Composite SERS (Surface Enhanced Raman Scattering) substrate detection chip and preparation method and application thereof

Abstract

The invention relates to the technical field of surface enhanced Raman detection, in particular to a composite SERS (Surface Enhanced Raman Scattering) substrate detection chip as well as a preparation method and application thereof. The composite SERS substrate detection chip comprises a bottom sheet with a groove; the silver nanowires and the metal nanoparticles are located in the grooves, the silver nanowires form a loose net-shaped structure, and the metal nanoparticles are loaded on the silver nanowires. According to the composite SERS substrate detection chip disclosed by the invention, benzopyrene can be detected without introducing modified molecules.

Description

Technical Field

[0001] The present invention relates to the technical field of surface-enhanced Raman detection, and particularly relates to a composite SERS substrate detection chip, a preparation method thereof, and an application thereof. Background Art

[0002] Surface Enhanced Raman Spectroscopy (SERS) detection refers to a technique in which, when light is incident on certain specially prepared nano-substrates or sols, the Raman scattering signal of adsorbed molecules is greatly enhanced compared to ordinary Raman signals due to the enhancement of the electromagnetic field on the surface of the nanostructure. This technique has the advantages of rich fingerprint information, high sensitivity, being unaffected by water interference, and enabling real-time detection, and is widely used in various fields such as environmental detection, disease diagnosis, and biomedicine.

[0003] The key to SERS detection lies in constructing a suitable SERS substrate. Currently, SERS substrates are generally composed of nano-particles such as gold, silver, and copper, which can have a strong coupling effect with incident light, confine the energy of the incident light in the nano-gap, and obtain an electromagnetic field enhancement region. For petroleum substances such as polycyclic aromatic hydrocarbons that are difficult to adsorb, thiols or graphene and other modifier molecules are generally added to the SERS substrate, and their non-covalent bond forces are used to adsorb target molecules into the electromagnetic field enhancement region, ultimately achieving the detection of trace substances in water. Therefore, how to simply and conveniently prepare a suitable SERS substrate and fix target molecules in the electromagnetic field enhancement region is the current research focus.

[0004] Patent application CN 109060757 A discloses a surface-enhanced Raman scattering method for rapidly detecting microbial contamination using a paper-based enhanced substrate, and the method includes: providing a sol solution; preparing a paper-based enhanced substrate; and performing Raman spectroscopy testing using the paper-based enhanced substrate. The preparation method of the paper-based enhanced substrate includes: soaking a paper substrate in about 1% hydrochloric acid overnight, after cleaning, washing it with ultrapure water multiple times; drying the washed paper substrate with an inert gas; soaking the dried paper substrate with the provided sol solution; and freeze-drying the soaked paper substrate after cryopreservation at a low temperature, thereby obtaining the paper-based enhanced substrate.

[0005] Patent application CN 114674804 A discloses a preparation method of a wipeable SERS substrate for detecting pesticides, including the following steps: preparation of silver nanoparticles, dissolving silver nitrate in deionized water, heating and adding sodium citrate for reaction, cooling to room temperature, and setting aside; preparation of the SERS substrate: preparing a chitosan solution, freezing the chitosan solution overnight, freeze-drying to obtain a porous structure, washing to obtain a chitosan foam substrate, placing the chitosan foam substrate in a silver nano-ion suspension, ultrasonicating, and freeze-drying to obtain the target product.

[0006] Patent application CN 103257063 B discloses a water body polycyclic aromatic hydrocarbon detection reagent. The detection reagent consists of reagent A and reagent B. Reagent A is an aqueous solution of graphene / nano-precious metal composite; reagent B is a surfactant solution. It also discloses the application of a water body polycyclic aromatic hydrocarbon detection reagent, including the following steps: First, disperse reagent B in the polycyclic aromatic hydrocarbon solution; then add reagent A to the above mixture and mix evenly; finally, take an appropriate amount of the mixed solution for Raman detection to obtain the SERS spectrum of the polycyclic aromatic hydrocarbon. Summary of the Invention

[0007] The object of the present invention is to overcome the problems existing in the prior art, that is, the existing SERS substrate has to introduce a modification molecule in order to adsorb the target molecule into the nano-gap, which leads to easy interference with the detection of the target molecule, complex preparation process, and relatively high lower limit of detection concentration. The present invention provides a composite SERS substrate detection chip, its preparation method and application.

[0008] To achieve the above object, on the one hand, the present invention provides a composite SERS substrate detection chip, including:

[0009] A bottom plate with grooves; and

[0010] Silver nanowires and metal nanoparticles located in the grooves, and the silver nanowires form a loose network structure, and the metal nanoparticles are loaded on the silver nanowires.

[0011] Preferably, the diameter of the silver nanowires is 12 - 60 nm, and the length is 10 - 45 μm.

[0012] Preferably, the metal nanoparticles are gold nanoparticles or silver nanoparticles;

[0013] Preferably, the size of the metal nanoparticles is 5 - 60 nm.

[0014] Preferably, the bottom area of the groove is 1 - 17 mm 2 , and the height is 3 - 9 mm.

[0015] On the second aspect, the present invention provides a method for preparing a composite SERS detection chip, and the method includes the following steps:

[0016] (1) Provide a bottom plate with grooves;

[0017] (2) Inject a mixed solution containing silver nanowires and metal nanoparticles into the grooves, then place the bottom plate in liquid nitrogen, and then perform freeze-drying;

[0018] Among them, in the mixed solution containing silver nanowires and metal nanoparticles, the concentration of silver nanowires is 18 - 65 mg / mL, and the concentration of metal nanoparticles is 10 - 50 mg / mL.

[0019] Preferably, the bottom area of the groove is 1 - 17 mm 2 , and the height is 3 - 9 mm.

[0020] Preferably, the diameter of the silver nanowires is 12 - 60 nm, and the length is 10 - 45 μm.

[0021] Preferably, the metal nanoparticles are gold nanoparticles or silver nanoparticles;

[0022] Preferably, the size of the metal nanoparticles is 5 - 60 nm.

[0023] Preferably, in the solution containing silver nanowires and metal nanoparticles, the weight ratio of silver nanowires to metal nanoparticles is 1:0.5 - 1.5.

[0024] Preferably, the material of the negative film is a polymer material, silicon nitride, glass, silicon wafer or metal;

[0025] Preferably, the polymer material is selected from PMMA, PC, PVC or PET.

[0026] Preferably, the injection volume of the mixed solution containing silver nanowires and metal nanoparticles is in a volume ratio of 0.45 - 0.85:1 to the volume of the groove.

[0027] Preferably, the conditions for freeze-drying include: the pressure is 1 - 50 Pa, and the time is 6 - 30 h.

[0028] The third aspect of the present invention provides a composite SERS substrate detection chip prepared by the method described above.

[0029] The fourth aspect of the present invention provides the application of the composite SERS substrate detection chip described above in the detection of benzo[a]pyrene.

[0030] The fifth aspect of the present invention provides a method for detecting the concentration of benzo[a]pyrene, the method comprising: dropping the solution to be measured into the groove of the composite SERS substrate detection chip described above, and performing SERS detection using a Raman spectrometer.

[0031] Preferably, the volume of the solution to be measured is 5 - 10 μL.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The composite SERS substrate detection chip described in the present invention can detect benzo[a]pyrene without introducing modifier molecules. Specifically, the silver nanowires located in the grooves of the negative film form a network structure, and at the same time, metal nanoparticles are loaded on the silver nanowires. After the target is easily injected into the grooves, it can be quickly absorbed by the composite structure of the silver nanowires and the metal nanoparticles, and then collapse, fixing the target molecules in the nanogaps formed by the silver nanowires and the metal nanoparticles.

[0034] 2. The preparation method of the composite SERS substrate detection chip described in the present invention has the characteristics of simple steps, controllable size, and no need to use large and expensive instruments. Combining the freeze-drying technology, the silver nanowires self-assemble into a network structure, and at the same time, the metal nanoparticles adhere to the silver nanowires, facilitating the adsorption of the molecules to be detected in the solution.

[0035] 3. The composite SERS substrate detection chip described in the present invention is a solid material, which is convenient for storage and transportation, and is suitable for detecting trace petroleum substances in various water quality detection fields such as refinery sewage and domestic sewage. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a schematic flow chart of processing a solution to be detected using the composite SERS substrate detection chip described in the present invention;

[0037] Figure 2 is a characterization result diagram of the product prepared in Example 1 taken by a microscope;

[0038] Figure 3 is a characterization result diagram of the product prepared in Example 1 using SEM;

[0039] Figure 4 is a Raman signal diagram of benzo[a]pyrene at different concentrations under the SERS detection chip;

[0040] Figure 5 is a relationship diagram between the concentration of benzo[a]pyrene and the Raman signal intensity. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] The following further describes in detail the specific embodiments of the present invention with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only for explaining and illustrating the present invention, and are not used to limit the present invention.

[0042] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0043] On the one hand, the present invention provides a composite SERS substrate detection chip, comprising:

[0044] a bottom plate having a groove; and

[0045] silver nanowires and metal nanoparticles located in the groove, and the silver nanowires form a loose network structure, and the metal nanoparticles are loaded on the silver nanowires.

[0046] In the present invention, the silver nanowires located in the groove form a loose network structure through self-assembly, and can also serve as a carrier for metal nanoparticles. Based on the cooperation of silver nanowires and metals, more nano-gaps can be formed, providing a stronger and denser electromagnetic field enhancement region, thereby improving the detection sensitivity.

[0047] In a preferred embodiment, the silver nanowires have a diameter of 12 - 60 nm and a length of 10 - 45 μm.

[0048] In a preferred embodiment, the metal nanoparticles are gold nanoparticles or silver nanoparticles.

[0049] According to some specific embodiments of the present invention, the metal nanoparticles can be gold nanospheres, silver nanospheres, gold nanorods, silver nanocubes, gold nanotriangles, etc.

[0050] More preferably, the size of the metal nanoparticles is 5 - 60 nm.

[0051] In a preferred embodiment, the bottom area of the groove is 1 - 17 mm 2 , and the height is 3 - 9 mm.

[0052] On the second aspect, the present invention provides a method for preparing a composite SERS substrate detection chip, the method comprising the following steps:

[0053] (1) Providing a bottom plate having a groove;

[0054] (2) Injecting a mixed solution containing silver nanowires and metal nanoparticles into the groove, then placing the bottom plate in liquid nitrogen, and then performing freeze-drying;

[0055] Wherein, in the mixed solution containing silver nanowires and metal nanoparticles, the concentration of silver nanowires is 18 - 65 mg / mL, and the concentration of metal nanoparticles is 10 - 50 mg / mL.

[0056] In the method of the present invention, a solution containing silver nanowires and metal nanoparticles is injected into the groove of the negative film. The ultra-low temperature of liquid nitrogen can quickly turn the solution into a solid state, and then the solvent can be sublimated by freeze-drying technology without damaging the structure formed by the silver nanowires and metal nanoparticles. The present invention combines the freeze-drying technology to obtain a composite SERS detection chip, which does not involve large and expensive instruments such as vacuum coating machines and electron beam etching machines, and is convenient for further popularization and application.

[0057] In the present invention, the concentrations of silver nanowires and metal nanoparticles in the solution containing silver nanowires and metal nanoparticles are reasonably controlled. If the concentration is too low, the network structure is prone to collapse during freeze-drying. If the concentration is too high, the cost will increase. Therefore, the present invention limits the concentration of the silver nanowire solution to 18 - 65 mg / mL and the concentration of the metal nanoparticles to 10 - 50 mg / mL, which can not only form a stable network structure but also ensure the economic practicability of the method and effectively improve the detection sensitivity.

[0058] In a preferred embodiment, the bottom area of the groove is 1 - 17 mm 2 , and the height is 3 - 9 mm; specifically, the bottom area of the groove can be 1 mm 2 , 3 mm 2 , 5 mm 2 , 6 mm 2 , 7 mm 2 , 8 mm 2 , 9 mm 2 , 10 mm 2 , 12 mm 2 , 13 mm 2 , 14 mm 2 , 15 mm 2 , 16 mm 2 or 17 mm 2 ; the height of the groove can be 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm or 9 mm.

[0059] In a preferred embodiment, the diameter of the silver nanowires is 12 - 60 nm, more preferably 15 - 45 nm; the length is 10 - 45 μm, more preferably 15 - 40 μm.

[0060] In the present invention, the silver nanowires and metal nanoparticles can be commercially available products or self-made in the laboratory.

[0061] In some specific embodiments, the silver nanowires can be prepared according to the following procedures:

[0062] A1: Mix polyvinylpyrrolidone and water, and then add AgNO3 for mixing to obtain a mixture.

[0063] A2: Hydrothermally react the mixture, and then perform solid-liquid separation.

[0064] Preferably, the weight ratio of the dosage of polyvinylpyrrolidone to AgNO3 is 1:0.04 - 0.15.

[0065] Preferably, in step A2, the conditions of the hydrothermal reaction include: the temperature is 120 - 180 °C, and the time is 5 - 12 h.

[0066] Preferably, in step A2, the solid-liquid separation can be carried out by centrifugation.

[0067] In a preferred embodiment, the metal nanoparticles are gold nanoparticles or silver nanoparticles.

[0068] According to some specific embodiments of the present invention, the metal nanoparticles can be gold nanospheres, silver nanospheres, gold nanorods, silver nanocubes, gold nanotriangles, etc.

[0069] Preferably, the geometric size of the metal nanoparticles is 5 - 60 nm.

[0070] More preferably, the metal nanoparticles are gold nanorods with a diameter of 8 - 20 nm and a length of 20 - 60 nm.

[0071] According to some specific embodiments of the present invention, the gold nanorods can be prepared according to the following process:

[0072] B1: Mix cetyltrimethylammonium bromide (CTAB), HAuCl4 and water, and then add NaBH4 for mixing to obtain a first mixed solution;

[0073] B2: Mix CTAB, HAuCl4 and water, and then add ascorbic acid for mixing to obtain a second mixed solution;

[0074] B3: Mix the first mixed solution and the second mixed solution for reaction, then add NaCl to continue the reaction, perform solid-liquid separation, and wash.

[0075] Preferably, in step B1, the molar ratio of the dosages of CTAB, HAuCl4 and NaBH4 is 1:2 - 3:0.003 - 0.01.

[0076] Preferably, in step B2, the molar ratio of the dosages of CTAB, HAuCl4 and ascorbic acid is 1:0.01 - 0.04:0.01 - 0.07.

[0077] In a preferred embodiment, in step B3, when the first mixed solution and the second mixed solution are mixed, the dosage ratio of the first mixed solution to the second mixed solution is 1:120 - 250, where the dosage of the first mixed solution is calculated based on the molar amount of CTAB input when preparing the first mixed solution, and the dosage of the second mixed solution is calculated based on the molar amount of CTAB input when preparing the second mixed solution.

[0078] In a preferred embodiment, in the solution containing silver nanowires and metal nanoparticles, the weight ratio of silver nanowires to metal nanoparticles is 1:0.5 - 1.5.

[0079] In a preferred embodiment, the material of the negative film is a polymer material, silicon nitride, glass, silicon wafer or metal.

[0080] More preferably, the polymer material is selected from PMMA, PC, PVC or PET.

[0081] In a preferred embodiment, the injection volume of the mixed solution containing silver nanowires and metal nanoparticles and the volume of the groove are in a ratio of 0.45 - 0.85:1. If the injection volume of the mixed solution is too small, the number of added metal particles and silver nanowires will be small, resulting in a reduced detection effect; if the injection volume is too large, it is likely to overflow from the groove and is not easy to operate.

[0082] In a preferred embodiment, the conditions of freeze-drying include: the pressure is 1 - 50 Pa and the time is 6 - 30 h.

[0083] The third aspect of the present invention provides a composite SERS substrate detection chip prepared by the method described above.

[0084] The fourth aspect of the present invention provides the application of the composite SERS substrate detection chip described above in the detection of benzo[a]pyrene.

[0085] Combined with Figure 1 , the process of using the composite SERS detection chip of the present invention to process the solution to be detected during Raman detection includes: dropping the solution to be detected into the groove, and the solution to be detected quickly fills the entire groove. As the solvent volatilizes, the structure of silver nanowires and metal nanoparticles in the groove will collapse, and the target molecules will be wrapped in the nano-gaps between silver nanowires and metal nanoparticles.

[0086] The fifth aspect of the present invention provides a method for detecting the concentration of benzo[a]pyrene, which includes: dropping the solution to be detected into the groove of the composite SERS substrate detection chip described above, and performing SERS detection using a Raman spectrometer.

[0087] Preferably, the volume of the solution to be detected is 5 - 10 μL.

[0088] In a specific embodiment, benzo[a]pyrene can be quantitatively detected. The testing process includes the following steps:

[0089] S1; Drop the benzo[a]pyrene standard solution into the groove of the composite SERS substrate detection chip, dry it, and after the solvent has completely evaporated, perform SERS detection using a Raman spectrometer and record the peak intensity;

[0090] S2: Use the negative logarithm of the concentration as the abscissa and the peak intensity as the ordinate for fitting to obtain the relationship between the concentration and the peak intensity;

[0091] S3: Drop the solution to be tested into the groove of the composite SERS substrate detection chip, dry it, and after the solvent has completely evaporated, perform SERS detection using a Raman spectrometer and record the peak intensity. Then substitute the peak intensity into the relationship formula for calculation to obtain the concentration of benzo[a]pyrene in the solution to be tested.

[0092] The present invention will be described in detail below through examples, but the protection scope of the present invention is not limited thereto.

[0093] Example 1

[0094] Preparation of silver nanowires:

[0095] A1: Add 0.3 g of polyvinylpyrrolidone (PVP, MW = 55,000) to 40 mL of water and mix. After stirring for 35 minutes, a clear solution is formed, and then 0.022 g of AgNO3 is added and stirred vigorously for 15 minutes to obtain a mixture;

[0096] A2: Transfer the mixture to a hydrothermal reactor (volume 50 mL) and seal it for reaction at 150 °C for 8 hours; After the reaction, take out the suspension, centrifuge it at 3000 rpm for 5 minutes, repeat 4 times to remove the unreacted substances, and obtain silver nanowires with a diameter of 28 nm and a length of 30 μm.

[0097] Preparation of gold nanorods:

[0098] B1: Mix 6 mL of a CTAB solution with a concentration of 0.25 mol / L and 8 mL of an HAuCl4 solution with a concentration of 0.4 mol / L, and then quickly add 0.6 mL of a freshly prepared NaBH4 solution with a concentration of 0.015 mol / L. The solution changes from light yellow to bright brownish yellow, and continue to stir for 2 min to obtain a first mixture;

[0099] B2: Take a clean 50 mL round-bottom flask, add 5 mL of CTAB solution with a concentration of 0.22 mol / L, then dropwise add 3 mL of HAuCl4 solution with a concentration of 9 mmol / L. After it stabilizes, quickly add 0.65 mL of ascorbic acid solution with a concentration of 0.08 mol / L. Wait until the solution changes from yellow to colorless, and add pure water to make the total volume up to 25 mL to obtain the second mixed solution.

[0100] B3: Add 60 μL of the first mixed solution (the amount of CTAB required to prepare this solution is about 6.16×10 -3 mmol) to 25 mL of the second mixed solution (the amount of CTAB required to prepare this solution is 1.1 mmol), that is, the usage ratio of the first mixed solution to the second mixed solution is 1:178. After reacting for 4 hours, add 0.8 mL of NaCl solution with a concentration of 4 mol / L, and continue the constant-temperature reaction for 10 h. After centrifugal washing at 7000 r / min, gold nanorods with a diameter of 15 nm and a length of 45 nm are obtained.

[0101] Preparation of composite SERS substrate detection chip:

[0102] (1) Provide a negative film with grooves. The material of the negative film is PET, the shape of the negative film is square, the thickness of the negative film is 8 mm, the bottom area of the groove is 5 mm 2 , and the height of the groove is 6 mm;

[0103] (2) Mix the above silver nanowires, the above gold nanorods and water to obtain a mixed solution, and control the concentration of silver nanowires in the mixed solution to be 35 mg / ml and the concentration of gold nanorods to be 25 mg / ml;

[0104] Take 23 μL of the mixed solution and drop it into the groove of the negative film, then put the negative film into liquid nitrogen together. Further place it in a freeze dryer, and place it for 23 h under a vacuum degree of 8 Pa to obtain the composite SERS detection chip.

[0105] Example 2

[0106] Preparation of composite SERS substrate detection chip:

[0107] (1) Provide a negative film with grooves. The material of the negative film is PET, the shape of the negative film is square, the thickness of the negative film is 8 mm, the bottom area of the groove is 5 mm 2 , and the height of the groove is 6 mm;

[0108] (2) Mix the above silver nanowires (the same as in Example 1), the above gold nanorods (the same as in Example 1) and water to obtain a mixed solution, and control the concentration of silver nanowires in the mixed solution to be 45 mg / mL and the concentration of gold nanorods to be 25 mg / mL;

[0109] Take 23 μL of the mixed solution and drop it into the groove of the bottom plate, and then put the bottom plate into liquid nitrogen together. Further, place it in a freeze dryer and place it for 23 h under a vacuum of 8 Pa to obtain a composite SERS detection chip.

[0110] Example 3

[0111] Prepare a composite SERS substrate detection chip:

[0112] (1) Provide a bottom plate with a groove. The material of the bottom plate is PET, the shape of the bottom plate is square, the thickness of the bottom plate is 8 mm, and the bottom area of the groove is 5 mm 2 , and the height of the groove is 6 mm;

[0113] (2) Mix the above silver nanowires (same as Example 1), the above gold nanorods (same as Example 1) and water to obtain a mixed solution, and control the concentration of silver nanowires in the mixed solution to be 25 mg / mL and the concentration of gold nanorods to be 25 mg / mL;

[0114] Take 23 μL of the mixed solution and drop it into the groove of the bottom plate, and then put the bottom plate into liquid nitrogen together. Further, place it in a freeze dryer and place it for 23 h under a vacuum of 8 Pa to obtain a composite SERS detection chip.

[0115] Example 4

[0116] Prepare a composite SERS substrate detection chip:

[0117] (1) Provide a bottom plate with a groove. The material of the bottom plate is PET, the shape of the bottom plate is square, the thickness of the bottom plate is 8 mm, and the bottom area of the groove is 5 mm 2 , and the height of the groove is 6 mm;

[0118] (2) Mix the above silver nanowires (same as Example 1), the above gold nanorods (same as Example 1) and water to obtain a mixed solution, and control the concentration of silver nanowires in the mixed solution to be 35 mg / mL and the concentration of gold nanorods to be 35 mg / mL;

[0119] Take 23 μL of the mixed solution and drop it into the groove of the bottom plate, and then put the bottom plate into liquid nitrogen together. Further, place it in a freeze dryer and place it for 23 h under a vacuum of 8 Pa to obtain a composite SERS detection chip.

[0120] Example 5

[0121] Prepare a composite SERS substrate detection chip:

[0122] (1) Provide a bottom plate with a groove. The material of the bottom plate is PET, the shape of the bottom plate is square, the thickness of the bottom plate is 8 mm, and the bottom area of the groove is 6 mm2 , the height of the groove is 6 mm;

[0123] (2) Mix the above silver nanowires (same as in Example 1), the above gold nanorods (same as in Example 1) and water to obtain a mixed solution, and control the concentration of silver nanowires in the mixed solution to be 35 mg / mL and the concentration of gold nanorods to be 25 mg / mL;

[0124] Take 17 μL of the mixed solution and drop it into the groove of the negative film, then put the negative film into liquid nitrogen together. Further, put it into a freeze dryer and place it for 23 h under a vacuum degree of 8 Pa to obtain a composite SERS detection chip.

[0125] Example 6

[0126] Prepare a composite SERS substrate detection chip:

[0127] (1) Provide a negative film with a groove, the material of the negative film is PET, the shape of the negative film is square, the thickness of the negative film is 8 mm, and the bottom area of the groove is 6 mm 2 , the height of the groove is 6 mm;

[0128] (2) Mix the above silver nanowires (same as in Example 1), the above gold nanorods (same as in Example 1) and water to obtain a mixed solution, and control the concentration of silver nanowires in the mixed solution to be 35 mg / mL and the concentration of gold nanorods to be 25 mg / mL;

[0129] Take 27 μL of the mixed solution and drop it into the groove of the negative film, then put the negative film into liquid nitrogen together. Further, put it into a freeze dryer and place it for 23 h under a vacuum degree of 8 Pa to obtain a composite SERS detection chip.

[0130] Example 7

[0131] Prepare a composite SERS substrate detection chip:

[0132] (1) Provide a negative film with a groove, the material of the negative film is PET, the shape of the negative film is square, the thickness of the negative film is 8 mm, and the bottom area of the groove is 5 mm 2 , the height of the groove is 6 mm;

[0133] (2) Mix the above silver nanowires (same as in Example 1), silver nanospheres (purchased commercially, with a diameter of 45 nm) and water to obtain a mixed solution, and control the concentration of silver nanowires in the mixed solution to be 35 mg / mL and the concentration of silver nanospheres to be 25 mg / mL;

[0134] Take 23 μL of the mixed solution and drop it into the groove of the bottom plate, then put the bottom plate into liquid nitrogen together. Further, place it in a freeze dryer and keep it for 23 h under a vacuum of 8 Pa to obtain the composite SERS detection chip.

[0135] Example 8

[0136] Prepare a composite SERS substrate detection chip:

[0137] (1) Provide a bottom plate with a groove. The material of the bottom plate is PET, the shape of the bottom plate is square, the thickness of the bottom plate is 8 mm, the bottom area of the groove is 5 mm 2 , and the height of the groove is 6 mm;

[0138] (2) Prepare silver nanowires:

[0139] A1: Add 0.3 g of polyvinylpyrrolidone (PVP, MW = 55,000) to 40 mL of water and mix. After stirring for 35 minutes to form a clear solution, then add 0.03 g of AgNO3 and stir vigorously for 15 minutes to obtain a mixed material;

[0140] A2: Carry out a hydrothermal reaction on the mixed material, transfer it to a hydrothermal kettle (with a volume of 50 mL), and seal it at 150 °C for 8 hours; after the reaction, take out the suspension, centrifuge it at 3000 rpm for 5 minutes, and repeat 4 times to remove the unreacted substances to obtain silver nanowires with a diameter of 35 nm and a length of 38 μm.

[0141] Mix the above silver nanowires, the above gold nanorods (the same as in Example 1) and water to obtain a mixed solution, and control the concentration of silver nanowires in the mixed solution to be 40 mg / mL and the concentration of gold nanorods to be 30 mg / mL;

[0142] Take 20 μL of the mixed solution and drop it into the groove of the bottom plate, then put the bottom plate into liquid nitrogen together. Further, place it in a freeze dryer and keep it for 23 h under a vacuum of 8 Pa to obtain the composite SERS detection chip.

[0143] Example 9

[0144] Prepare a composite SERS detection chip:

[0145] (1) Provide a bottom plate with a groove. The material of the bottom plate is PET, the shape of the bottom plate is square, the thickness of the bottom plate is 8 mm, the bottom area of the groove is 5 mm 2 , and the height of the groove is 6 mm;

[0146] (2) Prepare gold nanorods:

[0147] B1: Mix 6 mL of a CTAB solution with a concentration of 0.25 mol / L and 8 mL of an HAuCl4 solution with a concentration of 0.4 mol / L, and then quickly add 0.6 mL of a freshly prepared NaBH4 solution with a concentration of 0.015 mol / L. The solution changes from light yellow to bright brownish yellow. Continue stirring for 2 min to obtain the first mixed solution.

[0148] B2: Take a clean 50 mL round-bottom flask, add 5 mL of a CTAB solution with a concentration of 0.22 mol / L, and then dropwise add 2 mL of an HAuCl4 solution with a concentration of 9 mmol / L. After it stabilizes, quickly add 0.65 mL of an ascorbic acid solution with a concentration of 0.08 mol / L. Wait for the solution to change from yellow to colorless, and then add pure water to make the total volume up to 25 mL to obtain the second mixed solution.

[0149] B3: Add 60 μL of the first mixed solution (the amount of CTAB required to prepare this solution is approximately 6.16×10 -3 mmol) to 25 mL of the second mixed solution (the amount of CTAB required to prepare this solution is 1.1 mmol), that is, the volume ratio of the first mixed solution to the second mixed solution is 1:178. After reacting for 4 h, add 0.8 mL of a NaCl solution with a concentration of 4 mol / L, and continue the constant-temperature reaction for 10 h. After centrifugal washing at 7000 r / min, gold nanorods with a diameter of 10 nm and a length of 35 nm are obtained.

[0150] Mix the above silver nanowires (same as in Example 1), the above gold nanorods and water to obtain a mixed solution, and control the concentration of silver nanowires in the mixed solution to be 30 mg / mL and the concentration of gold nanorods to be 20 mg / mL.

[0151] Take 25 μL of the mixed solution and drop it into the groove of the negative film, and then put the negative film into liquid nitrogen together. Further, place it in a freeze dryer, and place it for 23 h under a vacuum degree of 8 Pa to obtain a composite SERS detection chip.

[0152] Comparative Example 1

[0153] Prepare an SERS detection chip:

[0154] (1) Provide a negative film with a groove. The shape of the negative film is square, the thickness of the negative film is 8 mm, the bottom area of the groove is 5 mm 2 , and the height of the groove is 6 mm.

[0155] (2) Mix the above silver nanowires (same as in Example 1), the above gold nanorods (same as in Example 1) and water to obtain a mixed solution, and control the concentration of silver nanowires in the mixed solution to be 35 mg / mL and the concentration of gold nanorods to be 25 mg / mL.

[0156] Take 23 μL of the mixed solution and drop it into the groove of the substrate. After the solvent has completely evaporated, a SERS detection chip is obtained.

[0157] Comparative Example 2

[0158] Preparation of SERS detection chip:

[0159] (1) Provide a substrate with a groove. The substrate is square in shape, with a thickness of 8 mm and the bottom area of the groove is 5 mm 2 , and the height of the groove is 6 mm;

[0160] (2) Mix the above silver nanowires (the same as in Example 1), the above gold nanorods (the same as in Example 1) and water to obtain a mixed solution, and control the concentration of silver nanowires in the mixed solution to be 8 mg / mL and the concentration of gold nanorods to be 8 mg / mL;

[0161] Take 23 μL of the mixed solution and drop it into the groove of the substrate. After the solvent has completely evaporated, a SERS detection chip is obtained. Then put the substrate into liquid nitrogen together. Further, place it in a freeze dryer and place it for 23 h under a vacuum of 8 Pa. During the placement process, it is observed that the silver nanowires and gold nanorods collapse at the bottom of the groove. This may be due to the too low concentration of the silver nanowire solution, resulting in insufficient supporting force of the formed network structure.

[0162] Test Example 1

[0163] Use an Olympus CX31 microscope to take pictures of the chip prepared in Example 1, and the results are as Figure 2 shown; use SEM to characterize the chip prepared in Example 1, and the results are as Figure 3 shown.

[0164] It can be observed from Figure 3 that the silver nanowires cross each other to form a loose network structure, and at the same time, the gold nanorods are loaded on the network structure formed by the silver nanowires.

[0165] Test Example 2

[0166] Test the performance of the products prepared in the examples and comparative examples. The test process is as follows:

[0167] Drop 6 μL of an ethanol solution of benzo[a]pyrene with a concentration of 10 -6 M (i.e., 10 -6 mol / L) onto the sample to be tested. After the solvent has completely evaporated, use a Horiba Xplus confocal Raman spectrometer to test the substrate. Select a laser with a wavelength of 633 nm, a power of 0.5 mW, an integration time of 40 s, and an integration number of 2 times. Taking the intensity of benzo[a]pyrene molecules at 1385 cm -1 as an example, the results are shown in Table 1.

[0168] Table 1

[0169] Number Strength Number Strength Example 1 12879 Example 6 12743 Example 2 12913 Example 7 12893 Example 3 11674 Example 8 12145 Example 4 12657 Example 9 11376 Example 5 11294 Comparative Example 1 9768

[0170] As can be seen from Table 1, the composite SERS detection chip prepared in the examples has good signal amplification ability when applied to surface-enhanced Raman detection. The signal intensity of the SERS detection chip prepared in Comparative Example 1 is significantly lower than that of the examples. This may be because in this SERS detection chip, the silver nanowires and gold nanorods in the grooves are closely packed. After adding the solution to be measured, the target molecules can only adsorb on the surface of the silver nanowires and gold nanorods, and the number of molecules entering the nano-gap decreases, resulting in a decrease in the Raman signal.

[0171] Test Example 3

[0172] Prepare benzo[a]pyrene standard solutions with concentrations of 10 -9 mol / L, 10 -8 mol / L, 10 -7 mol / L, 10 -6 mol / L, and 10 -5 mol / L respectively. The corresponding Raman signal diagrams are as shown in the appendix Figure 4 . It can be seen from the figure that the characteristic peaks of pyrene molecules are mainly at 338 cm -1 , 1240 cm -1 , 1346 cm -1 , 1385 cm -1 , 1584 cm -1 , and 1624 cm -1 . It can be seen from the intensity of the characteristic peaks that as the concentration gradually decreases, the intensity of the characteristic peaks also gradually decreases. The composite SERS detection chip can achieve qualitative detection of benzo[a]pyrene molecules with a concentration ≥ 10 - 9 mol / L.

[0173] Furthermore, in order to achieve quantitative detection of benzo[a]pyrene molecules, based on the data in the appendix Figure 4 , select the intensity of the characteristic peak at 1385 cm -1 as the ordinate and the negative logarithm of the concentration as the abscissa to obtain Figure 5 . It can be seen from Figure 5 that the intensity of this characteristic peak and the logarithm of the concentration show a good functional relationship. The intensity of the characteristic peak y = -4179x + 38045, R 2 = 0.9981, where x is the negative logarithm of the concentration (i.e., -lgC). Based on this mathematical relationship, after measuring the intensity of the characteristic peak at 1385 cm -1 , the concentration of the solution can be calculated, thereby achieving quantitative detection of benzo[a]pyrene molecules.

[0174] Solutions with benzo[a]pyrene concentrations of 10 -6 mol / L, 10 -7 mol / L, and 10 -8 mol / L were prepared as test solutions. Three composite SERS detection chips prepared according to the method described in Example 1 were prepared. Then, the above test solutions were dropped into the grooves of the composite SERS detection chips. After the solvent had evaporated completely, the Raman spectra were measured, and the intensity of the characteristic peak at 1385 cm -1 was obtained. The concentration was calculated by substituting it into the above relational expression. The measured results were compared with the theoretical values of the test solutions, and the results are shown in Table 2.

[0175] Table 2

[0176] Theoretical value Characteristic peak intensity Measured value Error <![CDATA[10 -6 mol / L]]> 12846 <![CDATA[10 -6.03 mol / L]]> 6.7％ <![CDATA[10 -7 mol / L]]> 8876 <![CDATA[10 -6.98 mol / L]]> 4.7％ <![CDATA[10 -8 mol / L]]> 4529 <![CDATA[10 -8.02 mol / L]]> 4.5％

[0177] As can be seen from the results in Table 2, the benzo[a]pyrene concentration in the solution can be accurately measured using the composite SERS detection chip of the present invention.

[0178] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A composite SERS substrate detection chip, characterized in that, Comprising: A bottom film having grooves; And Silver nanowires and metal nanoparticles located within the grooves, and the silver nanowires form a loose network structure, and the metal nanoparticles are loaded on the silver nanowires.

2. The composite SERS substrate detection chip according to claim 1, wherein The silver nanowires have a diameter of 12 - 60 nm and a length of 10 - 45 μm.

3. The composite SERS substrate detection chip according to claim 1 or 2, characterized in that, The metal nanoparticles are gold nanoparticles or silver nanoparticles; Preferably, the size of the metal nanoparticles is 5 - 60 nm.

4. The composite SERS substrate detection chip according to any one of claims 1-3, characterized in that, The bottom area of the groove is 1-17 mm 2 , and the height is 3-9 mm.

5. A method for preparing a composite SERS substrate detection chip, characterized in that, The method comprises the following steps: (1) Providing a bottom film having grooves; (2) Injecting a mixed solution containing silver nanowires and metal nanoparticles into the grooves, then placing the bottom film in liquid nitrogen, and then performing freeze-drying; Wherein, in the mixed solution containing silver nanowires and metal nanoparticles, the concentration of silver nanowires is 18 - 65 mg / mL, and the concentration of metal nanoparticles is 10 - 50 mg / mL.

6. The method according to claim 5, characterized in that, The bottom area of the groove is 1-17 mm 2 , and the height is 3-9 mm.

7. The method according to claim 6 or 7, characterized in that, The silver nanowires have a diameter of 12 - 60 nm and a length of 10 - 45 μm.

8. The method according to any one of claims 5-7, characterized in that, The metal nanoparticles are gold nanoparticles or silver nanoparticles; Preferably, the size of the metal nanoparticles is 5 - 60 nm.

9. The method according to any one of claims 5 - 8, characterized in that In the solution containing silver nanowires and metal nanoparticles, the weight ratio of silver nanowires to metal nanoparticles is 1:0.5 - 1.

5.

10. The method according to claim 5, wherein The material of the bottom film is a polymer material, silicon nitride, glass, silicon wafer or metal; Preferably, the polymer material is selected from PMMA, PC, PVC or PET.

11. The method according to claim 5, wherein The injection volume of the mixed solution containing silver nanowires and metal nanoparticles is in a volume ratio of 0.45 - 0.85:1 to the volume of the grooves.

12. The method according to claim 5 or 11, characterized in that, The conditions for the freeze-drying include: a pressure of 1 - 50 Pa and a time of 6 - 30 h.

13. A composite SERS substrate detection chip prepared by the method according to any one of claims 5 - 12.

14. The application of the composite SERS substrate detection chip according to any one of claims 1 - 4 and 13 in the detection of benzo[a]pyrene.

15. A method for detecting the concentration of benzo[a]pyrene, characterized in that, The method comprises: dropping a test solution into the grooves of the composite SERS substrate detection chip according to any one of claims 1 - 4 and 13, and performing SERS detection using a Raman spectrometer.

16. The method according to claim 15, characterized in that, The volume of the test solution is 5 - 10 μL.

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