A method for manufacturing a surface-enhanced raman scattering substrate for detecting a virus macromolecule

Abstract

A method for manufacturing a surface-enhanced Raman scattering substrate for detecting virus macromolecules, in which a high-refractive spherical open cavity array with uniform noble metal nanoparticles on the inner surface is manufactured by using a dielectric nanoparticle monolayer close-packed array as a template, the coupling effect of surface plasmon resonance of noble metal particles and high-order magnetic resonance of high-refractive spherical cavities is utilized to form a uniform electric field enhanced hot zone on the inner surface of the cavity; after virus molecules enter the high-refractive spherical cavity with adsorbed noble metal particles, the virus molecule surface directly contacts the noble metal particles adsorbed on the inner surface of the high-refractive spherical cavity in a large area, that is, the virus molecules, the noble metal particles and the inner surface of the spherical cavity are coupled to overlap the electric field hot zone, which greatly improves the Raman signal intensity of the virus molecules and realizes high-sensitivity detection of the virus molecules.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of Raman spectrum detection and nano-array structure manufacturing, in particular to a manufacturing method of a surface-enhanced Raman scattering substrate for virus macromolecule detection. BACKGROUND

[0002] Currently widely used virus detection technologies mainly include nucleic acid detection and serological detection. For nucleic acid detection, due to the difference in virus load in different samples, false positive and false negative results may occur, and contradictory detection results of the same case within a few days may mislead disease prevention and control and medical diagnosis. For serological detection, since many viruses can form partially identical antibodies, serological detection may also misdiagnose false positive and false negative viruses. In addition, nucleic acid detection and serological detection require complex equipment, skilled professional technicians and sufficient expensive reagents (primers, enzymes, buffers, polymerases, etc.), and are not suitable for extensive routine screening of virus molecules. Therefore, it is urgent to find a simple, economical and sensitive virus detection technology.

[0003] Raman spectrum detection can achieve precise analysis of a single small molecule, and theoretically can completely suppress false diagnosis due to low virus load. Since Raman spectrum detection is only for virus molecules, it can well eliminate false positive or false negative detection results due to similar antibody characteristics, and achieve precise detection of virus molecules. In addition, Raman spectrum detection does not require other reagents, biological calibration or other special treatment, and is considered as a method for rapid and accurate detection of viruses.

[0004] According to the principle of surface electric field enhanced Raman scattering, the enhanced electric field formed by surface plasmon resonance of noble metal structures can greatly improve the Raman signal intensity. The traditional Raman spectrum detection substrate (Chem. Soc. Rev., 2017, DOI: 10.1039 / c7cs00238f) uses a sharp spike or gap structure with a feature size of less than 10 nm to form a local enhanced electric field, and the electric field strength is particularly sensitive to the size change of the small structure, so it is difficult to manufacture a uniform size spike or form an enhanced electric field, which requires the use of expensive equipment and a large amount of time consumption. In addition, the enhanced electric field decays exponentially with the distance from the surface of the noble metal enhanced region, and the enhanced electric field in the spike or gap with a size of about 10 nm is difficult to fully contact with the virus molecules with a diameter of about 100 nm in a large area, so the traditional Raman substrate is difficult to achieve high sensitivity for virus molecule Raman characteristic spectrum detection. SUMMARY

[0005] In order to overcome the above-mentioned prior art defects, the purpose of the present application is to provide a manufacturing method of surface enhanced Raman scattering substrate for virus macromolecule detection, to manufacture high refractive index spherical open cavity particle array with uniform noble metal nanoparticles on the inner surface by using dielectric nanoparticle monolayer close-packed array as a template, to form a uniform electric field enhanced hot zone on the inner surface of the cavity by using the coupling effect of surface plasmon resonance of noble metal particles and high-order magnetic resonance of high refractive index spherical cavity; the virus molecules coincide with the coupling enhanced electric field hot zone of noble metal particles and high refractive index spherical cavity, greatly improve the Raman signal intensity of virus molecules, and realize high sensitivity detection of virus molecules.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0007] A manufacturing method of surface enhanced Raman scattering substrate for virus macromolecule detection, comprising the following steps:

[0008] 1) A layer of photoresist is prepared on the surface of the substrate; wherein the substrate surface is flat, which can be glass, transparent epoxy resin, silica gel, polyethylene terephthalate (PET) film, and the thickness of the substrate is 0.5-5mm;

[0009] 2) The photoresist layer is patterned to form a template of regionalized nanoparticle array structure;

[0010] 3) A close-packed monolayer of dielectric nanoparticles is manufactured on the photoresist layer;

[0011] 4) The diameter of the dielectric nanoparticles is uniformly reduced by using reactive ion etching method, and the spacing of the nanoparticles is regulated;

[0012] 5) The dielectric nanoparticle layer is directly irradiated by a light source, and the photoresist is partially exposed by passing through the particle layer;

[0013] 6) A high refractive index dielectric or semiconductor thin film is prepared on the surface of the dielectric nanoparticle monolayer array to form a core-shell particle array with a dielectric particle as the core and a high refractive index material as the shell;

[0014] 7) The exposed photoresist layer and the nanoparticles on its surface are removed by using a photoresist developer;

[0015] 8) A support layer is manufactured on the remaining photoresist and the upper surface of the dielectric particle;

[0016] 9) The photoresist is irradiated from the bottom side of the substrate, and the part of the photoresist layer in direct contact with the substrate is exposed to form an exposed area with a thickness of 50-200nm;

[0017] 10) removing the exposed photoresist layer with a developer, separating the core-shell particle array from the substrate, and turning the core-shell particle array with high refractive index film over to the support layer, so that the core-shell particle array in direct contact with the substrate in step 2) is the upper surface of the whole structure;

[0018] 11) partially removing the high refractive index film coated on the surface of the dielectric nanoparticles by reactive ion etching, exposing the internal dielectric nanoparticles, and removing the internal dielectric nanoparticles by using 3-7% dilute hydrochloric acid, dilute sulfuric acid, hydrofluoric acid, 0.1-1M sodium hydroxide solution, or heating and gasification at 200-400℃, to form a high refractive index open cavity particle array;

[0019] 12) manufacturing uniform noble metal particles on the inner diameter surface of the spherical cavity, and completing the manufacturing of the high refractive index spherical cavity particle array with uniform noble metal particles on the inner surface.

[0020] The photoresist in step 2) is EPG533, EPG535, AZ4620, AZ5355, or SU8-3010, and the thickness is 0.8-3μm.

[0021] The dielectric nanoparticles in step 3) are silica, polystyrene, or alumina particles, and the shape of the dielectric nanoparticles is spherical or ellipsoidal, and the diameter of the dielectric nanoparticles is 100-400nm, and the dielectric nanoparticle array in the photoresist pattern is manufactured by spin coating, single layer film transfer, pulling method, or Langmuir-Blodgett film method.

[0022] After the etching method in step 4), the distance between adjacent particles is 40-200nm.

[0023] After partial exposure in step 5), the thickness of the exposed photoresist layer formed is 100-500nm.

[0024] The high refractive index film material in step 6) is silicon, silicon carbide, titanium dioxide, or silicon nitride, and is manufactured by magnetron sputtering method, and the thickness of the high refractive index film is 40-100nm.

[0025] In step 7), the sample obtained in step 6) is placed in a developer for 60-90 minutes to remove the exposed photoresist layer in step 5).

[0026] The support layer material covered in step 8) is epoxy resin, silica gel, or photoresist, and is manufactured by direct coating or spin coating method, and the thickness of the support layer is 0.5-5mm.

[0027] The exposed photoresist layer with a thickness of 500-1000 nm in step 9) is placed in a developing solution for 60-90 minutes after the exposure treatment to form the substrate separation in step 10) so that the high-refractive spherical cavity particle array is transferred to the support layer.

[0028] The method for uniformly distributing noble metal particles on the inner surface of the spherical cavity in step 12) uses electrostatic attraction. First, the material of the noble metal particles is gold or silver, which is prepared by directly reducing chloroauric acid or silver nitrate with sodium citrate to obtain gold or silver particles with an equivalent diameter of 15-30 nm and a negatively charged surface. The high-refractive spherical cavity array is suspended in a sealed container containing 50-200 μl of 3-aminopropyltrimethoxysilane (APTMS) and is continuously heated at 60-80 °C for 50-80 minutes, and then rinsed to complete the modification of the amino group. The high-refractive spherical cavity array sample is immersed in a gold or silver nanoparticle colloid and is left to stand at room temperature for 6-10 hours, and then rinsed to obtain a high-refractive spherical cavity array with gold or silver nanoparticles uniformly distributed on the inner surface.

[0029] The present application has the following advantages:

[0030] (1) The combination structure of noble metal and dielectric material in the present application can form a high-order magnetic resonance coupling resonance of plasmon and dielectric material to enhance the electric field. Therefore, the combination structure of noble metal and dielectric material is expected to become the development direction of a large-area uniform controllable Raman substrate independent of the gap.

[0031] (2) The combination of the surface plasmon resonance of the noble metal particles and the high-order magnetic resonance of the silicon spherical cavity forms a uniform electric field enhancement hot zone on the inner surface of the cavity. The electric field hot zone is the entire spherical inner surface. After the virus molecules enter the silicon spherical cavity with adsorbed noble metal particles, the virus molecules can directly contact the noble metal particles adsorbed on the inner surface of the silicon spherical cavity in a large area, i.e., the coupling of the virus molecules and the noble metal particles and the silicon spherical cavity coincides with the electric field hot zone, greatly improving the Raman signal strength of the virus molecules and realizing high-sensitivity detection of the virus molecules. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 A cross-sectional schematic diagram of preparing a photoresist on a substrate.

[0033] Figure 2 A cross-sectional schematic diagram of patterning a photoresist.

[0034] Figure 3 A schematic diagram of preparing a monolayer close-packed silicon dioxide nanoparticle array on the surface of the patterned photoresist.

[0035] Figure 4 A schematic diagram of using reactive ion etching to increase the distance between adjacent particles of the silicon dioxide array.

[0036] Figure 5 Schematic diagram for exposing the upper surface of photoresist to UV light to expose part of the photoresist.

[0037] Figure 6 Schematic diagram for covering the surface of particle array with high refractive index film.

[0038] Figure 7 Schematic diagram for removing the outer high refractive index shell of dielectric nanoparticle in the pattern after removing the upper surface particles.

[0039] Figure 8 Schematic diagram for coating the surface of core-shell particle array with support layer.

[0040] Figure 9 Schematic diagram for exposing the part of photoresist directly contacted by substrate to UV light.

[0041] Figure 10 Schematic diagram for separating the substrate from the core-shell particle array by removing part of the photoresist layer.

[0042] Figure 11 Schematic diagram for removing part of the upper surface high refractive index shell, exposing the internal dielectric nanoparticle, and removing the internal dielectric nanoparticle.

[0043] Figure 12 Schematic diagram for manufacturing uniform noble metal nanoparticle array on the inner surface of spherical cavity.

[0044] Figure 13 Axonometric view of a functional unit of the Raman detection substrate of the present embodiment.

[0045] Figure 14 Scanning electron microscope topography of the silicon spherical cavity particle array of the present embodiment with gold particles adsorbed on the surface.

[0046] Figure 15 Distribution of the electric field hot zone near the surface of gold particles on the inner surface of the silicon spherical cavity of the present embodiment. DETAILED DESCRIPTION

[0047] The technical solutions of the present application will be described clearly and completely below in combination with the drawings and embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0048] Embodiment I, a method for manufacturing a surface-enhanced Raman scattering substrate for detecting viral macromolecules, comprising the following steps:

[0049] 1) Refer to Figure 1, first with a thickness of 1 mm as the substrate 1, and sequentially using acetone, ethanol and deionized water for ultrasonic cleaning, nitrogen blowing dry and drying in a 150 °C air drying oven for 1 hour, then spin coating a layer of photoresist EPG533 as the sacrificial layer 2 on the surface of the substrate 1, with a thickness of 980 nm;

[0050] 2) Refer to Figure 2 , using a photoetching machine to expose through a mask, and after developing, a template of a square cell pattern array structure is formed on the photoresist layer 2;

[0051] 3) Refer to Figure 3 , prepare silica nanosphere particles with a diameter D = 360 ± 10 nm, after ultrasonic cleaning, disperse the silica nanosphere particles in n-butanol at a mass fraction of 15%, then ultrasonic for 30 minutes to make it uniformly dispersed, take 10 μl of particles and drop on the surface of the water solution in a 60 mm diameter culture dish to form a continuous and dense single layer of silica nanosphere particle film; using the single layer film transfer method, transfer the silica nanosphere particle film on the water surface to the surface of the patterned photoresist layer 2 and place it on a hot plate at a temperature of 50 °C for drying, to obtain a closely arranged single layer of silica nanosphere particle array 3 on the patterned photoresist layer 2;

[0052] 4) Refer to Figure 4 , use a reactive ion etching machine to reduce the particle diameter of the particle array 3 to 310 ± 10 nm to obtain sample one;

[0053] 5) Refer to Figure 5 , use a UV light source to directly irradiate the sample one for 5 seconds to expose the photoresist layer 2 and form an exposed photoresist layer 4 with a thickness of 200 nm;

[0054] 6) Refer to Figure 6 , use a magnetron sputtering technique to deposit a 80 nm silicon film 5 on the surface of the particle array 3 to form a core-shell particle array with a silica core and a silicon shell, to obtain sample two;

[0055] 7) Refer to Figure 7 , immerse the sample two in a 0.5 wt% sodium hydroxide solution for 5 minutes to remove the exposed photoresist layer 4 and the core-shell particles on its surface;

[0056] 8) Refer to Figure 8 , apply a colloidal substance prepared by mixing polydimethylsiloxane (PDMS) and a curing agent at a mass fraction of 10:1 to the surface of the photoresist layer 2, then remove the air bubbles in the PDMS with a vacuum pump, and place it in an oven at 80 °C for 2 hours to cure and form a support layer 6, to obtain sample three;

[0057] 9) Refer to Figure 9The sample three is turned over, the sample four is obtained by irradiating the sample three with UV light for 5 seconds from the side of the substrate 1, so that the photoresist layer 2 is exposed to about 200 nm of the glass slide, and the exposed photoresist layer 7 can be dissolved and removed to form a sample four;

[0058] 10) Refer to Figure 10 The sample four is placed in a sodium hydroxide solution with a concentration of 0.5 wt% for ultrasonic cleaning for 5 minutes, the substrate 1 is removed, and a core-shell particle array with a PDMS support layer 6 is formed to obtain a sample five;

[0059] 11) Refer to Figure 11 The sample five is placed in a reaction dry etching machine to remove the silicon film 5 exposed on the upper surface, and is immersed in a hydrofluoric acid solution with a volume fraction of 5% for 10 seconds to remove the internal silicon dioxide particles, so that a silicon open sphere high refractive index cavity array with a PDMS support layer is formed to obtain a sample six;

[0060] 12) Refer to Figure 12 The sample six is placed in a sealed container containing 100 μl of 3-aminopropyltrimethoxysilane (APTMS) and heated at 80°C for 1 hour to modify the amino group on the inner surface of the hollow silicon spherical particle, and gold nanoparticles with a diameter of about 20 nm are formed by using sodium citrate to directly reduce chloroauric acid. The amino-modified sample is placed in the gold particle colloid for 8 hours, and then taken out, washed with ultrapure water for 3 times, and placed in an oven at 80°C for 30 minutes, so that a silicon spherical high refractive index open cavity array with uniformly adsorbed gold particles 8 on the surface is formed.

[0061] Figure 13 The silicon spherical high refractive index open cavity array with uniformly distributed gold particles on the inner surface in this embodiment is a detection unit of the silicon spherical high refractive index open cavity array with uniformly distributed gold particles on the inner surface, and the structure formed in this embodiment is as shown in Figure 14 The silicon spherical high refractive index open cavity array with uniformly distributed gold particles on the inner surface is formed, and since the etching depth of the silicon spherical shell in step 11) in the embodiment is about 100 nm, a bowl-like array structure is formed. Figure 14 The upper left corner of the drawing shows a single particle, and the spherical open cavity structure with gold particles modified on the inner surface can be clearly seen; the electric field distribution of the above-mentioned particle structure is analyzed by using FDTD, and when the wavelength of the incident light is set to 633 nm, as shown in Figure 15 The electric field hot zone near the surface of the gold particles is formed by the coupling of the surface plasmon resonance of the gold particles and the 3-order magnetic resonance of the silicon spherical open cavity, and this resonance electric field enhancement hot zone is independent of the nanoscale gap. Moreover, the topographic characteristics of the inner concave surface can fully contact the virus molecules with a diameter of about 100 nm, so that high-sensitivity detection of the virus molecule signal can be realized. In addition, the strength of the electric field hot zone is optimized by adjusting the size and arrangement of the nanoparticles in this embodiment.

[0062] The above-mentioned are only embodiments of the present application, and the common knowledge of specific structures and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the present application, a number of modifications and improvements can be made. These should also be considered as the protection scope of the present application, the protection scope of the present application should be subject to the content of its claims, and the specific embodiments in the description can be used to explain the content of the claims.

Claims

1. A method for manufacturing a surface-enhanced Raman scattering substrate for detection of a virus macromolecule, characterized by, The method comprises the following steps: 1) preparing a photoresist layer on the surface of a substrate; 2) patterning the photoresist layer to form a template for the regionized nano-particle array structure; 3) manufacturing closely arranged single-layer dielectric nano-particles on the photoresist layer; In step 3), the dielectric nano-particles are silica, polystyrene or alumina particles; the dielectric nano-particles are spherical or ellipsoidal; the diameter of the dielectric nano-particles is 100-400 nm; the dielectric nano-particle array in the photoresist pattern is manufactured by spin coating, single-layer film transfer, pulling or Langmuir-Blodgett film method; 4) uniformly reducing the diameter of the dielectric nano-particles by reactive ion etching to control the distance between the nano-particles; After the etching in step 4), the distance between the adjacent nano-particles is 40-200 nm; 5) directly irradiating the dielectric nano-particle layer with a light source to expose the photoresist layer through the nano-particle layer; 6) preparing a high-refractive-index dielectric or semiconductor thin film on the surface of the single-layer dielectric nano-particle array to form a core-shell nano-particle array with a dielectric particle core and a high-refractive-index material shell; In step 6), the high-refractive-index thin film material is silicon, silicon carbide, titanium dioxide or silicon nitride, which is manufactured by magnetron sputtering; the thickness of the high-refractive-index thin film is 40-100 nm; 7) removing the exposed photoresist layer and the nano-particles on the surface thereof by using a photoresist developer; 8) manufacturing a support layer on the remaining photoresist and the surface of the dielectric particles; 9) irradiating the photoresist from the bottom side of the substrate to expose the part of the photoresist layer in direct contact with the substrate, 10) removing the exposed photoresist layer by using a developer to separate the core-shell nano-particle array from the substrate, thereby turning the core-shell nano-particle array with the high-refractive-index thin film to the support layer, so that the core-shell nano-particle array in direct contact with the substrate in step 2) is the upper surface of the entire structure; 11) partially removing the high-refractive-index thin film coated on the surface of the dielectric nano-particle by using reactive ion etching to expose the internal dielectric nano-particle; the exposed internal dielectric nano-particle has a height of one quarter to one half of its diameter; the internal dielectric spherical particle is removed to form a spherical cavity array of high-refractive-index material; 12) manufacturing uniform noble metal particles on the inner diameter surface of the spherical cavity to complete the manufacturing of the high-refractive-index spherical cavity particle array with the noble metal particles uniformly distributed on the inner surface; In step 12), the noble metal particles are uniformly distributed on the inner surface of the spherical cavity by electrostatic attraction; first, the material of the noble metal particles is gold or silver, which is prepared by directly reducing chloroauric acid or silver nitrate with sodium citrate to obtain gold or silver particles with an equivalent diameter of 15-30 nm and a negatively charged surface; the high-refractive-index spherical cavity array is suspended in a sealed container containing 50-200 ml of 3-aminopropyltrimethoxysilane (APTMS) and is continuously heated at 60-80°C for 50-80 minutes to complete the modification of the amino group after rinsing; the high-refractive-index spherical cavity array sample is immersed in a gold or silver nano-particle colloid and is left to stand at room temperature for 6-10 hours to obtain the high-refractive-index spherical cavity array with the gold or silver nano-particles uniformly distributed on the inner surface after rinsing.

2. The method of claim 1, wherein: The photoresist in step 2) is EPG533, EPG535, AZ4620, AZ5355 or SU8-3010, and the thickness is 0.8-3mm.

3. The method of claim 1, wherein: The thickness of the exposed photoresist layer formed in step 5) is 100-500nm.

4. The method of claim 1, wherein: In step 7), the sample obtained in step 6) is placed in a developing solution for 60-90 minutes to remove the exposed photoresist layer in step 5).

5. The method of claim 1, wherein: In step 8), the covering support layer material is epoxy resin or silica gel, which is prepared by direct coating or spin coating, and the thickness of the support layer is 0.5-5mm.

6. The method of claim 1, wherein: In step 9), the exposed photoresist layer with a thickness of 500-1000nm is placed in a developing solution for 60-90 minutes after exposure treatment to form step 10). In step 10), the substrate is separated to flip the high refractive index spherical cavity particle array to the support layer.

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