TWI452282B - A molecule carrier used for single molecule detection - Google Patents

Description

Molecular carrier for single molecule detection

The present invention relates to a molecular carrier for single molecule detection.

Single Molecule Detection (SMD) technology is different from general conventional detection technology, and the individual behavior of single molecules is observed. Single molecule detection technology is applied in the fields of environmental safety, biotechnology, sensors, food safety, etc. widely. Single molecule detection has reached the limit of molecular detection and has long been the goal pursued by people. Compared with the traditional analytical methods, the single-molecule detection system is in a non-equilibrium state of individual behavior or fluctuation behavior under equilibrium. Therefore, it is particularly suitable for studying chemical and biochemical reaction kinetics, biomolecular interaction, structure and function information. Early diagnosis of major diseases, pathological research and high-throughput drug screening.

At present, many methods are known for single molecule detection, and the structure of molecular carriers plays an important role in the development of single molecule detection technology and detection results. In the plurality of single molecule detection methods in the prior art, the molecular carrier structure coats the colloidal silver on the surface of the glass, the silver particles adhere to the surface of the glass through the colloid, and then the glass to which the silver particles are adhered is ultrasonically washed in the glass. The surface forms dispersed silver particles to form a molecular carrier. The analyte molecule is then placed on the surface of the molecular carrier to provide laser light radiation to the analyte molecule on its molecular support by a Raman detection system. The photons in the laser light collide with the molecules of the analyte, thereby changing the direction of the photons. Raw Raman scattering. In addition, the energy exchange between the photon and the molecule of the analyte changes the energy and frequency of the photon, so that the photon has structural information of the molecule of the analyte. The radiation signal from the molecule of the analyte is received by the sensor to form a Raman spectrum, and the molecule of the analyte is analyzed by a computer.

However, in the prior art, since the surface of the glass is a flat planar structure, the generated Raman scattering signal is not strong enough, so that the resolution of the single molecule detection is low, and is not suitable for detection of low concentration and micro sample. Therefore, the scope of application is limited.

In view of this, it is necessary to provide a molecular carrier capable of improving the resolution of single molecule detection.

A molecular carrier for single molecule detection, comprising a substrate, wherein a surface of the substrate is provided with a plurality of three-dimensional nanostructures and a metal layer is coated between the surface of the three-dimensional nanostructure and the adjacent three-dimensional nanostructure The surface of the substrate.

Compared with the prior art, the present invention resonates the metal surface plasma by the metal layer under the excitation of the external incident photoelectric field, and the surface layer enhances the Raman scattering because the metal layer is disposed on the surface of the three-dimensional nanostructure ( The role of SERS) is to enhance the radiation signal, which can improve the resolution and accuracy of single molecule detection.

10,20,30,40,50‧‧‧Molecular carrier

100,200,300,400,500‧‧‧Base

101,201,301,401,501‧‧‧metal layer

1001‧‧ Motherboard

102,202,302,402,502‧‧‧Three-dimensional nanostructure

108‧‧‧ mask layer

110‧‧‧Reactive etching gas

404‧‧‧First cylinder

406‧‧‧second cylinder

504‧‧‧First cylindrical space

506‧‧‧Second cylindrical space

FIG. 1 is a schematic structural view of a molecular carrier according to a first embodiment of the present invention.

2 is a cross-sectional view of the molecular carrier according to the first embodiment of the present invention taken along the line II-II.

3 is a scanning electron micrograph of a hemispherical three-dimensional nanostructure array according to a first embodiment of the present invention.

4 is a three-dimensional diagram including a plurality of patterns in a molecular carrier according to a first embodiment of the present invention; Schematic diagram of the structure of the nanostructure array.

Figure 5 is a flow chart of a single molecule detection method using a molecular carrier of the present invention.

FIG. 6 is a schematic diagram showing the preparation process of a three-dimensional nanostructure in a molecular carrier according to a first embodiment of the present invention.

Figure 7 is a scanning electron micrograph of a single layer of nanospheres arranged in a hexagonal close-packed surface on the surface of the substrate.

FIG. 8 is a scanning electron micrograph of a semi-ellipsoidal three-dimensional nanostructure array according to a second embodiment of the present invention.

FIG. 9 is a cross-sectional view showing a semi-ellipsoidal three-dimensional nanostructure array according to a second embodiment of the present invention.

FIG. 10 is a scanning electron micrograph of an inverted pyramidal three-dimensional nanostructure array according to a third embodiment of the present invention.

FIG. 11 is a cross-sectional view showing an inverted pyramidal three-dimensional nanostructure array according to a third embodiment of the present invention.

Figure 12 is a diagram showing the Raman spectrum obtained when different three-dimensional nanostructures are used for the detection of rhodamine molecules in the molecular carrier of the present invention.

FIG. 13 is a scanning electron micrograph of a double-layered cylindrical three-dimensional nanostructure array according to a fourth embodiment of the present invention.

FIG. 14 is a schematic structural view of a molecular carrier according to a fourth embodiment of the present invention.

Figure 15 is a cross-sectional view of the molecular carrier in the XV-XV direction according to a fourth embodiment of the present invention.

Figure 16 is a schematic view showing the structure of a molecular carrier according to a fifth embodiment of the present invention.

Figure 17 is a cross-sectional view of the molecular carrier according to a fifth embodiment of the present invention taken along XVII-XVII.

The invention will be further described in detail below with reference to the drawings and specific embodiments.

Referring to FIG. 1 , FIG. 2 and FIG. 3 , a first embodiment of the present invention provides a molecular carrier 10 for single molecule detection, the molecular carrier 10 comprising a substrate 100 and a plurality of three-dimensional nanostructures formed on the surface of the substrate 100 . 102 and a metal layer 101 disposed on a surface of the substrate 100 between the surface of the three-dimensional nanostructure 102 and the adjacent three-dimensional nanostructures 102.

The substrate 100 can be an insulating substrate or a semiconductor substrate. Specifically, the material of the substrate 100 may be tantalum, cerium oxide, tantalum nitride, quartz, glass, gallium nitride, gallium arsenide, sapphire, aluminum oxide or magnesium oxide. The shape of the substrate 100 is not limited, and only needs to have two oppositely disposed planes. In the embodiment, the shape of the substrate 100 is a flat shape. The size and thickness of the substrate 100 are not limited and can be selected according to the needs of actual single molecule detection. In this embodiment, the material of the substrate 100 is cerium oxide.

The three-dimensional nanostructure 102 is disposed on a surface of the substrate 100. The three-dimensional nanostructure 102 and the substrate 100 are integrally formed. The structure of the three-dimensional nanostructure 102 is not limited and may be a convex structure or a concave structure. The raised structure is a solid of protrusions extending outward from a surface of the substrate 100, the recessed structure being recessed inwardly from a surface of the substrate 100 to form a recessed space. The structure type of the three-dimensional nanostructure 102 can be controlled according to actual needs and experimental conditions. As shown in Figure 3, this In the embodiment, the three-dimensional nanostructure 102 is a semi-spherical convex structure having a diameter of 30 nm to 1000 nm and a height of 50 nm to 1000 nm. Preferably, the hemispherical convex structure has a bottom surface diameter of 50 nm to 200 nm and a height of 100 nm to 500 nm. The distance between each adjacent two hemispherical convex structures is equal, and may be 0 nm to 50 nm. The distance between the two hemispherical convex structures refers to the distance between the bottom surfaces of the hemispherical convex structures, and the distance between the hemispherical convex structures is zero nanometer refers to the two The hemispherical convex structure is tangent, and the bottom surfaces thereof are closely connected with no spaces in between. In this embodiment, the distance between the hemispherical three-dimensional nanostructures 102 is 10 nm.

The plurality of three-dimensional nanostructures 102 are disposed in an array on a surface of the substrate 100. The array form arrangement means that the plurality of three-dimensional nanostructures 102 can be arranged in an equidistant determinant arrangement, a concentric annular arrangement or a hexagonal dense arrangement. Moreover, the plurality of three-dimensional nanostructures 102 are arranged in an array to form a single pattern or a plurality of spaced apart patterns. The single pattern may be a triangle, a parallelogram, a body, a diamond, a square, a rectangle, or a circle. As shown in FIG. 4, the three-dimensional nanostructures 102 form four different patterns in an array.

The metal layer 101 covers the surface of the substrate 100 between the surface of the three-dimensional nanostructure 102 and the adjacent three-dimensional nanostructures 102. Specifically, the metal layer 101 is a continuous layered structure formed of a metal material, and may be a single layer layer structure or a plurality of layer structure. The metal layer 101 is deposited substantially uniformly on the surface of the substrate 100 between the surface of the plurality of three-dimensional nanostructures 102 and the adjacent three-dimensional nanostructures 102. A gap (Gap) is formed between the adjacent three-dimensional nanostructures 102, where surface plasma resonance exists on the surface of the metal layer 101, thereby generating Raman scattering enhancement. The metal layer 101 may be deposited on the three-dimensional layer by electron beam evaporation, ion beam sputtering, or the like. The surface of the nanostructure 102 and the surface of the substrate 100 between adjacent three-dimensional nanostructures 102. The metal layer 101 has a thickness of 2 nm to 200 nm. Preferably, the metal layer 101 has a uniform thickness. The material of the metal layer 101 is not limited and may be a metal such as gold, silver, copper, iron or aluminum. It can be understood that the material of the metal layer 101 in the embodiment is not limited to the above, and any metal material which is solid at normal temperature may be used. The metal layer 101 in this embodiment is preferably silver having a thickness of 20 nm.

Since the substrate 100 has a plurality of three-dimensional nanostructures 102, there are mainly several advantages. First, since the metal layer 102 in the molecular carrier 10 is directly formed on the surface of the substrate 100, no additional adhesive layer or other structure is required. The metal layer can be easily removed by etching or the like, and then a metal layer of different materials is deposited according to the needs of single molecule detection. The substrate 100 can be reused, and the metal layer 101 can be used according to the actual needs of detecting a single molecule. Freely replacing, does not affect the three-dimensional nanostructure 102 on the surface of the substrate 100, that is, a "free platform"; secondly, the metal layer 101 directly covers the surface of the three-dimensional nanostructure 102, The three-dimensional nanostructure 102 has a large surface area, so that the nano metal particles in the metal layer 101 can be firmly adhered to the surface of the three-dimensional nanostructure 102 and adjacent three-dimensional layers without an adhesive layer. The surface of the substrate 100 between the nanostructures 102, when the molecular carrier 10 is used to detect a single molecule, can reduce other chemical factors such as adhesion layers during the detection process The interference of the raw layer prevents the conductive medium of the adhesive layer from affecting the resonance distribution of the surface plasma; again, since the metal layer 101 is disposed on the surface of the three-dimensional nanostructure 102, the metal surface plasma is generated under the excitation of the external incident photoelectric magnetic field. Resonance absorption, while the three-dimensional nanostructure acts as a surface-enhanced Raman scattering, which increases the SERS enhancement factor and enhances Raman scattering. The SERS enhancement factor is related to the spacing between the three-dimensional nanostructures 102, the smaller the distance between the three-dimensional nanostructures 102, the greater the SERS enhancement factor. The theoretical value of the SERS enhancement factor can be from 10 ⁵ to 10 ¹⁵ , so that a better single molecule detection result can be obtained. The molecular carrier 10 of the present embodiment has a SERS enhancement factor greater than 10 ¹⁰ .

Referring to FIG. 5 and FIG. 6 together, the present invention further provides a single molecule detection method using the molecular carrier 10, the detection method mainly includes the following steps: Step (S11), providing a molecular carrier, the molecular carrier a substrate comprising a plurality of three-dimensional nanostructures on a surface thereof, a metal layer formed on a surface of the substrate between the surface of the three-dimensional nanostructure and the adjacent three-dimensional nanostructure, the metal layer being attached to the substrate The surface of the substrate; step (S12), assembling the analyte molecules on the surface of the metal layer away from the substrate; and (S13), detecting the molecules of the analyte assembled on the substrate by using a detector.

Specifically, in step (S11), a molecular carrier 10 is provided.

The preparation method of the molecular carrier 10 mainly includes: a step (S111), providing a mother board 1001; a step (S112), forming a three-dimensional nanostructure 102 on the surface of the mother board 1001 to form the substrate 100; and step (S113) A metal layer 101 is formed on the surface of the substrate 100 to form the molecular carrier 10.

In the step (S111), the mother board 1001 may be an insulating material or a semiconductor material. The material of the mother board 1001 in this embodiment is cerium oxide. The mother board 1001 has a thickness of 200 micrometers to 300 micrometers. The size, thickness, and shape of the motherboard 1001 are not limited, and may be selected according to actual needs.

Further, a surface of the mother board 1001 may be subjected to a hydrophilic treatment.

First, the surface of the mother board 1001 is cleaned and cleaned using a clean room standard process. Then, in a solution having a temperature of 30 ° C to 100 ° C and a volume ratio of NH ₃ ‧H ₂ O:H ₂ O ₂ :H ₂ O=x:y:z, the bath is warmed for 30 minutes to 60 minutes for the mother The surface of the plate 1001 was subjected to a hydrophilic treatment, followed by rinsing with deionized water 2 to 3 times. Where x is 0.2 to 2, y is 0.2 to 2, and z is 1 to 20. Finally, the surface of the mother board 1001 was blown dry with nitrogen.

Further, the surface of the mother board 1001 may be subjected to a second hydrophilic treatment, which specifically includes the following steps: the hydrophilically treated mother board 1001 is in a 2 wt% to 5 wt% sodium lauryl sulfate solution (SDS). Soak for 2 hours to 24 hours. It will be appreciated that the surface of the mother substrate 1001 after soaking in the SDS facilitates the spreading of subsequent nanospheres and forms an ordered array of large area nanospheres.

In the step (S112), a three-dimensional nanostructure 102 is formed on the surface of the mother board 1001. The method of forming the substrate 100 specifically includes the following steps: step (S1121), forming a mask on any surface of the motherboard 1001. Film layer 108.

The mask layer 108 of the mother board 1001 is a layered structure formed by a single layer of nanospheres. It can be understood that by using a single layer of nanospheres as the mask layer 108, a convex structure can be prepared at a position corresponding to the nanospheres.

The forming of a single layer of nanospheres as the mask layer 108 on the surface of the mother board 1001 specifically includes the following steps: First, preparing a mixed liquid containing nano microspheres.

In this embodiment, 150 ml of pure water, 3 μl to 5 μl of 0.01 wt% to 10 wt% of nanospheres, and an equivalent weight of 0.1 wt% to 3 wt are sequentially added to a 15 cm diameter watch glass. After the % SDS was formed, the mixture was allowed to stand for 30 to 60 minutes. After the nanospheres are fully dispersed in the mixture, add 1 microliter to 3 microliters. The 4 wt% SDS is used to adjust the surface tension of the nanospheres to facilitate the formation of a single layer of nanosphere arrays. Wherein, the diameter of the nano microspheres may be from 60 nm to 500 nm, and specifically, the diameter of the nanospheres may be 100 nm, 200 nm, 300 nm or 400 nm, and the diameter deviation is 3 Nano ~ 5 nm. Preferred nanospheres have a diameter of 200 nm or 400 nm. The nanospheres may be polymer nanospheres or nanobelt microspheres or the like. The material of the polymer nanospheres may be polystyrene (PS) or polymethyl methacrylate (PMMA). It will be appreciated that the mixture in the watch glass can be scaled as desired.

Next, a single layer of nanosphere mixture is formed on one surface of the mother board 1001, and the single layer of nanospheres is disposed in an array on the surface of the mother board 1001.

In the present embodiment, a single-layer nanosphere solution is formed on the surface of the mother substrate 1001 by a pulling or spin coating method. The single-layer nano microspheres may be arranged in a hexagonal close-packed arrangement, a simple cubic arrangement or a concentric annular arrangement by controlling the speed of the pulling method or the rotational speed of the spin coating method.

The method for forming a single-layer nano microsphere solution on the surface of the mother substrate 1001 by the pulling method comprises the following steps: First, the hydrophilically treated mother substrate 1001 is slowly inclined along the side wall of the surface dish. In the mixture into the watch glass, the mother board 1001 is inclined at an angle of 9 to 15 . Then, the mother board 1001 is slowly lifted from the mixture of the watch glass. Among them, the above-mentioned sliding down and lifting speed are equivalent, both are 5 mm / hour ~ 10 mm / hour. In the process, the nanospheres in the solution of the nanospheres are self-assembled to form a single layer of nanospheres arranged in a hexagonal close-pack.

In this embodiment, a single layer of nanosphere solution is formed on the surface of the mother board 1001 by spin coating, which comprises the following steps: First, the hydrophilically treated mother board 1001 is in a 2 wt% sodium lauryl sulfate solution. Soak for 2 hours to 24 hours, remove the mother board after removal The surface of 1001 is coated with 3 microliters to 5 microliters of polystyrene. Next, spin-coat at a speed of 400 rpm to 500 rpm for 5 seconds to 30 seconds. Then, spin coating at a speed of 800 rpm to 1000 rpm for 30 seconds to 2 minutes. Again, the spin coating speed is increased to 1400 rpm to 1500 rpm, and spin coating is applied for 10 seconds to 20 seconds to remove excess microspheres at the edges. Finally, after drying the surface of the mother substrate 1001 on which the nanospheres are distributed, a single layer of nanospheres arranged in a hexagonal close-packed layer can be formed on the surface of the mother board 1001 to form the mask. Layer 108. Further, the surface of the mother board 1001 may be further baked after the mask layer 108 is formed. The baking temperature is 50 ° C to 100 ° C, and the baking time is 1 minute to 5 minutes.

In this embodiment, the diameter of the nanospheres may be 400 nm. Referring to FIG. 7, the nanospheres in the single-layer nano microspheres are arranged in the lowest energy arrangement, that is, the hexagonal close-packed arrangement. The single-layer nanospheres are densely packed and have the largest duty ratio. Any three adjacent nanospheres in the single-layer nanospheres are in an equilateral triangle. It can be understood that by controlling the surface tension of the nanosphere solution, the nanospheres in the single layer of nanospheres can be arranged in a simple cubic shape.

In step (S1122), the surface of the mother board 1001 is etched by the reactive etching gas 110, and a plurality of three-dimensional nanostructures 102 are formed on the surface of the mother board 1001.

The step of etching the surface of the mother substrate 1001 using the reactive etching gas 110 is performed in a microwave plasma system. The microwave plasma system is a Reaction-Ion-Etching (RIE) mode. The reactive etching gas 110 does not substantially react with the nanospheres, but the reactive etching gas 110 etches the surface of the mother substrate 1001 to form a plurality of three-dimensional nanostructures 102 to obtain the substrate 100.

In this embodiment, the surface of the mother substrate 1001 on which the single-layered nanospheres are formed is placed on the micro In the wave plasma system, an inductive power source of the microwave plasma system produces a reactive etching gas 110. The reactive etching gas 110 diffuses from the generation region and drifts to the surface of the mother board 1001 with a lower ion energy. The reactive etching gas etches the surface of the mother substrate 1001 between the single-layer nanospheres without reacting with the nanospheres to form the three-dimensional nanostructure 102. It can be understood that the spacing between the three-dimensional nanostructures 102 and the height of the three-dimensional nanostructures 102 can be controlled by controlling the etching time of the reactive etching gas 110.

In this embodiment, the working gas of the microwave plasma system includes sulfur hexafluoride (SF ₆ ) and argon (Ar) or sulfur hexafluoride (SF ₆ ) and oxygen (O ₂ ). Among them, the rate of sulphur hexafluoride is 10 standard milliliters per minute ~ 60 standard conditions per minute, the access rate of argon or oxygen is 4 standard milliliters per minute ~ 20 standard conditions per minute. The working gas forms a gas pressure of 2 Pa to 10 Pa. The plasma system has a power of 40 watts to 70 watts. The etching time using the reactive etching gas 110 is 1 minute to 2.5 minutes. Preferably, the ratio of the power of the microwave plasma system to the gas pressure of the working gas of the microwave plasma system is less than 20:1.

Further, other gases such as trifluoromethane (CHF ₃ ), tetrafluoromethane (CF ₄ ), or a mixed gas thereof may be added to the reactive etching gas 110 to adjust the etching rate. The flow rate of the trifluoromethane (CHF ₃ ), tetrafluoromethane (CF ₄ ) or a mixed gas thereof may be 20 standard milliliters per minute to ~40 standard milliliters per minute.

It can be understood that by controlling the etching conditions and the etching atmosphere, different convex three-dimensional nanostructures 102, such as semi-ellipsoidal convex structures, and the like can be obtained. If the mask layer 108 is a continuous film having a plurality of openings, a recessed three-dimensional nanostructure 102 such as a hemispherical recessed structure, a semi-ellipsoidal recessed structure, an inverted pyramidal recessed structure, or the like can be obtained.

In step (S1123), the mask layer 108 is removed to obtain the substrate 100.

Using a non-toxic or low-toxic environmentally friendly agent such as tetrahydrofuran (THF), acetone, methyl ethyl ketone, cyclohexane, n-hexane, methanol or absolute ethanol as a stripping agent to dissolve the nanospheres, the residual of the nanospheres can be removed and retained. A three-dimensional nanostructure 102 on the surface of the mother board 1001.

In this example, the polystyrene nanospheres were removed by ultrasonic cleaning in methyl ethyl ketone.

In step S113, a metal layer 101 is formed on the surface of the substrate 100 between the surface of the three-dimensional nanostructure 102 and the adjacent three-dimensional nanostructure 102 to form the molecular carrier 10.

The metal layer 101 may be vertically vapor-deposited on the surface of the substrate 100 by means of electron beam evaporation, ion beam sputtering or the like. Since the three-dimensional nanostructure 102 is formed on the surface of the substrate 100, a metal thin film is formed on the surface of the substrate 100 in the gap between the three-dimensional nanostructure 102 and the adjacent three-dimensional nanostructure 102, thereby forming the molecular carrier 10 . The metal layer 101 has a thickness of 2 nm to 200 nm, and the material of the metal layer 101 is not limited, and may be metal such as gold, silver, copper, iron or aluminum. The thickness of the metal layer 101 in this embodiment is preferably 20 nm.

In step S12, the analyte molecules are assembled on the surface of the metal layer 101 away from the substrate.

The assembling the analyte molecule mainly comprises the following steps: first, providing a solution of the analyte molecule, wherein the molecular concentration of the analyte solution may be 10 ^-7 mmol/L to 10 ^-12 mmol/L, which may be according to actual conditions. The preparation has a molecular concentration of 10 ^-10 mmol/L; in the second embodiment, the molecular carrier 10 formed with the metal layer 101 is immersed in the solution to be tested, and the soaking time can be 2 min to 60 min, preferably 10 min, the analyte molecules are uniformly dispersed on the surface of the metal layer 101; finally, the molecular carrier 10 is taken out, and the molecular carrier is washed 5 to 15 times with water or ethanol, and then dried. The molecular carrier 10 is blown dry by a device such as a hair dryer or the like to evaporate residual water or ethanol, and the molecules of the analyte are assembled on the surface of the metal layer 101.

Step S13, detecting the molecules of the analyte by using a detector.

The molecular carrier 10 assembled with the analyte molecule is placed in a detecting device, and the molecule of the analyte is detected by a detector such as a Raman spectrometer. In this embodiment, the detection parameter of the Raman spectrometer is He-Ne: the excitation wavelength is 633 nm, the excitation time is 10 sec, the device power is 9.0 mW, and the operating power is 9.0 mW×0.05×1.

The single molecule detection method provided by the invention has the following advantages: First, the prior art single molecule detection method is to deposit an adhesive layer on a substrate, and then form a metal nanostructure as a molecular carrier on the adhesive layer, so the adhesive The layer has a certain influence on the single molecule detection, and the three-dimensional nanostructure of the present invention is directly formed on the substrate by reactive ion etching, and the metal layer is directly deposited on the surface of the substrate, thereby preventing the chemical such as the adhesion layer. The factor has an influence on the detection result. Secondly, the molecular carrier in the single molecule detection method of the present invention has a three-dimensional nanostructure, and the metal layer in the molecular carrier is directly deposited on the surface of the substrate, whereas in the prior art, the metal nanostructure must pass through the adhesion. The layer is fixed on the surface of the substrate, so that the result of the single molecule detection is affected; again, the shape, size, spacing, etc. of the three-dimensional nanostructure can be conveniently controlled by controlling the preparation conditions, etc., and the operability is high; Fourth, by providing a metal layer on the surface of the three-dimensional nanostructure, the analysis of single molecule detection can be improved. , For particular dyes, biological molecules, fluorescent materials and other substances biphenyl six generations can not be detected by conventional detection methods also can be detected using the present methods.

Referring to FIG. 8 to FIG. 9 together, a second embodiment of the present invention provides a molecular carrier 20 including a substrate 200, a plurality of three-dimensional nanostructures 202 formed on the surface of the substrate 200, and the three-dimensional nanostructures 202 disposed on the surface of the substrate 200. The metal layer 201 on the surface of the substrate 200 between the surface of the nanostructure 202 and the adjacent three-dimensional nanostructure 202. The structure of the molecular carrier 20 is substantially the same as that of the molecular carrier 20 described in the first embodiment, except that the three-dimensional nanostructure 202 in the molecular carrier 20 is a convex semi-ellipsoidal structure.

The bottom surface of the semi-ellipsoidal three-dimensional nanostructure 202 is circular, and has a diameter of 50 nm to 1000 nm and a height of 50 nm to 1000 nm. Preferably, the semi-ellipsoidal convex structure has a bottom surface diameter of 50 nm to 200 nm and a height of 100 nm to 500 nm. The distance between each adjacent two semi-ellipsoidal convex structures is equal, and the distance between the two semi-ellipsoidal convex structures refers to the bottom surface of the semi-ellipsoidal convex structure The distance between 0 nm and 50 nm. In this embodiment, the distance between the hemispherical three-dimensional nanostructures 202 is 40 nm.

The metal layer 201 is deposited on the surface of the three-dimensional nanostructure 202 and the surface of the substrate 200 between adjacent three-dimensional nanostructures 202. Specifically, the metal layer 201 is a single layered layer structure or a plurality of layered structures. The metal layer 201 is deposited substantially uniformly on the surface of the substrate 200 between the surface of the plurality of three-dimensional nanostructures 202 and the adjacent three-dimensional nanostructures 202. The theoretical value of the SERS enhancement factor of the molecular carrier 20 may be 10 ⁵ to 10 ¹⁵ , and the molecular carrier 20 of the present embodiment has a SERS enhancement factor of about 10 ⁶ .

Referring to FIG. 10 to FIG. 11 together, a third embodiment of the present invention provides a molecular carrier 30. The molecular carrier 30 includes a substrate 300, a plurality of three-dimensional nanostructures 302 formed on the surface of the substrate 300, and the three-dimensional nanostructures 302 disposed on the surface of the substrate 300. A metal layer 301 on the surface of the substrate 300 between the surface of the nanostructure 302 and the adjacent three-dimensional nanostructure 302. Molecular carrier 30 The structure is substantially the same as that of the molecular carrier 30 described in the first embodiment, except that the three-dimensional nanostructure 202 in the molecular carrier 30 is a depressed inverted pyramid structure.

The inverted inverted pyramid structure means that the surface of the substrate 300 is recessed inwardly to form a recessed space in an inverted pyramid shape. The shape of the bottom surface of the inverted pyramid-shaped three-dimensional nanostructure 302 is not limited, and may be other geometric shapes such as a triangle, a rectangle, and a square. The height of the surface of the three-dimensional nanostructure 302 recessed into the substrate 300 is 50 nm to 1000 nm, and the angle α formed by the top end of the inverted pyramid three-dimensional nanostructure 302 may be 15 to 70 degrees. In this embodiment, the bottom surface of the three-dimensional nanostructure 302 is an equilateral triangle, and the side length of the equilateral triangle is 50 nm to 1000 nm. Preferably, the inverted pyramidal three-dimensional nanostructure 302 has a bottom side length of 50 nm to 200 nm, a height of the recessed base surface of 100 nm to 500 nm, and an angle α formed by the tip end is 30. degree. The distance between each adjacent two inverted pyramid three-dimensional nanostructures 302 is equal, and the distance between each two inverted pyramid three-dimensional nanostructures 302 refers to the bottom surface of the inverted pyramid three-dimensional nanostructure The distance between 0 nm and 50 nm.

The metal layer 301 is deposited on the surface of the inverted pyramidal three-dimensional nanostructure 302 and the surface of the substrate 300 between adjacent three-dimensional nanostructures 302. Specifically, the metal layer 301 is a single layer layer structure or a plurality of layer structure. The metal layer 301 is deposited substantially uniformly on the surface of the substrate 300 between the surface of the plurality of three-dimensional nanostructures 302 and the adjacent three-dimensional nanostructures 302. The theoretical value of the SERS enhancement factor of the molecular carrier 30 may be 10 ⁵ to 10 ¹⁵ , and the molecular carrier 30 of the present embodiment has a SERS enhancement factor of about 10 ⁸ .

Please refer to FIG. 12, which is a three-dimensional nanostructure of the molecular carrier in the embodiment. It is used to detect the Raman spectrum of rhodamine molecules when it is a hemispherical, inverted pyramidal or semi-ellipsoidal structure.

Referring to FIG. 13 , FIG. 14 and FIG. 15 , a fourth embodiment of the present invention provides a molecular carrier 40 for single molecule detection, the molecular carrier 40 comprising a substrate 400 and a plurality of three-dimensional nanostructures disposed on the substrate 400 . 402, and a metal layer 401 of the substrate 400 disposed between the surface of the three-dimensional nanostructure 402 and the adjacent three-dimensional nanostructure 402. The metal layer 401 is attached to the surface of the substrate 400 between the three-dimensional nanostructure 402 and the three-dimensional nanostructure 402. The molecular carrier 40 of the second embodiment of the present invention has substantially the same structure as the molecular carrier 10 of the first embodiment, except that the three-dimensional nanostructure 402 in the molecular carrier 40 is a stepped structure.

The stepped structure is disposed on a surface of the substrate 400. The stepped structure is a stepped convex structure. The stepped raised structure is an entity of stepped protrusions that extend outwardly from the surface of the substrate 400. The stepped convex structure may be a plurality of layered structures, such as a plurality of triangular prisms, a plurality of quadrangular pyramids, a plurality of layers of hexagonal prisms or a plurality of layers of cylinders. Preferably, the stepped convex structure is a plurality of layered cylindrical structures. The maximum dimension of the stepped convex structure is 1000 nm or less, that is, its length, width and height are less than or equal to 1000 nm. Preferably, the stepped raised structure has a length, a width and a height ranging from 10 nm to 500 nm.

In this embodiment, the three-dimensional nanostructure 402 is a double-layered cylindrical structure with a stepped protrusion. Specifically, the three-dimensional nanostructure 402 includes a first cylinder 404 and a second cylinder 406 disposed on a surface of the first cylinder 404. The first cylinder 404 is disposed adjacent to the substrate 400. The side of the first cylinder 404 is perpendicular to the surface of the substrate 400. The side of the second cylinder 406 is perpendicular to the upper surface of the first cylinder 404, which refers to the surface of the second cylinder 406 away from the substrate 400. The first cylinder 404 A stepped raised structure is formed with the second cylinder 406, and the second cylinder 406 is disposed within the range of the first cylinder 404. Preferably, the first cylinder 404 is disposed coaxially with the second cylinder 406. The first cylinder 404 and the second cylinder 406 are of a unitary structure, that is, the second cylinder 406 is a cylindrical structure extending from the top surface of the first cylinder 404.

The diameter of the bottom surface of the first cylinder 404 is larger than the diameter of the bottom surface of the second cylinder 406. The bottom surface of the first cylinder 404 has a diameter of 30 nm to 1000 nm and a height of 50 nm to 1000 nm. Preferably, the first cylinder 404 has a bottom surface diameter of 50 nm to 200 nm and a height of 100 nm to 500 nm. The bottom surface of the second cylinder 406 has a diameter of 10 nm to 500 nm and a height of 20 nm to 500 nm. Preferably, the second cylinder 406 has a bottom surface diameter of 20 nm to 200 nm and a height of 100 nm to 300 nm. The dimensions of the first cylinder 404 and the second cylinder 406 can be prepared according to actual needs. In this embodiment, the first cylinder 404 is disposed coaxially with the second cylinder 406. The bottom surface of the first cylinder 404 has a diameter of 380 nm and a height of 105 nm. The bottom surface of the second cylinder 406 has a diameter of 280 nm and a height of 55 nm. The distance between the adjacent first cylinders 404 may be 0 nm to 50 nm; the distance between the adjacent two second cylinders 406 is 10 nm to 100 nm.

The preparation method of the double-layered cylindrical three-dimensional nanostructure 402 is basically the same as the preparation method of the three-dimensional nanostructure 102 in the first embodiment, except that the surface of the mother board is etched by using a reactive etching gas. At the same time, the mask layer is etched. By controlling the etching time and the etching direction, on the one hand, the reactive etching gas etches the surface of the mother board between the single-layer nano microspheres to form a first cylinder 404; The reactive etching gas simultaneously etches a single layer of nanospheres on the surface of the mother board to form smaller diameter nanospheres, that is, each nanosphere in the single layer of nanospheres is Etching reduction to the first a cylindrical microsphere 404 having a smaller diameter, wherein the reactive etching gas can further etch the first cylinder 404 to form the second cylinder 406, thereby forming the plurality of stepped three-dimensional nanoparticles Structure 402.

The metal layer 401 is deposited on the surface of the substrate 400 between the surface of the three-dimensional nanostructure 402 and the adjacent three-dimensional nanostructure 402. Specifically, the metal layer 401 is a single layer layer structure or a plurality of layer structure formed by spreading a plurality of dispersed nano metal particles. The nano metal particles are dispersed on the surface of the substrate 400 between the surface of the plurality of three-dimensional nanostructures 402 and the adjacent three-dimensional nanostructures 402.

With respect to the first embodiment, the molecular carrier 40 provided by the second embodiment of the present invention has two distances between adjacent double-layered cylindrical structures because the three-dimensional nanostructure 402 is a convex double-layered cylindrical structure. Different gaps (Gap), that is, a gap is formed between adjacent first cylinders 404, and another gap is formed between adjacent second cylinders 406. Therefore, when the molecular carrier is used for single molecule detection, under the excitation of the laser emitted by the detector, the metal layer 401 at the gap between the adjacent first cylinders 404 generates surface electromagnetic coupling resonance while the second cylinder The metal layer 401 at the gap between 406 generates electromagnetic coupling resonance, which enhances the Raman scattering of the surface of the metal layer, thereby further improving the SERS enhancement factor, enhancing the Raman spectrum, and improving the resolution of the single molecule detection, Single molecule detection results are more accurate.

Referring to FIG. 16 and FIG. 17, a fifth embodiment of the present invention provides a molecular carrier 50 for single molecule detection, the molecular carrier 50 comprising a substrate 500, a plurality of three-dimensional nanostructures 502 disposed on the substrate 500, and a setting A metal layer 501 of the substrate 500 between the surface of the three-dimensional nanostructure 502 and the adjacent three-dimensional nanostructure 502. The molecular carrier 50 according to the fifth embodiment of the present invention has substantially the same structure as the molecular carrier 50 of the fourth embodiment, except that the three-dimensional nanostructure 502 in the molecular carrier 50 is 502. It is a stepped recessed structure.

The stepped recessed structure is a space of a stepped recess formed by recessing from the surface of the substrate 500 into the base 500. The stepped recess structure may be a plurality of layered structures, such as a plurality of layers of triangular prisms, a plurality of layers of quadrangular prisms, a plurality of layers of hexagonal prisms or a plurality of layers of cylinders. Preferably, the stepped recessed structure is a plurality of layered cylindrical structures. The stepped recessed structure is a plurality of layers of cylindrical structures, which means that the space of the stepped recesses is a plurality of layers of cylindrical shapes. The maximum dimension of the stepped recessed structure is 1000 nm or less, that is, its length, width and height are less than or equal to 1000 nm. Preferably, the stepped recess structure has a length, a width and a height ranging from 10 nm to 500 nm.

In this embodiment, the shape of the three-dimensional nanostructure 502 is a double-layered cylindrical structure, and the cylindrical structure is a cylindrical structure space, specifically including a first cylindrical space 504, and a first cylindrical space 504 is connected to the second cylindrical space 506. The first cylindrical space 504 is disposed coaxially with the second cylindrical space 506. The first cylindrical space 504 is disposed adjacent to a surface of the substrate 500. The diameter of the first cylindrical space 504 is greater than the diameter of the second cylindrical space 506. The first cylindrical space 504 has a diameter of 30 nm to 1000 nm and a height of 50 nm to 1000 nm. The second cylindrical space 506 has a diameter of 10 nm to 500 nm and a height of 20 nm to 500 nm. The dimensions of the second cylindrical space 506 and the second cylindrical space 506 can be prepared according to actual needs.

The surfaces of the plurality of three-dimensional nanostructures 502 on the substrate 500 are arranged in an array. The arrangement in the form of an array means that the plurality of three-dimensional nanostructures 502 can be arranged in a simple cubic arrangement, a concentric annular arrangement or a hexagonal dense arrangement, and the plurality of three-dimensional nanometers arranged in an array form. Structure 502 can form a single pattern or a plurality of patterns. The distance between the adjacent two three-dimensional nanostructures 502 equal. Specifically, the distance between the adjacent first cylindrical spaces 504 may be from 1 nm to 1000 nm, preferably from 10 nm to 50 nm; between the adjacent two second cylindrical spaces 506 The distance is from 15 nm to 900 nm, preferably from 20 nm to 100 nm. The form of the surface of the plurality of three-dimensional nanostructures 502 on the substrate 500 and the distance between the adjacent two three-dimensional nanostructures 502 can be prepared according to actual needs. In this embodiment, the plurality of three-dimensional nanostructures 502 are arranged in a hexagonal close-packed form to form a single square pattern.

The method for preparing the three-dimensional nanostructure 502 of the double-layered cylindrical space is substantially the same as the method for preparing the three-dimensional nanostructure 402 of the fourth embodiment, except that the mask layer has a plurality of openings. Continuous film. The reactive etching gas etches the surface of the substrate in the opening while etching the mask layer. In one aspect, the reactive etching gas etches a surface of the substrate of the opening to form a first cylindrical space 504; on the other hand, the reactive etching gas simultaneously faces a surface of the substrate The mask layer is etched to make the opening larger, so that the reactive etching gas etches a larger range to the substrate, thereby forming the first cylindrical space 504, and finally preparing a step at a position corresponding to the opening. a concave structure. It can be understood that the spacing between the three-dimensional nanostructures 502 can be controlled by controlling the etching time of the reactive etching gas, and the dimensions of the first cylindrical space 504 and the second cylindrical space 506 in the three-dimensional nanostructure 502 can also be controlled. The continuous film having a plurality of openings can be prepared by nanoimprinting, template deposition, or the like.

The molecular carrier 50 provided by the fifth embodiment of the present invention functions substantially the same as the molecular carrier 40 provided by the fourth embodiment. Since the three-dimensional nanostructure 502 is a double-layered cylindrical space, the double-layered cylindrical space has two different gaps, that is, the first cylindrical space 504 forms a gap, and the second cylindrical space 506 forms another gap. . Therefore, when the molecular carrier is used for single molecule detection, the metal layer in the first cylindrical space 504 generates surface electromagnetic coupling resonance under the excitation of the external incident photoelectric magnetic field, and the metal layer of the second cylindrical space 506 generates electromagnetic The coupler resonance enhances the Raman scattering, so the SERS enhancement factor can be further improved, the resolution of the single molecule detection is improved, and the single molecule detection result is more accurate.

In summary, the present invention has indeed met the requirements of the invention patent, and has filed a patent application according to law. However, the above description is only a preferred embodiment of the present invention, and the claim of the present invention cannot be limited thereby. Equivalent modifications or variations made by those skilled in the art in light of the spirit of the invention are intended to be included in the following claims.

10‧‧‧Molecular carrier

100‧‧‧Base

101‧‧‧metal layer

102‧‧‧Three-dimensional nanostructure

Claims (16)

A molecular carrier for single molecule detection, comprising a substrate, wherein the substrate is provided with a plurality of three-dimensional nanostructures on one surface and a metal layer coated on the surface of the three-dimensional nanostructure and adjacent three-dimensional nanostructures The plurality of three-dimensional nanostructures form an array between the surfaces of the substrate and are integrally formed with the substrate. The molecular carrier for single molecule detection according to Item 1, wherein the three-dimensional nanostructure is a convex structure or a concave structure. The molecular carrier for single molecule detection according to claim 2, wherein the distance between the adjacent three-dimensional nanostructures is from 0 nm to 50 nm. The molecular carrier for single molecule detection according to claim 2, wherein the three-dimensional nanostructure is a hemispherical structure, a semi-ellipsoidal structure or an inverted pyramidal structure. The molecular carrier for single molecule detection according to claim 2, wherein the three-dimensional nanostructure is a stepped structure. The molecular carrier for single molecule detection according to claim 5, wherein the stepped structure has a maximum size of 1000 nm or less. The molecular carrier for single molecule detection according to claim 5, wherein the stepped structure is a plurality of triangular prisms, a plurality of quadrangular pyramids, a plurality of layers of hexagonal prisms or a plurality of layers of cylinders. The molecular carrier for single molecule detection according to Item 5, wherein the three-dimensional nanostructure comprises a first cylinder and a second cylinder disposed on the upper surface of the first cylinder, and the first cylinder The diameter is larger than the diameter of the second cylinder, and the first cylinder and the second cylinder are integrally formed and coaxially disposed. The molecular carrier for single molecule detection according to claim 5, wherein the three-dimensional nanostructure comprises a first cylindrical space, and a second cylindrical space communicating with the first cylindrical space The first cylindrical space is disposed coaxially with the second cylindrical space, the first cylindrical space is disposed near a surface of the substrate and the diameter of the first cylindrical space is larger than the diameter of the second cylindrical space. The molecular carrier for single molecule detection according to Item 1, wherein the plurality of three-dimensional nanostructures are arranged in a simple cubic arrangement, a concentric annular arrangement or a hexagonal dense arrangement. The surface of the substrate. The molecular carrier for single molecule detection according to claim 1, wherein the plurality of three-dimensional nanostructures form a single pattern or a plurality of patterns. The molecular carrier for single molecule detection according to Item 1, wherein the metal layer is a single layered layer structure or a plurality of layered structures. The molecular carrier according to claim 1, wherein the metal layer is a continuous layered structure formed of a metal material. The molecular carrier for single molecule detection according to claim 13, wherein the metal layer is deposited on a surface of the three-dimensional nanostructure and a surface of the substrate between adjacent three-dimensional nanostructures. The molecular carrier for single molecule detection according to the above item 1, wherein the metal layer has a thickness of from 2 nm to 200 nm. The molecular carrier for single molecule detection according to claim 1, wherein the molecular carrier surface enhanced Raman scattering has an enhancement factor of 10 ⁵ to 10 ¹⁵ .