group includes SH, ═ S, -NH2、＝NH、≡N-NO2At least one of, -COOH, -OH and alkyl.
By denoting-SH or ═ S as A, -NH2B1, NH B2, and N B3, -NO2C, -COOH, D, -OH, E, alkyl, F, and the remainder of the functional molecule, X.
The functional molecule may be an a-X-a combination: such as 1, 2-ethanedithiol, 1, 6-hexanedithiol, p-mercaptoterephthalic acid, bis (mercaptoacetic) ethylene glycol, bis-mercapto-polyethylene glycol, p-mercaptothiophenol, 2,4, 6-trimercapto-1, 3, 5-triazine, 2-mercaptobenzothiazole, etc.; A-X-B1 combination: such as mercaptoethylamine, mercaptopropylamine, mercaptobutylamine, p-mercaptoaniline, m-mercaptoaniline, orthomercaptoaniline, 5-amino-2-mercaptobenzimidazole, 2-amino-benzothiazole, etc.; A-X-B2 combination: such as 6-mercaptopurine, 8-mercaptoadenine, 2-mercaptobenzimidazole-4-carboxylic acid, methyl 2-mercaptobenzimidazole-4-carboxylate, 5- (1H-pyrrol-1-yl) -2-mercaptobenzimidazole, 5-ethoxy-2-mercaptobenzimidazole, 5-bromo-2-mercaptobenzimidazole, 2-mercaptobenzimidazole-5-sodium sulfonate dihydrate, 3-cyanoacetophenone, 5, 6-dichlorobenzimidazole-2-thiol, 5-chlorobenzimidazole-2-thiol, 2-mercapto-5-methoxybenzimidazole, 4-amino-6-mercaptopyrazole [3,4-d ] pyrimidine, 1, 2-ethylenethiourea, methimazole, 2-mercaptoimidazole, etc.; A-X-B3 combination: such as methylene blue, 2-mercapto-5-methylbenzimidazole, sodium mercaptoimidazolepropanesulfonate, and the like; A-X-C combination: p-nitrothiophenol, 2-mercapto-5-nitrobenzimidazole, and the like; A-X-D combination: thioglycolic acid, mercaptopropionic acid, a-mercaptoisobutyric acid, 4-mercaptobenzoic acid, mercaptoisobutyric acid, 2-mercaptosuccinic acid, 5-mercapto-1, 2,3, 4-tetrazoleacetic acid, 2-mercaptobenzothiazoleacetic acid, dimercaptosuccinic acid, etc.; A-X-E combination: mercaptoethanol, mercaptopropanol, mercaptobutanol, mercaptopentanol, 4-hydroxythiophenol, and the like; A-X-F combination: mercaptoethane, mercaptobutane, mercaptopentane, mercaptohexane, 3-hexanethiol, thiophenol, etc.; A-X combination: 4-mercaptophenylboronic acid, p-mercaptobenzenesulfonic acid, 4-tolylthiophenol, methyl mercaptopropionate, and the like.
B1-X-B1 combination: such as 1, 6-hexanediamine, p-aminophenylamine, m-aminophenylamine, o-aminophenylamine, melamine, 2- (4-aminophenyl) ethylamine, 4,5, 6-triaminopyrimidine sulfate, acridine orange hydrochloride, acriflavine hydrochloride, 3, 5-diaminoacridine hydrochloride, bromomethylphenanthine, basic fuchsin, crystal violet, 3, 6-diaminoacridine, bismark brown Y, 6-benzylaminopurine, N-benzyl-aminopurine, ethidium bromide, 4-amino-5-imidazolecarboxamide hydrochloride, rhodamine 110, thionine acetate, etc.; B1-X-B2 combination: such as cresyl violet acetate, adenine, 8-aza-adenine, 4-aminopyrazolo [3,4-d ] pyrimidine, 6-aminopurine, 2-iodo-6-aminopurine, 2-fluoro-6-aminopurine, 2-bromo-6-aminopurine, adenine hydrochloride, basic blue, etc.; B1-X-B3 combination: such as chlorpromazine hydrochloride, silver sulfadiazine (I), 9-amino-acridine, amsacrine hydrochloride, 9-amine-1, 2,3, 4-tetrahydrochloride hydrochloride, etc.; B1-X-C combination: such as p-nitroaniline, m-nitroaniline, o-nitroaniline, 2-ethoxy-6-nitro-9-aminoacridine, etc.; B1-X-D combination: l-glutamine, amino acids, and the like; B1-X-E combination: tyrosine, (2R) -3- (6-aminopurine-9-yl) propane-1, 2-diol, 2-amino-hydroxybenzene-4-sulfonamide, methyl amino (hydroxyimino) acetate, phthalimido hydroxybutyric acid, methyl amino (hydroxyimino) acetate, and the like; B1-X-F combination: methylamine, ethylamine, propylamine, etc.; B1-X combination: aniline, 9-aminofluorene hydrochloride, 2-amino-1, 1, 3-tricyanopropene, formamide, and the like.
B2-X-B2 combination: such as N6-benzoyladenine, 1, 3-diiminoisoindoline, etc.; B2-X-B3 combination: such as 6-cyanopurine, etc.; B2-X-C combination: 3-nitrophthalimide, and the like; B2-X-D combination: imino acids, etc.; B2-X-E combination: such as zeatin and the like; B2-X-F combination: ethylenimine, propyleneimine, and the like; B2-X combination: phthalimide, 4-bromophthalimide, and the like.
B3-X-B3 combination: rhodamine B, rhodamine 6G, tertiary amines, etc.; B3-X-C combination; B3-X-D combination: 4-maleimidobenzoic acid, etc.; B3-X-E combination; B3-X-F combination: ethyl imine, etc.; B3-X is combined with dodecyl tertiary amine, etc.
Of course, the functional molecule may also be a derivative of the above-mentioned substances.
Most preferably, the functional molecule is one or more of the molecules having A-X-A, A-X-B1, A-X-B2, B1-X-B1 and B1-X-B2.
Preferably, the functional molecule is bonded on the surface of the nanoparticle aggregate through a covalent bond, and the covalent bond has a firm action and is not easy to fall off.
Preferably, the depth range of the pits is 30nm to 2 μm, and the diameter range of the mouth part is 50nm to 4 μm; the density of the pits is 108～1010Per cm2The minimum spacing distance between two adjacent pits of the substrate is 1-50 nm, preferably 5-50 nm, and more preferably 10-30 nm.
Preferably, the nanoparticle aggregates are formed by nanoparticles by self-assembly within the pits.
Further preferably, the substrate and/or the nanoparticles are hydrophobically modified and then self-assembled.
Preferably, the particle size of the nanoparticles is 2nm to 800nm, preferably 30nm to 120 nm.
Preferably, the material of the metal nanoparticles includes one or more of gold, silver, copper, platinum and aluminum, or the metal nanoparticles are of an alloy structure or a core-shell structure.
Further, the alloy structure may be an alloy structure of any two or three of gold, silver, copper, platinum, and aluminum. For example, the alloy structure includes an AuAg, AgCu, AuC, AuPt, AgPt and the like having SERS activity.
Core-shell structures include core-shell structures having two components, e.g., Au @ Ag, Ag @ Au, Au @ Pt, Fe3O4@Au，Fe3O4@Ag，Au@SiO2，Ag@SiO2Etc., wherein @ the former is the core and the latter is the shell.
Preferably, the metal nanoparticles have a regular or irregular shape. For example, the shape of the metal nanoparticles includes a sphere, a block, a plate, a rod, or the like, and is not limited thereto.
In the present invention, the metal nanoparticles may be used in the form of a dispersion, and the dispersion may further be a metal nanoparticle sol. The metal nanoparticles can be synthesized by a wet process, the morphology and size of the metal nanoparticles can also be conveniently regulated, and reference can be made to the following processes and conditions, but not limited to the following document 1: angew. chem. int. ed.45, 3414.
Preferably, the concentration of the metal nanoparticles in the dispersion is 1 × 109 1X 10 to one/mL11one/mL.
In the present invention, the concentration of the metal nanoparticles may be adjusted by adding a solvent, and the solvent used may be a conventional solvent in the art.
Preferably, the substrate includes an inorganic substrate, an organic substrate or an inorganic/organic composite substrate, such as glass, single crystal silicon, plastic, polytetrafluoroethylene material, polystyrene material, metal and metal oxide, etc.
In the invention, the pits are distributed at intervals on the whole surface of the substrate, namely, a gap is formed between the pits instead of being connected into a whole.
In the present invention, the substrate having a plurality of pits on the surface may be a substrate having pits of the same specification or pits of different specifications, and a substrate having pits of a plurality of specifications is preferably used.
The specifications of the pit are defined by the circumferential outline shape of the pit, the volume of the pit and the opening area of the pit, and when any one of the circumferential outline shape of the two pits, the volume of the pit and the opening area of the pit is different, the two specifications are considered.
Further preferably, the number of said pits per square centimeter of area is N, the N pits having at least N/10 gauge, still further preferably at least N/8 gauge, more preferably at least N/6 gauge, most preferably at least N/3 gauge.
According to the invention, the pits are preferably arranged on the surface of the substrate in an array manner, and the pits have various specifications, so that the SERS chip is microscopically disordered, and the conventional understanding of people on an excellent SERS substrate is broken through. As can be seen from the foregoing, since SERS substrate performance is closely related to structure, researchers have consistently strived to obtain uniform nanostructures j.phys.chem.c 111,6720 in pursuit of repeatable SERS substrates; ACS appl. Indeed, uniform nanostructures can ensure good reproducibility, but the inventors of the present application found, in long-term research and extensive practice, that energy resonance is very likely to occur between nanostructure units with similar structures, and energy accumulated at nanoparticle gaps ("hot spots") is dissipated, resulting in a great decrease in SERS activity at the "hot spots". It may be based on this factor that the SERS activity of some SERS substrates with too high structural similarity in the prior art is not prominent. The inventor of the present application makes specifications of a plurality of pits different to make the specifications of the pits as much as possible, so that sizes and/or shapes of a plurality of nano-structure units limited in the pits are not completely the same, and thus interaction between the nano-structure units with the same structure can be avoided, adverse effects on plasma localization caused by the interaction are eliminated, and SERS activity of the SERS unit when the SERS unit is applied as a SERS substrate is greatly enhanced. On the other hand, statistically, over a large area (1 μm)2) Have very close overall performance of the nanostructure elements (about 100 or more) and thus have macroscopically uniform characteristics, making the SERS chip very homogeneousAnd the method is uniform, so that the reliability of the SERS test result can be ensured, and the method can be well applied to quantitative detection.
Preferably, the density of the pits is 108～1010Per cm2A substrate.
Preferably, the minimum spacing distance between two adjacent pits is 1-50 nm, more preferably 5-50 nm, and still more preferably 10-30 nm.
In the present invention, the minimum spaced distance between two adjacent pits refers to a minimum distance among a plurality of distances between an arbitrary point on the upper edge of one pit and an arbitrary point on the upper edge of an adjacent one pit.
Preferably, the depth of the pits is in the range of 30nm to 2 μm, preferably 30nm to 300nm, more preferably 40nm to 300nm, and still more preferably 40nm to 200 nm.
In the present invention, the depth of the dimple refers to the maximum distance from the surface of the dimple where the upper edge of the dimple is located to the bottom surface of the dimple.
The diameter of the mouth is preferably in the range of 50nm to 4 μm, preferably 30nm to 4 μm, more preferably 30nm to 500nm, and still more preferably 40nm to 200 nm.
In the invention, the diameter of the opening part of the pit refers to the largest distance in a plurality of distances between any two points on the upper edge of the pit, and when the surface surrounded by the upper edge of the pit is circular, the diameter of the pit is the diameter of the circle; when the surface enclosed by the upper edges of the pits is square, the diameter of each pit is the diagonal line of the square; when the surface enclosed by the upper edges of the pits is triangular, the diameter of each pit is the longest side of the triangle; when the surface enclosed by the upper edge of the pit is in an ellipse shape, the diameter of the pit is the major axis of the ellipse.
According to the invention, by controlling the minimum distance between the pits and/or the density of the pits and/or the diameter of the opening of the pits, high-density stacking of the nano-structure units can be realized, and the SERS effect can be further enhanced. Furthermore, the invention can make the diameter of the pit and the metal nano particle as small as possible, thereby making the activity of the chip better and making the stability, uniformity and repeatability better.
Preferably, the pits are made by plasma etching, uv etching, chemical etching, laser etching, mechanical drilling, mechanical punching, nanosphere printing or electrochemical methods.
Further preferably, the plurality of pits have a plurality of specifications by controlling the preparation parameters.
For example, the substrate having a plurality of pits on the surface can be prepared by nanosphere printing or electrochemical method, and the following references are specifically and not limited to document 2: j.am.chem.soc.127, 3710; chem.Commun.53, 7949.
Among them, the process of electrochemically preparing a substrate having nano-pores is very easy and has been commercialized (e.g., AAO template). And the relative controllability of the nanosphere printing is stronger, and more pore structure parameters can be prepared. Compared with other nanostructure processing methods (such as EBL, nano-imprinting and the like), the two methods have the advantages of high resolution, strong operability and low cost, and are very suitable for preparing the substrate.
Preferably, the nanostructure element further comprises an antibody layer attached to the functional molecule layer.
Further preferably, the antibody layer consists of one or more antibodies.
The invention also provides a preparation method of the SERS chip, which is characterized by comprising the following steps: the method comprises the following steps:
(1) providing a SERS substrate, wherein the SERS substrate comprises a substrate, a plurality of pits arranged on the surface of the substrate, and nano particle aggregates arranged in the pits;
(2) and connecting functional molecules on the surface of the nanoparticle aggregate to form a functional molecular layer, wherein the functional molecular layer is used for fixing molecules to be detected on the surface of the nanoparticle aggregate.
Preferably, the preparation method of the SERS chip further includes a step of attaching an antibody to the antibody layer formed by the functional molecule layer.
The type of the antibody to be linked in the present invention is various, and specifically, an appropriate antibody can be selected and linked according to the antigen to be detected.
The invention also provides an application of the SERS chip in antigen detection.
The invention relates to surface functionalization of an enhanced substrate applied to a surface enhanced Raman spectroscopy technology. The method specifically comprises the step of further processing an enhanced substrate of the surface enhanced Raman spectroscopy technology, namely modifying a layer of ordered molecular film on the surface of a substrate with a special structure to form a functionalized SERS substrate, wherein the ordered molecular film is just like a layer of molecular arm and can grab target molecules to be grabbed, and different target molecules can be grabbed by adjusting the length of the arm and the size of a hand so as to be applied to different detection fields. The method belongs to the technical field of SERS detection.
The chip of the invention can be used in a plurality of different detection fields, such as food safety, national defense safety, biological medicine, environmental safety and the like.
Non-limiting examples of samples typically used in the methods of the invention include: human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, sputum, bronchial washes, bronchial aspirates, urine, semen, lymph, and various external secretions of the respiratory, intestinal, and genito-urinary tracts, tears, saliva, milk, leukocytes, myelomas, and the like; biological fluids, such as cell culture supernatants; a tissue specimen that may or may not be fixed; and a cytological specimen which may or may not be fixed. The sample used in the method of the invention will vary based on the assay format and the nature of the tissue, cells, extract or other material, particularly biological material, to be assayed. Methods for preparing protein extracts from cells or samples are well known in the art and can be readily modified to obtain samples compatible with the methods of the invention.
Due to the implementation of the technical scheme, compared with the prior art, the invention has the following advantages:
the invention modifies one or more functional molecules on the substrate, so that the chip has different functions, thereby being applicable to different detection fields. The preparation process of the chip is simple, the efficiency is high, the cost is low, the high-performance SERS chip can be produced on a large scale, and the commercialization requirement can be well met. The SERS chip prepared by the invention has the advantages of high repeatability, uniform hot spot, stable property, large-area growth and high sensitivity.
Drawings
Fig. 1 schematically illustrates a principle of manufacturing a functionalized SERS substrate, where molecules with unique properties are connected to the surface of the substrate through chemical bond interaction on the substrate, so that the substrate has special properties, and has specificity to selectively bind to a target analyte during detection of the analyte;
FIG. 2 is a Raman spectrum obtained in example 6, wherein a is 10-6mol/L Pb2+(ii) a b is 10-7mol/L Pb2+(ii) a c is 10-8mol/L Pb2+(ii) a d is 10-9mol/L Pb2+；
FIG. 3 is a logarithmic graph of the concentration versus Raman intensity for example 6;
FIG. 4 is a Raman spectrum obtained in example 7, wherein a is 10-5mol/L Zn2+(ii) a b is 10-6mol/L Zn2+C is 10-7mol/L Zn2+D is 10-8mol/L Zn2+E is 10-9mol/L Zn2+F is blank;
fig. 5 is a logarithmic graph of the concentration corresponding to the raman intensity in example 7.
Detailed Description
In order to make the present invention clearer, the present invention is further described with reference to the drawings and the embodiments, and it should be understood that the present embodiment is not intended to limit the scope of the present invention. Methods and conditions not described in detail in the present invention are conventional in the art.
Example 1
General functionalization, SERS-active carboxylation of substrate surface: the prepared SERS substrate was placed in a 1mL borax buffer (pH 9, 1 × 10)-3mol/L), 10. mu.L of 1X 10 was added-2And (3) soaking the substrate in mercaptoacetic acid (mol/L) at room temperature for 2h, taking out the substrate, and washing the substrate with a large amount of water to obtain the SERS substrate with the mercaptoacetic acid functionalized surface. The substrate can be used to detect difficult and difficultThe substrate has molecules that interact with and react with the carboxyl groups.
Example 2
General functionalization, the substrate surface has SERS-active carboxylation: the prepared SERS substrate was placed in a 1mL borax buffer (pH 9, 1 × 10)-3mol/L), 10. mu.L of 1X 10 was added-2And soaking the mercaptobenzoic acid in mol/L for 2 hours at room temperature, taking out, and washing with a large amount of water to obtain the SERS substrate with the mercaptobenzoic acid functionalized on the surface. The substrate can be used for detecting molecules which are difficult to interact with the substrate and can react with carboxyl, the molecules to be detected interact with mercaptobenzoic acid, the SERS signal of the mercaptobenzoic acid is changed, and the structure and the concentration of a substance to be detected are judged by utilizing the difference between the original SERS signal and the new SERS signal.
Example 3
The special functionalization is used for biological antigen detection: the prepared SERS substrate was placed in a 1mL borax buffer (pH 9, 1 × 10)-3mol/L), 10. mu.L of 1X 10 was added-2And soaking the mercaptoaniline in mol/L for 2 hours at room temperature, taking out, and washing with a large amount of water to obtain the SERS substrate with the mercaptoaniline functionalized surface. The mixture was placed in 1mL of borax buffer (pH 9, 1X 10)-3mol/L), adding 10 mu L of 1mg/mL goat anti-rabbit IgG, shaking and incubating for 2h at room temperature, removing supernatant, washing with 1mL borax buffer solution, adding 10 mu L of BSA (bovine serum albumin) (1%), blocking active sites, shaking and incubating for 2h at room temperature, washing with borax buffer solution twice, and storing at 4 ℃ in borax buffer solution for later use. The functionalized substrate can be used for detecting sheep antigens with different concentrations, and the structure and the concentration of a substance to be detected can be judged according to the change of front and back SERS signals.
Example 4
The special functionalization is used for biological antigen detection: the prepared SERS substrate was placed in a 1mL borax buffer (pH 9, 1 × 10)-3mol/L), 10. mu.L of 1X 10 was added-2And (3) soaking 2-amino-6-hydroxy-8-mercaptopurine in mol/L for 2h at room temperature, taking out, and washing with a large amount of water to obtain the surface functionalized SERS substrate. The resulting mixture was placed in 1mL of borax buffer (pH 9, 1X 10-3mol/L), 10. mu.L of 1mg/mL goat anti-rabbit IgG was added,incubate with shaking at room temperature for 2h, remove supernatant, rinse with 1mL borax buffer, add 10. mu.L BSA (bovine serum albumin) (1%) to it, block the active site, incubate with shaking at room temperature for 2h, after two times borax wash buffer, store in borax buffer resuspension at 4 ℃ for use. The functionalized substrate can be used for detecting sheep antigens with different concentrations, and the structure and the concentration of a substance to be detected can be judged according to the change of front and back SERS signals.
Example 5
Preparing a carboxyl heavy metal particle marker: 1 μ L of 1X 10 gold sol (1 ml, 0.002 mg/ml) was added-3mol/L mercaptobenzoic acid, shaking for 20min at room temperature, centrifuging and washing for 3 times, and dispersing in 1ml of water.
Example 6
And (3) heavy metal particle detection: several portions of the substrate of example 1 were added with 0.5ml of the marker of example 5, and then 0.5ml of Pb was added to the substrate of example 1, respectively2+After soaking the solution for 20min, the solution is cleaned by a large amount of water, dried and tested by Raman.
Example 7
And (3) heavy metal particle detection: several portions of the substrate of example 1 were added with 0.5ml of the marker of example 5, and then 0.5ml of Zn was added to the substrate of example 12+After soaking the solution for 20min, the solution is cleaned by a large amount of water, dried and tested by Raman.

Claims (14)

1. A SERS chip comprises a SERS substrate and a plurality of nano-structure units arranged on the surface of the SERS substrate, wherein each nano-structure unit comprises a plurality of pits arranged on the surface of the SERS substrate and a metal nano-particle aggregate arranged in each pit; the metal nanoparticle aggregate is formed by self-assembling metal nanoparticles in the pit, and specifically, the self-assembling is carried out after the hydrophobic modification is carried out on the SERS substrate and/or the metal nanoparticles; the particle diameter of the metal nanoparticles2 nm-800 nm, the diameter range of the opening part of the pit is 50 nm-4 mu m, the depth range of the pit is 30 nm-2 mu m, and the density of the pit is 108～1010Per cm2Substrate and 10 per square centimeter of substrate8～1010Each pit has 107～109The minimum spacing distance between two adjacent pits is 1-50 nm, the pits are arrayed on the surface of the SERS substrate, the pits have various specifications so that the SERS chip can show a micro-disordered state in a microscopic mode, the specifications of the pits are limited by the circumferential profile shape of the pits, the volume of the pits and the opening area of the pits, and when any one of the circumferential profile shape of the pits, the volume of the pits and the opening area of the pits is different, the pits are regarded as two specifications.
2. The SERS chip according to claim 1, wherein: the functional molecule layer is composed of one or more functional molecules, the functional molecules comprise at least a first functional group and a second functional group, the first functional group is used for connecting the metal nanoparticle aggregate, and the second functional group is used for connecting molecules to be detected.
3. The SERS chip according to claim 2, wherein: the first functional group comprises-SH, ═ S, -NH2At least one of NH and ≡ N; the second functional group includes SH, ═ S, -NH2、＝NH、≡N、-NO2At least one of, -COOH, -OH and alkyl.
4. The SERS chip according to claim 1, wherein: the functional molecule is bonded on the surface of the metal nanoparticle aggregate through a covalent bond.
5. The SERS chip according to any of claims 1 to 4, wherein: the minimum spacing distance between two adjacent pits is 5-50 nm.
6. The SERS chip according to claim 5, wherein: the minimum spacing distance between two adjacent pits is 10-30 nm.
7. The SERS chip according to claim 5, wherein: the particle size of the metal nano-particles is 30 nm-120 nm.
8. The SERS chip according to claim 5, wherein: the material of the metal nano-particles comprises one or more of gold, silver, copper, platinum and aluminum.
9. The SERS chip according to claim 5, wherein: the metal nano particles are in an alloy structure or a core-shell structure.
10. The SERS chip according to any of claims 1 to 4, wherein: the SERS substrate comprises an inorganic substrate, an organic substrate or an inorganic/organic composite substrate.
11. The SERS chip according to any of claims 1 to 4, wherein: the pits are made by ultraviolet etching, chemical etching, laser etching, nanosphere printing or electrochemical methods.
12. The SERS chip according to any of claims 1 to 4, wherein: the nanostructure element further comprises an antibody layer attached to the functional molecular layer.
13. The SERS chip according to claim 12, wherein: the antibody layer consists of one or more antibodies.
14. Use of the SERS chip according to any of claims 12 to 13 for antigen detection.
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