WO2021046741A1 - Substrate material for high-efficiency surface-enhanced raman scattering and preparation method - Google Patents

Substrate material for high-efficiency surface-enhanced raman scattering and preparation method Download PDF

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WO2021046741A1
WO2021046741A1 PCT/CN2019/105288 CN2019105288W WO2021046741A1 WO 2021046741 A1 WO2021046741 A1 WO 2021046741A1 CN 2019105288 W CN2019105288 W CN 2019105288W WO 2021046741 A1 WO2021046741 A1 WO 2021046741A1
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solution
stirring
gold
enhanced raman
volume ratio
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French (fr)
Chinese (zh)
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熊良钟
张茂峰
熊清爵
陈敏
阮志燕
王振
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亳州市新健康科技有限公司
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering

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  • the invention relates to the technical field of Raman scattering, in particular to a high-efficiency surface-enhanced Raman scattering base material and a preparation method.
  • kidney damage characterized by kidney damage is one of the most challenging health problems worldwide.
  • urine routine tests, osmotic pressure, blood creatinine, urea nitrogen, and endogenous creatinine clearance are generally used to determine kidney damage.
  • NAG enzyme N-acetyl- ⁇ -D-glucosaminidase
  • KIM-1 kidney injury molecule 1
  • the content of -1 can change within 4-6 hours after kidney injury. It can quickly, sensitively and specifically reflect the damage and recovery process of various kidney diseases. It can become a reliable biological marker for detecting early kidney injury. Therefore, High-precision detection of KIM-1 content in urine is of great significance for the diagnosis and treatment of acute and chronic kidney diseases characterized by kidney damage.
  • the main method for determining the KIM-1 content in urine is the ELISA method with a sensitivity of pg/ml. Therefore, this method cannot achieve the quantitative detection of KIM-1 content in urine within a large dynamic range.
  • SERS Surface Enhanced Raman Scattering
  • SERS detection can be completed quickly within a few minutes. Therefore, surface-enhanced Raman scattering has been widely used in various chemical sensing, biological analysis, biosensing, and early cancer diagnosis.
  • the current surface-enhanced Raman scattering substrate materials mostly use smooth macroscopic glass, gold, and silver. Or the bimetal film as the base material does not have sufficient plasma "hot spots", which leads to the limited sensitivity of the surface enhanced Raman technology.
  • the current selection of SERS detection substrate material is used to bond Manra detection molecules, of which the best effect is the P-ATP molecule, the sensitivity is ng/ml-pg/ml, although the P-ATP is bonded After the molecule, the sensitivity of SERS detection has been improved, but the detection sensitivity and detection range width required by many detected objects are higher.
  • the purpose of the present invention is to provide an efficient surface-enhanced Raman scattering substrate material and a preparation method, which can achieve high-sensitivity detection of KIM-1 content in urine, up to fg/ml level, thereby achieving a KIM-1 with a large dynamic range Assay.
  • One aspect of the present invention is to provide a high-efficiency surface-enhanced Raman scattering substrate including:
  • Nano-shell the nano-shell is made of nano-silver material, the nano-shell is evenly and completely wrapped on the surface of the nano-core, and the outer surface of the nano-shell is bonded with the Raman detection molecule vitamin K4.
  • the vitamin K4 introduces a sulfhydryl group through diazotization thiolysis.
  • the diameter of the nano-core is 19-26 nm, and the length is 80-96 nm.
  • the thickness of the nano-shell is 2-18 nm.
  • Another aspect of the present invention is to provide a method for preparing a high-efficiency surface-enhanced Raman scattering base material, which includes the following steps:
  • the vitamin K4 ethanol solution is diazotized and mercaptolyzed to introduce sulfhydryl groups, and then added to the gold core silver shell nanorod suspension to obtain a mixture, and the mixture is gently shaken to obtain vitamin K4 labeled gold core silver shell nanorods.
  • step 1) is deionized water
  • the concentration of the HAuCl 4 solution is 25 mM, and the dilution factor is 50 times;
  • the concentration of the CTAB solution is 0.2M
  • the volume ratio of the HAuCl 4 solution to the CTAB solution is 1:50;
  • step 2) the concentration of the NaBH 4 solution is 0.01M, the temperature is 25-30°C, and it is ready to use;
  • the volume ratio of the NaBH 4 solution to the HAuCl 4 solution is 1:6;
  • the stirring in step 2) is magnetic stirring, the speed of the magnetic stirring is 1200rpm, the time is 2min, the standing time is 30min, and the temperature is 25-30°C;
  • step 3 the dissolving temperature is 50°C, and the cooling temperature is 28-30°C;
  • the volume ratio of the water in step 3) to the NaBH 4 solution in step 2) is 1250:3;
  • the volume ratio of CTAB, the mass of sodium oleate and the volume of silver nitrate solution in step 3) to the HAuCl4 solution in step 1) is 70g: 12.34g: 180ml: 1ml;
  • the concentration of the silver nitrate solution is 4.0 M, and the holding time is 1 min;
  • the concentration of the HAuCl 4 solution is 1.0 mM, the concentration of the HCl solution is 37 wt%, and the concentration of the ascorbic acid solution is 0.064 M;
  • the volume ratio of the HAuCl 4 solution, the HCl solution and the ascorbic acid solution in step 4) to the water in step 3) is 250:2.1:1.25:250;
  • the first stirring in step 4) is magnetic stirring, the speed is 700 rpm, and the duration is 90 min;
  • the second stirring in step 4) is magnetic stirring, the speed is 400rpm, and the duration is 15min;
  • the third stirring in step 4) is magnetic stirring, the speed is 1200rpm, and the duration is 30s;
  • the volume ratio of the added amount of the seed solution in step 5) to the HAuCl 4 solution in step 1) is 4:1;
  • the stirring in step 5) is magnetic stirring, the speed is 1500rpm, and the duration is 30s;
  • step 5 the standing temperature is 28-30°C, and the time is 8-12h;
  • the speed of centrifugation mentioned in step 5 is 8000r/min, and the duration is 10min
  • step 5 the concentration of the CTAC solution is 80 mM, and the volume ratio of the CTAC solution to the seed solution is 1:2;
  • step 6 the concentration of the CTAC solution is 80 mM, and the volume ratio of the CTAC solution to the seed solution is 1:2;
  • step 2 the water in step 2 is deionized water, and the dilution factor is 8 times;
  • the concentration of the nitric acid solution is 10 mM, and the volume ratio of the silver nitrate solution to the gold nanorod solution is (1:0.4)-(1:5);
  • the volume ratio of the ascorbic acid solution to the silver nitrate solution is 1:1;
  • the frequency of the ultrasonic treatment is 100KHz, and the treatment time is 2min;
  • the storage temperature in the water bath is 63-68°C, and the time is 4h;
  • the rotation speed of the centrifugation is 8000r/min, and the time is 10min;
  • the solution used for the dispersion is deionized water, and the volume ratio of the deionized water to the gold nanorod solution is 1:2.
  • step 3 the volume ratio of the vitamin K4 ethanol solution to the gold core silver shell nanorod suspension is 1:500;
  • the gentle shaking time is 2h.
  • the invention also provides the use of the high-efficiency surface-enhanced Raman scattering base material in preparing the surface-enhanced Raman scattering base material for detecting kidney damage factors.
  • kidney injury factor is a glycoprotein immune substance.
  • the high-efficiency surface-enhanced Raman scattering substrate material provided by the present invention no longer uses the macroscopic materials widely cited in the prior art, but chooses nano-scale metal particles.
  • the local electromagnetic field of the nano-scale metal particles can be significantly enhanced, which is different from the traditional gold Compared with nanoparticles, gold-core-silver-shell nanoparticles can further enhance electromagnetic signals.
  • vitamin K4 is bonded to the surface of the gold-core-silver shell, so that the electromagnetic coupling between two nano-metal particles at the junction of two nano-metal particles can produce up to
  • the SERS enhancement factor (EF) of 10 14 can greatly enhance the detection sensitivity.
  • the invention also provides a method for preparing a highly efficient surface-enhanced Raman scattering substrate material.
  • the preparation method can well adjust the thickness of the silver shell by controlling the concentration of silver nitrate and ascorbic acid in the silver shell growth solution. Thickness of the silver shell.
  • Figure 1A is a transmission electron microscopy image of gold nanorods
  • Figure 1B- Figure 1F are transmission electron microscopy images of gold core-silver shell nanorods with silver shells of different thicknesses prepared in 0.2, 0.5, 1.0, 2.0 and 2.5 mL silver nitrate solutions;
  • Figure 2 shows the UV-NIR extinction spectra of gold nanorods and silver shells of different thicknesses prepared in 0.2, 0.5, 1.0, 2.0 and 2.5 mL silver nitrate solutions;
  • Figure 4 is a graph of the Raman intensity varying with the thickness of the silver shell at a spectrum of 1080 cm -1;
  • Figure 5 shows the high-sensitivity recognition ability of low-concentration KIM-1 in human urine based on the single-layer array film of gold-core-silver-shell nanorods;
  • Figure 6 shows that the SERS intensity has a biomarker concentration at 1145 cm -1 , and the inset shows its wide dynamic detection range.
  • This embodiment provides a substrate material based on surface-enhanced Raman technology and a preparation method thereof, and the structure of the substrate material is:
  • Gold nanorod core with a diameter of 23nm and a length of 89.2nm;
  • the gold core silver shell nanorod is prepared by the following preparation method, and the preparation method includes the following steps:
  • step 6) Repeat step 5) three times, and store the precipitate in CTAC solution to obtain a gold nanorod solution;
  • This embodiment provides a substrate material based on surface-enhanced Raman technology and a preparation method thereof, and the structure of the substrate material is:
  • the core of gold nanorods has a diameter of 21.7 and a length of 93.8;
  • the gold core silver shell nanorod is prepared by the following preparation method, and the preparation method includes the following steps:
  • step 6) Repeat step 5) three times, and store the precipitate in the CTAC solution to obtain the gold nanorod solution.
  • This embodiment provides a substrate material based on surface-enhanced Raman technology and a preparation method thereof, and the structure of the substrate material is:
  • the core of gold nanorods has a diameter of 24.3nm and a length of 84.6nm;
  • the gold core silver shell nanorod is prepared by the following preparation method, and the preparation method includes the following steps:
  • step 6) Repeat step 5) three times, and store the precipitate in the CTAC solution to obtain the gold nanorod solution.
  • This embodiment provides a substrate material based on surface-enhanced Raman technology and a preparation method thereof, and the structure of the substrate material is:
  • the core of gold nanorods has a diameter of 22.8nm and a length of 90.4nm;
  • the gold core silver shell nanorod is prepared by the following preparation method, and the preparation method includes the following steps:
  • step 6) Repeat step 5) three times, and store the precipitate in the CTAC solution to obtain the gold nanorod solution.
  • This embodiment provides a substrate material based on surface-enhanced Raman technology and a preparation method thereof, and the structure of the substrate material is:
  • the core of gold nanorods has a diameter of 24.0nm and a length of 91.5nm;
  • the gold core silver shell nanorod is prepared by the following preparation method, and the preparation method includes the following steps:
  • step 6) Repeat step 5) three times, and store the precipitate in the CTAC solution to obtain the gold nanorod solution.
  • the single-layer array films of gold core silver shell nanorods and gold nanorods in Examples 1-5 were respectively produced:
  • the SERS intensity is the best when the diameter of the gold nanorod is 22nm, the length is 90nm, and the thickness of the silver nanoshell is 16nm, which can reach 100 times that of the gold nanorod as the base material.
  • the vitamin K4 labeled gold core silver shell nanorods of the present invention replace the base material in the traditional SERS, and are used for the analysis of standard KIM1 biomarkers in artificial urine.
  • the gold core silver shell nanorod solution was functionalized with 150 mM cysteamine solution. Then, 12 ⁇ L of 25% wt glutaraldehyde solution was added to functionalize the gold core silver shell nanorod solution. Add 12mL of 20 ⁇ g/mL KIM1 capture antibody solution to the transformed gold core silver shell nanorod solution and store it at 4°C for 12 hours. Next, add 12mL of 1% BSA solution to the stored solution and let it stand for 1 hour. In order to block non-specific binding to the active site, a substrate structure solution is obtained.
  • the substrate solution was equally divided into 6 parts, and 6 kinds of urine to be tested were added to the 6 parts of substrate solution respectively, and incubated at room temperature for 1 hour to obtain the KIM1 urine to be tested.

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Abstract

A surface-enhanced Raman technology-based substrate material and a preparation method therefor. The substrate material of the surface-enhanced Raman technology comprises: a nanocore composed of gold nanorods; and a nanoshell composed of a nano silver material, wherein the nanoshell evenly and completely wraps the surface of the nanocore, and the outer surface of the nanoshell is combined with vitamin K4. The substrate material is used as a probe for surface-enhanced Raman detection and may significantly enhance the sensitivity of detection, and implement the high-sensitivity detection of KIM-1 content in urine, thereby achieving KIM-1 content determination of a large dynamic range.

Description

一种高效表面增强拉曼散射基底材料及制备方法High-efficiency surface enhanced Raman scattering base material and preparation method 技术领域Technical field
本发明涉及拉曼散射技术领域，特别涉及一种高效表面增强拉曼散射基底材料及制备方法。The invention relates to the technical field of Raman scattering, in particular to a high-efficiency surface-enhanced Raman scattering base material and a preparation method.
背景技术Background technique
目前，以肾脏损伤为特征的急慢性肾脏疾病是世界范围内具有挑战性的健康问题之一，判断肾脏损伤一般采用化验尿常规、渗透压、血肌酐、尿素氮、内生肌酐清除率等几项指标，但是这些指标增高的时候，很多肾脏损伤已经非常严重或者已经产生了不可逆的损害，因此，很多学者指出可以把尿N-乙酰-β-D-氨基葡萄糖苷酶(NAG酶)活性作为损伤的早期指标，但该指标一般在肾病发作8-16小时才显现，目前，经研究证明，尿液中肾损伤分子1(KIM-1)含量对早期诊断急性肾损伤具有显著特异性，KIM-1含量在肾损伤4-6小时就可以出现改变，能迅速、灵敏、特异地反映各种肾脏疾病的损伤及恢复过程，可成为一种检测早期肾损伤的可靠生物学标记物，因此，对于尿液中KIM-1含量高精度的检测对于以肾脏损伤为特征的急慢性肾脏疾病的诊断和治疗有很大意义。At present, acute and chronic kidney disease characterized by kidney damage is one of the most challenging health problems worldwide. To determine kidney damage, urine routine tests, osmotic pressure, blood creatinine, urea nitrogen, and endogenous creatinine clearance are generally used to determine kidney damage. However, when these indicators increase, many kidney damages have been severe or irreversible damage has occurred. Therefore, many scholars have pointed out that urine N-acetyl-β-D-glucosaminidase (NAG enzyme) activity can be used as An early indicator of injury, but this indicator generally does not appear until 8-16 hours after the onset of kidney disease. At present, studies have shown that the content of kidney injury molecule 1 (KIM-1) in urine has significant specificity for the early diagnosis of acute kidney injury. The content of -1 can change within 4-6 hours after kidney injury. It can quickly, sensitively and specifically reflect the damage and recovery process of various kidney diseases. It can become a reliable biological marker for detecting early kidney injury. Therefore, High-precision detection of KIM-1 content in urine is of great significance for the diagnosis and treatment of acute and chronic kidney diseases characterized by kidney damage.
目前，尿液中KIM-1含量的测定方法主要是ELISA方法，灵敏度为pg/ml，因此，该方法无法实现大动态范围内定量检测尿液中的KIM-1含量。At present, the main method for determining the KIM-1 content in urine is the ELISA method with a sensitivity of pg/ml. Therefore, this method cannot achieve the quantitative detection of KIM-1 content in urine within a large dynamic range.
表面增强拉曼散射(SERS)技术由于其高灵敏度，窄线宽和指纹效应，已成为一种有吸引力且功能强大的分析技术，可实现多重生物传感。表面增强拉曼散射具有可达14到15个数量级的卓越的拉曼增强效率，使其能够可靠、准确地检测超痕量甚至单分子水平的分析物。此外，SERS检测可以在几分钟内快速完成。因此,表面增强拉曼散射已经广泛应用于 各种化学传感、生物分析、生物传感和早期癌症诊断等领域，但是，目前表面增强拉曼散射基底材料大都选用光滑的宏观玻璃、金、银或双金属膜作为基底材料，并不具有充足的等离子体“热点”，这导致表面增强拉曼技术的灵敏度有限。Surface Enhanced Raman Scattering (SERS) technology has become an attractive and powerful analysis technology due to its high sensitivity, narrow line width and fingerprint effect, which can realize multiple biosensing. Surface-enhanced Raman scattering has an excellent Raman enhancement efficiency of up to 14 to 15 orders of magnitude, enabling it to reliably and accurately detect ultra-trace or even single-molecule analytes. In addition, SERS detection can be completed quickly within a few minutes. Therefore, surface-enhanced Raman scattering has been widely used in various chemical sensing, biological analysis, biosensing, and early cancer diagnosis. However, the current surface-enhanced Raman scattering substrate materials mostly use smooth macroscopic glass, gold, and silver. Or the bimetal film as the base material does not have sufficient plasma "hot spots", which leads to the limited sensitivity of the surface enhanced Raman technology.
为了提高SERS检测的灵敏度，目前选用在SERS检测的基底材料上键合曼拉检测分子，其中效果最佳的为P-ATP分子，灵敏度为ng/ml-pg/ml，虽然键合P-ATP分子后，SERS检测的灵敏度有所提升，但是很多被检测物所需要的检测灵敏度以及检测范围宽度要求更高In order to improve the sensitivity of SERS detection, the current selection of SERS detection substrate material is used to bond Manra detection molecules, of which the best effect is the P-ATP molecule, the sensitivity is ng/ml-pg/ml, although the P-ATP is bonded After the molecule, the sensitivity of SERS detection has been improved, but the detection sensitivity and detection range width required by many detected objects are higher.
发明内容Summary of the invention
本发明的目的在于提供一种高效表面增强拉曼散射基底材料及制备方法，实现尿液中KIM-1含量的高敏感度检测，可达fg/ml级，从而达到大动态范围的KIM-1含量测定。The purpose of the present invention is to provide an efficient surface-enhanced Raman scattering substrate material and a preparation method, which can achieve high-sensitivity detection of KIM-1 content in urine, up to fg/ml level, thereby achieving a KIM-1 with a large dynamic range Assay.
本发明的一个方面在于，提供一种高效表面增强拉曼散射基底材包括：One aspect of the present invention is to provide a high-efficiency surface-enhanced Raman scattering substrate including:
纳米内核，所述纳米内核是由金纳米棒构成；和A nano-core, the nano-core is composed of gold nanorods; and
纳米外壳，所述纳米外壳是由纳米银材料构成，所述纳米外壳均匀完全的包裹于所述纳米内核表面，所述纳米外壳外表面键合有拉曼检测分子维生素K4。Nano-shell, the nano-shell is made of nano-silver material, the nano-shell is evenly and completely wrapped on the surface of the nano-core, and the outer surface of the nano-shell is bonded with the Raman detection molecule vitamin K4.
进一步地，所述维生素K4经重氮化巯解引入巯基。Further, the vitamin K4 introduces a sulfhydryl group through diazotization thiolysis.
进一步地，所述纳米内核的直径为19-26nm，长度为80-96nm。Further, the diameter of the nano-core is 19-26 nm, and the length is 80-96 nm.
进一步地，所述纳米外壳的厚度为2-18nm。Further, the thickness of the nano-shell is 2-18 nm.
本发明的另一个方面在于，提供一种高效表面增强拉曼散射基底材料的制备方法，包括以下步骤：Another aspect of the present invention is to provide a method for preparing a high-efficiency surface-enhanced Raman scattering base material, which includes the following steps:
(1)金纳米棒的制备；(1) Preparation of gold nanorods;
1)用水对HAuCl 4溶液进行稀释得到稀释液，向稀释液中的加入 CTAB溶液得到溶液一； 1) Dilute the HAuCl 4 solution with water to obtain a diluent, and add CTAB solution to the diluent to obtain solution one;
2)将溶液一快速注入NaBH 4溶液中，搅拌得到种子溶液，静置； 2) Quickly inject the solution into the NaBH 4 solution, stir to obtain the seed solution, and let it stand;
3)将CTAB和油酸钠溶解于水中，冷却后加入硝酸银溶液，保温后得到溶液二；3) Dissolve CTAB and sodium oleate in water, add silver nitrate solution after cooling, and obtain solution 2 after heat preservation;
4)一边搅拌一边向溶液二中注射HAuCl 4溶液，继续第一次搅拌，改变第二次搅拌速度，一边搅拌一边加入HCl溶液，继续第二次搅拌，最后加入抗坏血酸溶液，进行第三次搅拌，得到生长液； 4) While stirring, inject the HAuCl 4 solution into the second solution, continue the first stirring, change the second stirring speed, add the HCl solution while stirring, continue the second stirring, and finally add the ascorbic acid solution, and perform the third stirring , Get the growth solution;
5)将种子溶液注入生长液中得到混合溶液，将混合溶液静置，将静置后的混合溶液进行离心，收集沉淀物，将沉淀物分散于CTAC溶液中；5) Inject the seed solution into the growth solution to obtain a mixed solution, leave the mixed solution to stand, centrifuge the settled mixed solution, collect the precipitate, and disperse the precipitate in the CTAC solution;
6)将步骤5)重复三次，将所述得沉淀物储存在CTAC溶液中，得到金纳米棒溶液；6) Repeat step 5) three times, and store the precipitate in CTAC solution to obtain a gold nanorod solution;
(2)金核银壳纳米棒的制备；(2) Preparation of gold core silver shell nanorods;
将金纳米棒溶液用水稀释，向稀释液中加入硝酸银溶液，进行超声波处理后加入抗坏血酸溶液，经水浴保存、离心和分散得到金核银壳纳米棒悬浮液；Dilute the gold nanorod solution with water, add silver nitrate solution to the diluent, add ascorbic acid solution after ultrasonic treatment, store in a water bath, centrifuge and disperse to obtain a gold core silver shell nanorod suspension;
(3)拉曼检测分子的键合；(3) Raman detects the bonding of molecules;
将维生素K4乙醇溶液经重氮化巯解引入巯基，再添加到金核银壳纳米棒悬浮液中得到混合物，温和摇动混合物，得到拉维生素K4标记的金核银壳纳米棒。The vitamin K4 ethanol solution is diazotized and mercaptolyzed to introduce sulfhydryl groups, and then added to the gold core silver shell nanorod suspension to obtain a mixture, and the mixture is gently shaken to obtain vitamin K4 labeled gold core silver shell nanorods.
进一步地，步骤1)中所述水为去离子水；Further, the water in step 1) is deionized water;
所述HAuCl 4溶液的浓度为25mM，所述稀释的倍数为50倍； The concentration of the HAuCl 4 solution is 25 mM, and the dilution factor is 50 times;
所述CTAB溶液的浓度为0.2M；The concentration of the CTAB solution is 0.2M;
所述HAuCl 4溶液与所述CTAB溶液的体积比为1:50； The volume ratio of the HAuCl 4 solution to the CTAB solution is 1:50;
步骤2)中所述NaBH 4溶液的浓度为0.01M，温度为25-30℃，现用现配； In step 2), the concentration of the NaBH 4 solution is 0.01M, the temperature is 25-30°C, and it is ready to use;
所述NaBH 4溶液与所述HAuCl 4溶液的体积比为1：6； The volume ratio of the NaBH 4 solution to the HAuCl 4 solution is 1:6;
步骤2)中所述搅拌为磁力搅拌，所述磁力搅拌的速度为1200rpm，时间为2min，所述静置时间为30min，温度为25-30℃；The stirring in step 2) is magnetic stirring, the speed of the magnetic stirring is 1200rpm, the time is 2min, the standing time is 30min, and the temperature is 25-30°C;
步骤3)中所述溶解温度为50℃，所述冷却温度为28-30℃；In step 3), the dissolving temperature is 50°C, and the cooling temperature is 28-30°C;
步骤3)中所述水与步骤2)中所述NaBH 4溶液的体积比为1250：3； The volume ratio of the water in step 3) to the NaBH 4 solution in step 2) is 1250:3;
步骤3)中所述CTAB、油酸钠质量和硝酸银溶液体积与步骤1)中所述HAuCl4溶液的体积比为70g：12.34g：180ml：1ml；The volume ratio of CTAB, the mass of sodium oleate and the volume of silver nitrate solution in step 3) to the HAuCl4 solution in step 1) is 70g: 12.34g: 180ml: 1ml;
所述硝酸银溶液得浓度为4.0M，所述保温的时间为1min；The concentration of the silver nitrate solution is 4.0 M, and the holding time is 1 min;
步骤4)中所述HAuCl 4溶液的浓度为1.0mM，所述HCl溶液的浓度为37wt％，所述抗坏血酸溶液得浓度为0.064M； In step 4), the concentration of the HAuCl 4 solution is 1.0 mM, the concentration of the HCl solution is 37 wt%, and the concentration of the ascorbic acid solution is 0.064 M;
步骤4)中所述HAuCl 4溶液、HCl溶液和所述抗坏血酸溶液与步骤3)中所述水的体积比为250:2.1:1.25:250； The volume ratio of the HAuCl 4 solution, the HCl solution and the ascorbic acid solution in step 4) to the water in step 3) is 250:2.1:1.25:250;
步骤4)中所述第一次搅拌为磁力搅拌，速度为700rpm，持续时间为90min；The first stirring in step 4) is magnetic stirring, the speed is 700 rpm, and the duration is 90 min;
步骤4)中所述第二次搅拌为磁力搅拌，速度为400rpm，持续时间为15min；The second stirring in step 4) is magnetic stirring, the speed is 400rpm, and the duration is 15min;
步骤4)中所述第三次搅拌为磁力搅拌，速度为1200rpm，持续时间为30s；The third stirring in step 4) is magnetic stirring, the speed is 1200rpm, and the duration is 30s;
步骤5)中所述种子溶液的加入量与步骤1)中所述HAuCl 4溶液的体积比为4:1； The volume ratio of the added amount of the seed solution in step 5) to the HAuCl 4 solution in step 1) is 4:1;
步骤5)中所述搅拌为磁力搅拌，速度为1500rpm，持续时间为30s；The stirring in step 5) is magnetic stirring, the speed is 1500rpm, and the duration is 30s;
步骤5)中所述静置的温度为28-30℃，时间为8-12h；In step 5), the standing temperature is 28-30°C, and the time is 8-12h;
步骤5)中所述离心的转速为8000r/min，持续时间为10minThe speed of centrifugation mentioned in step 5) is 8000r/min, and the duration is 10min
步骤5)中所述CTAC溶液的浓度为80mM，所述CTAC溶液与所述种子溶液的体积比为1:2；In step 5), the concentration of the CTAC solution is 80 mM, and the volume ratio of the CTAC solution to the seed solution is 1:2;
步骤6)中所述CTAC溶液的浓度为80mM，所述CTAC溶液与所述种子溶液的体积比为1:2；In step 6), the concentration of the CTAC solution is 80 mM, and the volume ratio of the CTAC solution to the seed solution is 1:2;
进一步地，步骤2中所述水为去离子水，所述稀释的倍数为8倍；Further, the water in step 2 is deionized water, and the dilution factor is 8 times;
所述硝酸溶液的浓度为10mM，所述硝酸银溶液与所述金纳米棒溶液的体积比为(1:0.4)-(1:5)；The concentration of the nitric acid solution is 10 mM, and the volume ratio of the silver nitrate solution to the gold nanorod solution is (1:0.4)-(1:5);
所述抗坏血酸溶液的体积与所述硝酸银溶液的体积比为1:1；The volume ratio of the ascorbic acid solution to the silver nitrate solution is 1:1;
所述超声波处理的频率为100KHz，处理时间为2min；The frequency of the ultrasonic treatment is 100KHz, and the treatment time is 2min;
所述水浴保存的温度为63-68℃，时间为4h；The storage temperature in the water bath is 63-68°C, and the time is 4h;
所述离心的转速为8000r/min，时间为10min；The rotation speed of the centrifugation is 8000r/min, and the time is 10min;
所述分散所用的溶液为去离子水，所述去离子水与所述金纳米棒溶液体积比为1:2.The solution used for the dispersion is deionized water, and the volume ratio of the deionized water to the gold nanorod solution is 1:2.
进一步地，步骤3中所述维生素K4乙醇溶液与所述金核银壳纳米棒悬浊液的体积比为1:500；Further, in step 3, the volume ratio of the vitamin K4 ethanol solution to the gold core silver shell nanorod suspension is 1:500;
所述温和摇动的时间为2h。The gentle shaking time is 2h.
本发明还提供了高效表面增强拉曼散射基底材料在制备检测肾损伤因子的表面增强拉曼散射基底材料中的用途。The invention also provides the use of the high-efficiency surface-enhanced Raman scattering base material in preparing the surface-enhanced Raman scattering base material for detecting kidney damage factors.
进一步地，所述肾损伤因子为糖蛋白类免疫物质。Further, the kidney injury factor is a glycoprotein immune substance.
本发明的有益之处在于：The advantages of the present invention are:
本发明提供的一种高效表面增强拉曼散射基底材料不再使用现有技术中广泛引用的宏观材料，而是选择纳米级金属颗粒，纳米级金属颗粒局部电磁场可以显著增强，与传统上的金纳米颗粒相比较，金核银壳纳米颗粒可以进一步增强电磁信号，同时在金核银壳表面键合维生素K4，使得两个纳米金属颗粒交界处两个纳米金属颗粒之间的电磁耦合可以产生高达10 14的SERS增强因子(EF)，这种信号的增强可以大大增强检测灵敏度。 The high-efficiency surface-enhanced Raman scattering substrate material provided by the present invention no longer uses the macroscopic materials widely cited in the prior art, but chooses nano-scale metal particles. The local electromagnetic field of the nano-scale metal particles can be significantly enhanced, which is different from the traditional gold Compared with nanoparticles, gold-core-silver-shell nanoparticles can further enhance electromagnetic signals. At the same time, vitamin K4 is bonded to the surface of the gold-core-silver shell, so that the electromagnetic coupling between two nano-metal particles at the junction of two nano-metal particles can produce up to The SERS enhancement factor (EF) of 10 14 can greatly enhance the detection sensitivity.
本发明还提供的一种高效表面增强拉曼散射基底材料得制备方法，该制备方法通过控制银壳生长液中硝酸银和抗坏血酸的浓度，可以很好地调节银壳的厚度，根据需求制备不同厚度的银壳。The invention also provides a method for preparing a highly efficient surface-enhanced Raman scattering substrate material. The preparation method can well adjust the thickness of the silver shell by controlling the concentration of silver nitrate and ascorbic acid in the silver shell growth solution. Thickness of the silver shell.
应当理解，前述大体的描述和后续详尽的描述均为示例性说明和解释，并不应当用作对本发明所要求保护内容的限制。It should be understood that the foregoing general description and the subsequent detailed description are exemplary descriptions and explanations, and should not be used as a limitation on the content claimed by the present invention.
附图说明Description of the drawings
参考随附的附图，本发明更多的目的、功能和优点将通过本发明实施方式的如下描述得以阐明，其中：With reference to the accompanying drawings, more objectives, functions and advantages of the present invention will be clarified by the following description of the embodiments of the present invention, in which:
图1A为金纳米棒透射电镜图像；图1B-图1F为在0.2、0.5、1.0、2.0和2.5mL硝酸银溶液中制备的不同厚度银壳的金核银壳纳米棒透射电镜图像；Figure 1A is a transmission electron microscopy image of gold nanorods; Figure 1B-Figure 1F are transmission electron microscopy images of gold core-silver shell nanorods with silver shells of different thicknesses prepared in 0.2, 0.5, 1.0, 2.0 and 2.5 mL silver nitrate solutions;
图2为金纳米棒和在0.2、0.5、1.0、2.0和2.5mL硝酸银溶液中制备的不同厚度银壳的金核银壳纳米棒UV-NIR消光光谱；Figure 2 shows the UV-NIR extinction spectra of gold nanorods and silver shells of different thicknesses prepared in 0.2, 0.5, 1.0, 2.0 and 2.5 mL silver nitrate solutions;
图3为在实施例1-5中金核银壳纳米棒和金纳米棒的单层列阵膜上吸收的维生素K4的拉曼光谱；3 is the Raman spectrum of vitamin K4 absorbed on the single-layer array film of gold core silver shell nanorods and gold nanorods in Examples 1-5;
图4为在光谱为1080cm -1处拉曼强度随银壳厚度变化的图； Figure 4 is a graph of the Raman intensity varying with the thickness of the silver shell at a spectrum of 1080 cm -1;
图5为基于金核银壳纳米棒单层阵列膜对人体尿液中低浓度KIM-1的高灵敏度识别能力；Figure 5 shows the high-sensitivity recognition ability of low-concentration KIM-1 in human urine based on the single-layer array film of gold-core-silver-shell nanorods;
图6为SERS强度在1145cm -1处具有生物标记物浓度，插图显示其宽动态检测范围。 Figure 6 shows that the SERS intensity has a biomarker concentration at 1145 cm -1 , and the inset shows its wide dynamic detection range.
具体实施方式detailed description
通过参考示范性实施例，本发明的目的和功能以及用于实现这些目的和功能的方法将得以阐明。然而，本发明并不受限于以下所公开的示范性实施例；可以通过不同形式来对其加以实现。说明书的实质仅仅是帮助相关领域技术人员综合理解本发明的具体细节。By referring to exemplary embodiments, the objects and functions of the present invention and methods for achieving these objects and functions will be clarified. However, the present invention is not limited to the exemplary embodiments disclosed below; it can be implemented in different forms. The essence of the description is only to help those skilled in the relevant art to comprehensively understand the specific details of the present invention.
在下文中，将参考附图描述本发明的实施例。在附图中，相同的附图标记代表相同或类似的部件，或者相同或类似的步骤。Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the drawings, the same reference signs represent the same or similar components or the same or similar steps.
实施例1Example 1
本实施例提供了一种基于表面增强拉曼技术的基底材料及其制备方法，该基底材料结构为：This embodiment provides a substrate material based on surface-enhanced Raman technology and a preparation method thereof, and the structure of the substrate material is:
金纳米棒内核，直径为23nm，长度为89.2nm；Gold nanorod core with a diameter of 23nm and a length of 89.2nm;
银纳米外壳，厚度为4nm；Silver nano shell with a thickness of 4nm;
该金核银壳纳米棒由以下制备方法制得，该制备方法包括以下步骤：The gold core silver shell nanorod is prepared by the following preparation method, and the preparation method includes the following steps:
1、金纳米棒的制备1. Preparation of gold nanorods
1)在20mL的玻璃瓶中将0.1mL浓度为25mM HAuCl 4溶液用去离子水稀释至5mL，向稀释液中加入5mL 0.2M的CTAB溶液，得到溶液一； 1) Dilute 0.1 mL of 25 mM HAuCl 4 solution in a 20 mL glass bottle with deionized water to 5 mL, and add 5 mL of 0.2M CTAB solution to the diluent to obtain solution one;
2)将0.6mL0.01M的NaBH 4溶液快速注入溶液一中，NaBH 4溶液现用现配，对混合溶液进行磁力搅拌，速度为1200rpm，搅拌2min，最后获得的种子溶液在30℃静置30min待用； 2) Quickly inject 0.6 mL of 0.01M NaBH 4 solution into solution 1. The NaBH 4 solution is ready to use, and the mixed solution is magnetically stirred at a speed of 1200 rpm for 2 minutes. The finally obtained seed solution is allowed to stand at 30°C for 30 minutes stand-by;
3)将7.0g CTAB和1.234g油酸钠溶解于水中250mL 50℃水中，自然冷却到30℃，然后加入18ml,4.0mM的硝酸银溶液，保温1min，得到溶液二；3) Dissolve 7.0g CTAB and 1.234g sodium oleate in 250mL water at 50°C, cool to 30°C naturally, then add 18ml, 4.0mM silver nitrate solution and keep it for 1min to obtain solution two;
4)一边磁力搅拌一边向溶液二中注射250ml 1.0mM的HAuCl 4溶液，在700rpm速度下搅拌90min，改变第二次磁力搅拌速度为400rpm，一边搅拌一边加入2.1ml 37wt％的HCl溶液，搅拌15min，最后加入1.25ml 0.064M的抗坏血酸溶液，进行第三次搅拌，搅拌速度为1200rpm，搅拌30s，得到生长液； 4) While magnetic stirring, inject 250ml 1.0mM HAuCl 4 solution into solution 2, stir at 700rpm for 90min, change the second magnetic stirring speed to 400rpm, add 2.1ml 37wt% HCl solution while stirring, and stir for 15min Finally, add 1.25ml of 0.064M ascorbic acid solution and stir for the third time at a stirring speed of 1200rpm for 30s to obtain a growth solution;
5)将0.4mL种子溶液注入生长液中1500rpm速度下搅拌30s，最后，将混合液在30℃条件下静置10h，将静置后的混合液在8000r/min 的速度速度下离心10min，收集沉淀物，将沉淀物分散于80mM的CTAC溶液中；5) Inject 0.4 mL of seed solution into the growth solution and stir at 1500 rpm for 30 seconds. Finally, the mixture is allowed to stand at 30°C for 10 hours, and the static mixture is centrifuged at a speed of 8000 r/min for 10 minutes and collected Precipitate, disperse the precipitate in 80mM CTAC solution;
6)将步骤5)重复三次，将所述得沉淀物储存在CTAC溶液中，得到金纳米棒溶液；6) Repeat step 5) three times, and store the precipitate in CTAC solution to obtain a gold nanorod solution;
2、金核银壳纳米棒的制备2. Preparation of gold core silver shell nanorods
将0.5mL金纳米棒溶液用水稀释至4ml，向稀释液中加入0.2ml 10mM的硝酸银溶液，在100KHz频率下超声波处理2min，后加入0.2ml 0.1M的抗坏血酸溶液，经65℃水浴保存4h、8000r/min离心10min，收集沉淀物分散到1ml去离子水中，得到金核银壳纳米棒悬浮液。Dilute 0.5ml gold nanorod solution to 4ml with water, add 0.2ml 10mM silver nitrate solution to the diluent, ultrasonic treatment at 100KHz frequency for 2min, then add 0.2ml 0.1M ascorbic acid solution, store in 65℃ water bath for 4h, Centrifuge at 8000r/min for 10min, collect the precipitate and disperse it into 1ml of deionized water to obtain a gold core silver shell nanorod suspension.
3、拉曼检测分子的键合3. Raman detects the bonding of molecules
将2μL,5mM的维生素K4乙醇溶液经重氮化巯解引入巯基后添加到1.0ml金核银壳纳米棒悬浮液中得到混合物，温和摇动混合物2h，得到维生素K4标记的金核银壳纳米棒溶液。Add 2μL, 5mM vitamin K4 ethanol solution by diazotization to sulfhydryl to introduce sulfhydryl groups and add it to 1.0ml gold core silver shell nanorod suspension to obtain the mixture. Shake the mixture gently for 2 hours to obtain vitamin K4 labeled gold core silver shell nanorods. Solution.
实施例2Example 2
本实施例提供了一种基于表面增强拉曼技术的基底材料及其制备方法，该基底材料结构为：This embodiment provides a substrate material based on surface-enhanced Raman technology and a preparation method thereof, and the structure of the substrate material is:
金纳米棒内核，直径为21.7，长度为93.8；The core of gold nanorods has a diameter of 21.7 and a length of 93.8;
银纳米外壳，厚度为6nm；Silver nano shell with a thickness of 6nm;
该金核银壳纳米棒由以下制备方法制得，该制备方法包括以下步骤：The gold core silver shell nanorod is prepared by the following preparation method, and the preparation method includes the following steps:
1、金纳米棒的制备1. Preparation of gold nanorods
1)在20mL的玻璃瓶中将0.1mL浓度为25mM HAuCl 4溶液用去离子水稀释至5mL，向稀释液中加入5mL 0.2M的CTAB溶液，得到溶液一； 1) Dilute 0.1 mL of 25 mM HAuCl 4 solution in a 20 mL glass bottle with deionized water to 5 mL, and add 5 mL of 0.2M CTAB solution to the diluent to obtain solution one;
2)将0.6mL0.01M的NaBH 4溶液快速注入溶液一中，NaBH 4溶液现用现配，对混合溶液进行磁力搅拌，速度为1200rpm，搅拌2min，最 后获得的种子溶液在30℃静置30min待用； 2) Quickly inject 0.6 mL of 0.01M NaBH 4 solution into solution 1. The NaBH 4 solution is ready to use, and the mixed solution is magnetically stirred at a speed of 1200 rpm for 2 minutes. The finally obtained seed solution is allowed to stand at 30°C for 30 minutes stand-by;
3)将7.0g CTAB和1.234g油酸钠溶解于水中250mL 50℃水中，自然冷却到30℃，然后加入18ml,4.0mM的硝酸银溶液，保温1min，得到溶液二；3) Dissolve 7.0g CTAB and 1.234g sodium oleate in 250mL water at 50°C, cool to 30°C naturally, then add 18ml, 4.0mM silver nitrate solution and keep it for 1min to obtain solution two;
4)一边磁力搅拌一边向溶液二中注射250ml 1.0mM的HAuCl 4溶液，在700rpm速度下搅拌90min，改变第二次磁力搅拌速度为400rpm，一边搅拌一边加入2.1ml 37wt％的HCl溶液，搅拌15min，最后加入1.25ml 0.064M的抗坏血酸溶液，进行第三次搅拌，搅拌速度为1200rpm，搅拌30s，得到生长液； 4) While magnetic stirring, inject 250ml 1.0mM HAuCl 4 solution into solution 2, stir at 700rpm for 90min, change the second magnetic stirring speed to 400rpm, add 2.1ml 37wt% HCl solution while stirring, and stir for 15min Finally, add 1.25ml of 0.064M ascorbic acid solution and stir for the third time at a stirring speed of 1200rpm for 30s to obtain a growth solution;
5)将0.4mL种子溶液注入生长液中1500rpm速度下搅拌30s，最后，将混合液在30℃条件下静置10h，将静置后的混合液在8000r/min的速度速度下离心10min，收集沉淀物，将沉淀物分散于80mM的CTAC溶液中；5) Inject 0.4 mL of seed solution into the growth solution and stir at 1500 rpm for 30 seconds. Finally, the mixture is allowed to stand at 30°C for 10 hours, and the static mixture is centrifuged at a speed of 8000 r/min for 10 minutes, and collected Precipitate, disperse the precipitate in 80mM CTAC solution;
6)将步骤5)重复三次，将所述得沉淀物储存在CTAC溶液中，得到金纳米棒溶液。6) Repeat step 5) three times, and store the precipitate in the CTAC solution to obtain the gold nanorod solution.
2、金核银壳纳米棒的制备2. Preparation of gold core silver shell nanorods
将0.5mL金纳米棒溶液用水稀释至4mL，向稀释液中加入0.5ml 10mM的硝酸银溶液，在100KHz频率下超声波处理2min，后加入0.5 0.1M的抗坏血酸溶液，经65℃水浴保存4h、8000r/min离心10min，收集沉淀物分散到1ml去离子水中，得到金核银壳纳米棒悬浮液。Dilute 0.5mL gold nanorod solution to 4mL with water, add 0.5ml 10mM silver nitrate solution to the diluent, ultrasonic treatment at 100KHz frequency for 2min, then add 0.5 0.1M ascorbic acid solution, store in 65℃ water bath for 4h, 8000r Centrifuge for 10 min/min, collect the precipitate and disperse it into 1 ml of deionized water to obtain a gold core silver shell nanorod suspension.
3、拉曼检测分子的键合3. Raman detects the bonding of molecules
将2μL,5mM的维生素K4乙醇溶液经重氮化巯解引入巯基后添加到1.0ml金核银壳纳米棒悬浮液中得到混合物，温和摇动混合物2h，得到维生素K4标记的金核银壳纳米棒溶液。Add 2μL, 5mM vitamin K4 ethanol solution by diazotization to sulfhydryl to introduce sulfhydryl groups and add it to 1.0ml gold core silver shell nanorod suspension to obtain the mixture. Shake the mixture gently for 2 hours to obtain vitamin K4 labeled gold core silver shell nanorods. Solution.
实施例3Example 3
本实施例提供了一种基于表面增强拉曼技术的基底材料及其制备方法，该基底材料结构为：This embodiment provides a substrate material based on surface-enhanced Raman technology and a preparation method thereof, and the structure of the substrate material is:
金纳米棒内核，直径为24.3nm，长度为84.6nm；The core of gold nanorods has a diameter of 24.3nm and a length of 84.6nm;
银纳米外壳，厚度为8nm；Silver nano shell with a thickness of 8nm;
该金核银壳纳米棒由以下制备方法制得，该制备方法包括以下步骤：The gold core silver shell nanorod is prepared by the following preparation method, and the preparation method includes the following steps:
1、金纳米棒的制备1. Preparation of gold nanorods
1)在20mL的玻璃瓶中将0.1mL浓度为25mM HAuCl 4溶液用去离子水稀释至5mL，向稀释液中加入5mL 0.2M的CTAB溶液，得到溶液一； 1) Dilute 0.1 mL of 25 mM HAuCl 4 solution in a 20 mL glass bottle with deionized water to 5 mL, and add 5 mL of 0.2M CTAB solution to the diluent to obtain solution one;
2)将0.6mL0.01M的NaBH 4溶液快速注入溶液一中，NaBH 4溶液现用现配，对混合溶液进行磁力搅拌，速度为1200rpm，搅拌2min，最后获得的种子溶液在30℃静置30min待用； 2) Quickly inject 0.6 mL of 0.01M NaBH 4 solution into solution 1. The NaBH 4 solution is ready to use, and the mixed solution is magnetically stirred at a speed of 1200 rpm for 2 minutes. The finally obtained seed solution is allowed to stand at 30°C for 30 minutes stand-by;
3)将7.0g CTAB和1.234g油酸钠溶解于水中250mL 50℃水中，自然冷却到30℃，然后加入18ml,4.0mM的硝酸银溶液，保温1min，得到溶液二；3) Dissolve 7.0g CTAB and 1.234g sodium oleate in 250mL water at 50°C, cool to 30°C naturally, then add 18ml, 4.0mM silver nitrate solution and keep it for 1min to obtain solution two;
4)一边磁力搅拌一边向溶液二中注射250ml 1.0mM的HAuCl 4溶液，在700rpm速度下搅拌90min，改变第二次磁力搅拌速度为400rpm，一边搅拌一边加入2.1ml 37wt％的HCl溶液，搅拌15min，最后加入1.25ml 0.064M的抗坏血酸溶液，进行第三次搅拌，搅拌速度为1200rpm，搅拌30s，得到生长液； 4) While magnetic stirring, inject 250ml 1.0mM HAuCl 4 solution into solution 2, stir at 700rpm for 90min, change the second magnetic stirring speed to 400rpm, add 2.1ml 37wt% HCl solution while stirring, and stir for 15min Finally, add 1.25ml of 0.064M ascorbic acid solution and stir for the third time at a stirring speed of 1200rpm for 30s to obtain a growth solution;
5)将0.4mL种子溶液注入生长液中1500rpm速度下搅拌30s，最后，将混合液在30℃条件下静置10h，将静置后的混合液在8000r/min的速度速度下离心10min，收集沉淀物，将沉淀物分散于80mM的CTAC溶液中；5) Inject 0.4 mL of seed solution into the growth solution and stir at 1500 rpm for 30 seconds. Finally, the mixture is allowed to stand at 30°C for 10 hours, and the static mixture is centrifuged at a speed of 8000 r/min for 10 minutes, and collected Precipitate, disperse the precipitate in 80mM CTAC solution;
6)将步骤5)重复三次，将所述得沉淀物储存在CTAC溶液中，得 到金纳米棒溶液。6) Repeat step 5) three times, and store the precipitate in the CTAC solution to obtain the gold nanorod solution.
2、金核银壳纳米棒的制备2. Preparation of gold core silver shell nanorods
将0.5mL金纳米棒溶液用水稀释至4ml，向稀释液中加入1.0ml 10mM的硝酸银溶液，在100KHz频率下超声波处理2min，后加入1.0ml 0.1M的抗坏血酸溶液，经65℃水浴保存4h、8000r/min离心10min，收集沉淀物分散到1ml去离子水中，得到金核银壳纳米棒悬浮液。Dilute 0.5mL gold nanorod solution to 4ml with water, add 1.0ml 10mM silver nitrate solution to the diluent, ultrasonic treatment at 100KHz frequency for 2min, then add 1.0ml 0.1M ascorbic acid solution, store in 65℃ water bath for 4h, Centrifuge at 8000r/min for 10min, collect the precipitate and disperse it in 1ml deionized water to obtain a gold core silver shell nanorod suspension.
3、拉曼检测分子的键合3. Raman detects the bonding of molecules
将2μL,5mM的维生素K4乙醇溶液经重氮化巯解引入巯基后添加到1.0ml金核银壳纳米棒悬浮液中得到混合物，温和摇动混合物2h，得到维生素K4标记的金核银壳纳米棒溶液。Add 2μL, 5mM vitamin K4 ethanol solution by diazotization to sulfhydryl to introduce sulfhydryl groups and add it to 1.0ml gold core silver shell nanorod suspension to obtain the mixture. Shake the mixture gently for 2 hours to obtain vitamin K4 labeled gold core silver shell nanorods. Solution.
实施例4Example 4
本实施例提供了一种基于表面增强拉曼技术的基底材料及其制备方法，该基底材料结构为：This embodiment provides a substrate material based on surface-enhanced Raman technology and a preparation method thereof, and the structure of the substrate material is:
金纳米棒内核，直径为22.8nm，长度为90.4nm；The core of gold nanorods has a diameter of 22.8nm and a length of 90.4nm;
银纳米外壳，厚度为10nm；Silver nano shell with a thickness of 10nm;
该金核银壳纳米棒由以下制备方法制得，该制备方法包括以下步骤：The gold core silver shell nanorod is prepared by the following preparation method, and the preparation method includes the following steps:
1、金纳米棒的制备1. Preparation of gold nanorods
1)在20mL的玻璃瓶中将0.1mL浓度为25mM HAuCl 4溶液用去离子水稀释至5mL，向稀释液中加入5mL 0.2M的CTAB溶液，得到溶液一； 1) Dilute 0.1 mL of 25 mM HAuCl 4 solution in a 20 mL glass bottle with deionized water to 5 mL, and add 5 mL of 0.2M CTAB solution to the diluent to obtain solution one;
2)将0.6mL0.01M的NaBH 4溶液快速注入溶液一中，NaBH 4溶液现用现配，对混合溶液进行磁力搅拌，速度为1200rpm，搅拌2min，最后获得的种子溶液在30℃静置30min待用； 2) Quickly inject 0.6 mL of 0.01M NaBH 4 solution into solution 1. The NaBH 4 solution is ready to use, and the mixed solution is magnetically stirred at a speed of 1200 rpm for 2 minutes. The finally obtained seed solution is allowed to stand at 30°C for 30 minutes stand-by;
3)将7.0g CTAB和1.234g油酸钠溶解于水中250mL 50℃水中，自然冷却到30℃，然后加入18ml,4.0mM的硝酸银溶液，保温1min，得 到溶液二；3) Dissolve 7.0g CTAB and 1.234g sodium oleate in 250mL water at 50°C, cool it naturally to 30°C, then add 18ml, 4.0mM silver nitrate solution, keep it warm for 1min, and get solution two;
4)一边磁力搅拌一边向溶液二中注射250ml 1.0mM的HAuCl 4溶液，在700rpm速度下搅拌90min，改变第二次磁力搅拌速度为400rpm，一边搅拌一边加入2.1ml 37wt％的HCl溶液，搅拌15min，最后加入1.25ml 0.064M的抗坏血酸溶液，进行第三次搅拌，搅拌速度为1200rpm，搅拌30s，得到生长液； 4) While magnetic stirring, inject 250ml 1.0mM HAuCl 4 solution into solution 2, stir at 700rpm for 90min, change the second magnetic stirring speed to 400rpm, add 2.1ml 37wt% HCl solution while stirring, and stir for 15min Finally, add 1.25ml of 0.064M ascorbic acid solution and stir for the third time at a stirring speed of 1200rpm for 30s to obtain a growth solution;
5)将0.4mL种子溶液注入生长液中1500rpm速度下搅拌30s，最后，将混合液在28℃条件下静置10h，将静置后的混合液在8000r/min的速度速度下离心10min，收集沉淀物，将沉淀物分散于80mM的CTAC溶液中；5) Inject 0.4 mL of seed solution into the growth solution and stir at 1500 rpm for 30 seconds. Finally, the mixture is allowed to stand at 28°C for 10 hours, and the static mixture is centrifuged at a speed of 8000 r/min for 10 minutes, and collected Precipitate, disperse the precipitate in 80mM CTAC solution;
6)将步骤5)重复三次，将所述得沉淀物储存在CTAC溶液中，得到金纳米棒溶液。6) Repeat step 5) three times, and store the precipitate in the CTAC solution to obtain the gold nanorod solution.
2、金核银壳纳米棒的制备2. Preparation of gold core silver shell nanorods
将0.5mL金纳米棒溶液用水稀释至4ml，向稀释液中加入2.0ml 10mM的硝酸银溶液，在100KHz频率下超声波处理2min，后加入2.0ml 0.1M的抗坏血酸溶液，经65℃水浴保存4h、8000r/min离心10min，收集沉淀物分散到1ml去离子水中，得到金核银壳纳米棒悬浮液。Dilute 0.5mL gold nanorod solution to 4ml with water, add 2.0ml 10mM silver nitrate solution to the diluent, sonicate for 2min at 100KHz frequency, then add 2.0ml 0.1M ascorbic acid solution, store in 65℃ water bath for 4h, Centrifuge at 8000r/min for 10min, collect the precipitate and disperse it in 1ml deionized water to obtain a gold core silver shell nanorod suspension.
3、拉曼检测分子的键合3. Raman detects the bonding of molecules
将2μL,5mM的维生素K4乙醇溶液经重氮化巯解引入巯基后添加到1.0ml金核银壳纳米棒悬浮液中得到混合物，温和摇动混合物2h，得到维生素K4标记的金核银壳纳米棒溶液。Add 2μL, 5mM vitamin K4 ethanol solution by diazotization to sulfhydryl to introduce sulfhydryl groups and add it to 1.0ml gold core silver shell nanorod suspension to obtain the mixture. Shake the mixture gently for 2 hours to obtain vitamin K4 labeled gold core silver shell nanorods. Solution.
实施例5Example 5
本实施例提供了一种基于表面增强拉曼技术的基底材料及其制备方法，该基底材料结构为：This embodiment provides a substrate material based on surface-enhanced Raman technology and a preparation method thereof, and the structure of the substrate material is:
金纳米棒内核，直径为24.0nm，长度为91.5nm；The core of gold nanorods has a diameter of 24.0nm and a length of 91.5nm;
银纳米外壳，厚度为16nm；Silver nano shell with a thickness of 16nm;
该金核银壳纳米棒由以下制备方法制得，该制备方法包括以下步骤：The gold core silver shell nanorod is prepared by the following preparation method, and the preparation method includes the following steps:
1、金纳米棒的制备1. Preparation of gold nanorods
1)在20mL的玻璃瓶中将0.1mL浓度为25mM HAuCl 4溶液用去离子水稀释至5mL，向稀释液中加入5mL 0.2M的CTAB溶液，得到溶液一； 1) Dilute 0.1 mL of 25 mM HAuCl 4 solution in a 20 mL glass bottle with deionized water to 5 mL, and add 5 mL of 0.2M CTAB solution to the diluent to obtain solution one;
2)将0.6mL0.01M的NaBH 4溶液快速注入溶液一中，NaBH 4溶液现用现配，对混合溶液进行磁力搅拌，速度为1200rpm，搅拌2min，最后获得的种子溶液在30℃静置30min待用； 2) Quickly inject 0.6 mL of 0.01M NaBH 4 solution into solution 1. The NaBH 4 solution is ready to use, and the mixed solution is magnetically stirred at a speed of 1200 rpm for 2 minutes. The finally obtained seed solution is allowed to stand at 30°C for 30 minutes stand-by;
3)将7.0g CTAB和1.234g油酸钠溶解于水中250mL 50℃水中，自然冷却到30℃，然后加入18ml,4.0mM的硝酸银溶液，保温1min，得到溶液二；3) Dissolve 7.0g CTAB and 1.234g sodium oleate in 250mL water at 50°C, cool to 30°C naturally, then add 18ml, 4.0mM silver nitrate solution and keep it for 1min to obtain solution two;
4)一边磁力搅拌一边向溶液二中注射250ml 1.0mM的HAuCl 4溶液，在700rpm速度下搅拌90min，改变第二次磁力搅拌速度为400rpm，一边搅拌一边加入2.1ml 37wt％的HCl溶液，搅拌15min，最后加入1.25ml 0.064M的抗坏血酸溶液，进行第三次搅拌，搅拌速度为1200rpm，搅拌30s，得到生长液； 4) While magnetic stirring, inject 250ml 1.0mM HAuCl 4 solution into solution 2, stir at 700rpm for 90min, change the second magnetic stirring speed to 400rpm, add 2.1ml 37wt% HCl solution while stirring, and stir for 15min Finally, add 1.25ml of 0.064M ascorbic acid solution and stir for the third time at a stirring speed of 1200rpm for 30s to obtain a growth solution;
5)将0.4mL种子溶液注入生长液中1500rpm速度下搅拌30s，最后，将混合液在25℃条件下静置10h，将静置后的混合液在8000r/min的速度速度下离心10min，收集沉淀物，将沉淀物分散于80mM的CTAC溶液中；5) Inject 0.4 mL of seed solution into the growth solution and stir at 1500 rpm for 30 seconds. Finally, the mixture is allowed to stand at 25°C for 10 hours, and the static mixture is centrifuged at a speed of 8000 r/min for 10 minutes, and collected Precipitate, disperse the precipitate in 80mM CTAC solution;
6)将步骤5)重复三次，将所述得沉淀物储存在CTAC溶液中，得到金纳米棒溶液。6) Repeat step 5) three times, and store the precipitate in the CTAC solution to obtain the gold nanorod solution.
2、金核银壳纳米棒的制备2. Preparation of gold core silver shell nanorods
将0.5mL金纳米棒溶液用水稀释至4ml，向稀释液中加入2.5ml 10 mM的硝酸银溶液，在100KHz频率下超声波处理2min，后加入2.5ml 0.1M的抗坏血酸溶液，经65℃水浴保存4h、8000r/min离心10min，收集沉淀物分散到1ml去离子水中，得到金核银壳纳米棒悬浮液。Dilute 0.5ml gold nanorod solution to 4ml with water, add 2.5ml 10 mM silver nitrate solution to the diluent, ultrasonic treatment at 100KHz frequency for 2min, then add 2.5ml 0.1M ascorbic acid solution, store in 65℃ water bath for 4h , Centrifuge at 8000r/min for 10min, collect the precipitate and disperse it in 1ml of deionized water to obtain a gold core silver shell nanorod suspension.
3、拉曼检测分子的键合3. Raman detects the bonding of molecules
将2μL,5mM的维生素K4乙醇溶液经重氮化巯解引入巯基后添加到1.0ml金核银壳纳米棒悬浮液中得到混合物，温和摇动混合物2h，得到维生素K4标记的金核银壳纳米棒溶液。Add 2μL, 5mM vitamin K4 ethanol solution by diazotization to sulfhydryl to introduce the sulfhydryl group and add it to 1.0ml gold core silver shell nanorod suspension to obtain the mixture, gently shake the mixture for 2h to obtain vitamin K4 labeled gold core silver shell nanorod Solution.
实验例1Experimental example 1
按照以下方法，分别制作实施例1-5中金核银壳纳米棒和金纳米棒的单层列阵膜：According to the following methods, the single-layer array films of gold core silver shell nanorods and gold nanorods in Examples 1-5 were respectively produced:
将0.2mL已制备金悬浮液用蒸馏水稀释至2ml，在100KHz频率下超声处理10min后得到非聚集颗粒，然后，将非聚集颗粒放置在预处理过的硅片上，并在在一个密封的盒子里自组装成单层阵列薄膜，切成片，尺寸约为5mm×5mm。Dilute 0.2mL of the prepared gold suspension to 2ml with distilled water, and sonicate it at 100KHz for 10 minutes to obtain non-aggregated particles. Then, place the non-aggregated particles on the pretreated silicon wafer and place them in a sealed box. The inside is self-assembled into a single-layer array film and cut into slices with a size of about 5mm×5mm.
实验例2Experimental example 2
对实施例1-5中获得的金核银壳纳米棒进行以下测定：The gold-core-silver-shell nanorods obtained in Examples 1-5 were subjected to the following determinations:
(1)利用加速电压为200kv的场发射透射电镜(JEM-2100F,JEOL)获得透射电镜(TEM)图像，结果如图1所示；(1) Obtain a transmission electron microscope (TEM) image using a field emission transmission electron microscope (JEM-2100F, JEOL) with an accelerating voltage of 200kv, and the result is shown in Figure 1;
(2)用岛津紫外-1800紫外-可见分光光度计测定了紫外-可见消光光谱，结果如图2所示；(2) The UV-Visible extinction spectrum was measured with Shimadzu UV-1800 UV-Vis spectrophotometer, and the result is shown in Figure 2;
(3)采用Renishaw inVia共焦拉曼光谱仪，以20倍物镜和785nm激光为激发源，在徕卡显微镜上进行拉曼光谱采集。光谱在800-1800cm -1范围内，曝光时间为10秒，结果如图3-4所示。 (3) Using a Renishaw inVia confocal Raman spectrometer, using a 20x objective lens and a 785nm laser as the excitation source, the Raman spectrum was collected on a Leica microscope. The spectrum is in the range of 800-1800cm -1 and the exposure time is 10 seconds. The result is shown in Figure 3-4.
综合以上测定结果，可以得出，在金纳米棒直径为22nm，长度为90nm,银纳米壳厚度为16nm的情况下SERS强度最好，可达到金纳米作为基底 材料的100倍。Based on the above measurement results, it can be concluded that the SERS intensity is the best when the diameter of the gold nanorod is 22nm, the length is 90nm, and the thickness of the silver nanoshell is 16nm, which can reach 100 times that of the gold nanorod as the base material.
实验例3Experimental example 3
将本发明中的带维生素K4标记的金核银壳纳米棒替换传统SERS中的基底材料，用于人工尿液中标准KIM1生物标记物的分析。The vitamin K4 labeled gold core silver shell nanorods of the present invention replace the base material in the traditional SERS, and are used for the analysis of standard KIM1 biomarkers in artificial urine.
首先，取6.0ml带维生素K4标记的金核银壳纳米棒溶液，在7000rpm下离心10分钟并且分散到6.0mL去离子水中。接下来，将12μL，25％wt戊二醛溶液添加到6.0ml维生素K4标记的金核银壳纳米棒溶液中，轻度摇动1.5h，产物在6000rpm下离心10min，将沉淀物分散到6ml，9μg/mL的KIM1检测抗体溶液中，得到顶层结构溶液，将该混合溶液在4℃条件下保存12h，然后用1.0mL 1×PBS缓冲液离心纯化，在4℃条件下保存待用。First, take 6.0 ml of the vitamin K4 labeled gold core silver shell nanorod solution, centrifuge at 7000 rpm for 10 minutes, and disperse in 6.0 mL of deionized water. Next, add 12 μL of 25% wt glutaraldehyde solution to 6.0 ml of vitamin K4 labeled gold core silver shell nanorod solution, shake gently for 1.5 hours, centrifuge the product at 6000 rpm for 10 minutes, and disperse the precipitate to 6 ml. In 9μg/mL KIM1 detection antibody solution, the top structure solution was obtained. The mixed solution was stored at 4°C for 12h, and then purified by centrifugation with 1.0mL 1×PBS buffer, and stored at 4°C until use.
然后，用150mM的半胱胺溶液对金核银壳纳米棒溶液进行氨基功能化处理，然后，加入12μL，25％wt戊二醛溶液，使金核银壳纳米棒溶液功能化，之后在功能化后的金核银壳纳米棒溶液中加入12mL，20μg/mL KIM1的捕获抗体溶液，于4℃温度下保存12h，接下来，向保存后的溶液加入12mL，1％BSA溶液，静置1h以阻断非特异性结合活性位点，得到衬底结构溶液。Then, the gold core silver shell nanorod solution was functionalized with 150 mM cysteamine solution. Then, 12 μL of 25% wt glutaraldehyde solution was added to functionalize the gold core silver shell nanorod solution. Add 12mL of 20μg/mL KIM1 capture antibody solution to the transformed gold core silver shell nanorod solution and store it at 4°C for 12 hours. Next, add 12mL of 1% BSA solution to the stored solution and let it stand for 1 hour. In order to block non-specific binding to the active site, a substrate structure solution is obtained.
取6份人工尿液溶液各200μL，分别在6份溶液中加入浓度为1ng/mL、10pg/mL、100fg/mL、1fg/mL、0.1fg/mL和0fg/mL的KIM1，混合均匀后得到待测尿液，待用。Take 200μL each of 6 artificial urine solutions, and add KIM1 with the concentration of 1ng/mL, 10pg/mL, 100fg/mL, 1fg/mL, 0.1fg/mL and 0fg/mL to the 6 solutions respectively, and mix them evenly. Urine to be tested, ready for use.
将衬底溶液平均分为6份，分别在6份衬底溶液中加入6种待测尿液，在室温下温育1h，得到待测KIM1尿液。The substrate solution was equally divided into 6 parts, and 6 kinds of urine to be tested were added to the 6 parts of substrate solution respectively, and incubated at room temperature for 1 hour to obtain the KIM1 urine to be tested.
最后，在每份待测KIM1尿液中加入200μL顶层结构溶液，经特异性结合后形成待测的检测抗体-待测抗原-捕获抗体“夹心”结构溶液，进行SERS检测，如图5所示，,拉曼强度随KIM1浓度的增加呈单调下降 趋势，LOD从高于背景的3倍标准偏差低至0.1fg/mL；图6显示了1145cm -1峰值的拉曼强度随KIM1浓度的变化。图6中的插图显示了其较宽的线性动态响应范围，从1ng/mL到0.1fg/mL。 Finally, add 200μL of the top structure solution to each KIM1 urine to be tested, and form a "sandwich" structure solution of the detection antibody-antigen-to-be-tested-capture antibody to be tested after specific binding for SERS detection, as shown in Figure 5 ,, Raman intensity showed a monotonous downward trend with the increase of KIM1 concentration, and the LOD was as low as 0.1fg/mL from 3 times the standard deviation above the background; Figure 6 shows the Raman intensity of the peak at 1145 cm -1 as the KIM1 concentration changes. The inset in Figure 6 shows its wide linear dynamic response range, from 1ng/mL to 0.1fg/mL.
结合这里披露的本发明的说明和实践，本发明的其他实施例对于本领域技术人员都是易于想到和理解的。说明和实施例仅被认为是示例性的，本发明的真正范围和主旨均由权利要求所限定。In combination with the description and practice of the present invention disclosed herein, other embodiments of the present invention are easily conceived and understood by those skilled in the art. The description and embodiments are only considered to be exemplary, and the true scope and spirit of the present invention are defined by the claims.

Claims (10)

  1. 一种高效表面增强拉曼散射基底材料，包括：An efficient surface-enhanced Raman scattering substrate material, including:
    纳米内核，所述纳米内核是由金纳米棒构成；和A nano-core, the nano-core is composed of gold nanorods; and
    纳米外壳，所述纳米外壳是由纳米银材料构成，所述纳米外壳均匀完全的包裹于所述纳米内核表面，所述纳米外壳外表面结合有拉曼检测分子。Nano-shell, the nano-shell is made of nano-silver material, the nano-shell is evenly and completely wrapped on the surface of the nano-core, and the outer surface of the nano-shell is bound with Raman detection molecules.
  2. 根据权利要求1所述的表面增强拉曼散射基底材料，其特征在于，所述拉曼检测分子为维生素K4，所述维生素K4经重氮化巯解引入巯基。The surface-enhanced Raman scattering substrate material of claim 1, wherein the Raman detection molecule is vitamin K4, and the vitamin K4 introduces a sulfhydryl group through diazotization and sulfhydrylation.
  3. 根据权利要求1所述的表面增强拉曼散射基底材料，其特征在于，所述纳米内核的直径为19-26nm，长度为80-96nm。The surface-enhanced Raman scattering substrate material of claim 1, wherein the nano-core has a diameter of 19-26 nm and a length of 80-96 nm.
  4. 根据权利要求1所述的表面增强拉曼散射基底材料，其特征在于，所述纳米外壳的厚度为2-18nm。The surface-enhanced Raman scattering base material of claim 1, wherein the thickness of the nano-shell is 2-18 nm.
  5. 一种权利要求1-4所述的高效表面增强拉曼散射基底材料的制备方法，其特征在于，包括以下步骤：A method for preparing a high-efficiency surface-enhanced Raman scattering base material according to claims 1-4, characterized in that it comprises the following steps:
    (1)金纳米棒的制备；(1) Preparation of gold nanorods;
    1)用水对HAuCl 4溶液进行稀释得到稀释液，向稀释液中的加入CTAB溶液得到溶液一； 1) Dilute the HAuCl 4 solution with water to obtain a diluent, and add CTAB solution to the diluent to obtain solution one;
    2)将溶液一快速注入NaBH 4溶液中，搅拌得到种子溶液，静置； 2) Quickly inject the solution into the NaBH 4 solution, stir to obtain the seed solution, and let it stand;
    3)将CTAB和油酸钠溶解于水中，冷却后加入硝酸银溶液，保温后得到溶液二；3) Dissolve CTAB and sodium oleate in water, add silver nitrate solution after cooling, and obtain solution 2 after heat preservation;
    4)一边搅拌一边向溶液二中注射HAuCl 4溶液，继续第一次搅拌，改变第二次搅拌速度，一边搅拌一边加入HCl溶液，继续第二次搅拌，最后加入抗坏血酸溶液，进行第三次搅拌，得到生长液； 4) While stirring, inject the HAuCl 4 solution into the second solution, continue the first stirring, change the second stirring speed, add the HCl solution while stirring, continue the second stirring, and finally add the ascorbic acid solution, and perform the third stirring , Get the growth solution;
    5)将种子溶液注入生长液中得到混合溶液，将混合溶液静置，将静置后的混合溶液进行离心，收集沉淀物，将沉淀物分散于CTAC溶液中；5) Inject the seed solution into the growth solution to obtain a mixed solution, leave the mixed solution to stand, centrifuge the settled mixed solution, collect the precipitate, and disperse the precipitate in the CTAC solution;
    6)将步骤5)重复三次，将所述得沉淀物储存在CTAC溶液中，得到金纳米棒溶液；6) Repeat step 5) three times, and store the precipitate in CTAC solution to obtain a gold nanorod solution;
    (2)金核银壳纳米棒的制备；(2) Preparation of gold core silver shell nanorods;
    将金纳米棒溶液用水稀释，向稀释液中加入硝酸银溶液，进行超声波处理后加入抗坏血酸溶液，经水浴保存、离心和分散得到金核银壳纳米棒悬浮液；Dilute the gold nanorod solution with water, add silver nitrate solution to the diluent, add ascorbic acid solution after ultrasonic treatment, store in a water bath, centrifuge and disperse to obtain a gold core silver shell nanorod suspension;
    (3)拉曼检测分子的键合；(3) Raman detects the bonding of molecules;
    将维生素K4乙醇溶液经重氮化巯解引入巯基，再添加到金核银壳纳米棒悬浮液中得到混合物，温和摇动混合物，得到拉维生素K4标记的金核银壳纳米棒。The vitamin K4 ethanol solution is diazotized and mercaptolyzed to introduce sulfhydryl groups, and then added to the gold core silver shell nanorod suspension to obtain a mixture, and the mixture is gently shaken to obtain vitamin K4 labeled gold core silver shell nanorods.
  6. 根据权利要求5所述的一种高效表面增强拉曼散射基底材料制备方法，其特征在于，步骤1)中所述水为去离子水；The method for preparing a high-efficiency surface-enhanced Raman scattering base material according to claim 5, wherein the water in step 1) is deionized water;
    所述HAuCl 4溶液的浓度为25mM，所述稀释的倍数为50倍； The concentration of the HAuCl 4 solution is 25 mM, and the dilution factor is 50 times;
    所述CTAB溶液的浓度为0.2M；The concentration of the CTAB solution is 0.2M;
    所述HAuCl 4溶液与所述CTAB溶液的体积比为1:50； The volume ratio of the HAuCl 4 solution to the CTAB solution is 1:50;
    步骤2)中所述NaBH 4溶液的浓度为0.01M，现用现配； The concentration of the NaBH 4 solution in step 2) is 0.01M, and it is ready to use;
    所述NaBH 4溶液与所述HAuCl 4溶液的体积比为1：6； The volume ratio of the NaBH 4 solution to the HAuCl 4 solution is 1:6;
    步骤2)中所述搅拌为磁力搅拌，所述磁力搅拌的速度为1200rpm，时间为2min，所述静置时间为30min，温度为25-30℃；The stirring in step 2) is magnetic stirring, the speed of the magnetic stirring is 1200rpm, the time is 2min, the standing time is 30min, and the temperature is 25-30°C;
    步骤3)中所述溶解温度为50℃，所述冷却温度为28-30℃；In step 3), the dissolving temperature is 50°C, and the cooling temperature is 28-30°C;
    步骤3)中所述水与步骤2)中所述NaBH 4溶液的体积比为1250：3； The volume ratio of the water in step 3) to the NaBH 4 solution in step 2) is 1250:3;
    步骤3)中所述CTAB、油酸钠质量和硝酸银溶液体积与步骤1)中所述HAuCl 4溶液的体积比为70g：12.34g：180ml：1ml； The volume ratio of CTAB, the mass of sodium oleate and the volume of silver nitrate solution in step 3) to the HAuCl 4 solution in step 1) is 70g: 12.34g: 180ml: 1ml;
    所述硝酸银溶液得浓度为4.0M，所述保温的时间为1min；The concentration of the silver nitrate solution is 4.0 M, and the holding time is 1 min;
    步骤4)中所述HAuCl 4溶液的浓度为1.0mM，所述HCl溶液的浓度 为37wt％，所述抗坏血酸溶液得浓度为0.064M； In step 4), the concentration of the HAuCl 4 solution is 1.0 mM, the concentration of the HCl solution is 37 wt%, and the concentration of the ascorbic acid solution is 0.064 M;
    步骤4)中所述HAuCl 4溶液、HCl溶液和所述抗坏血酸溶液与步骤3)中所述水的体积比为250:2.1:1.25:250； The volume ratio of the HAuCl 4 solution, the HCl solution and the ascorbic acid solution in step 4) to the water in step 3) is 250:2.1:1.25:250;
    步骤4)中所述第一次搅拌为磁力搅拌，速度为700rpm，持续时间为90min；The first stirring in step 4) is magnetic stirring, the speed is 700 rpm, and the duration is 90 min;
    步骤4)中所述第二次搅拌为磁力搅拌，速度为400rpm，持续时间为15min；The second stirring in step 4) is magnetic stirring, the speed is 400rpm, and the duration is 15min;
    步骤4)中所述第三次搅拌为磁力搅拌，速度为1200rpm，持续时间为30s；The third stirring in step 4) is magnetic stirring, the speed is 1200rpm, and the duration is 30s;
    步骤5)中所述种子溶液的加入量与步骤1)中所述HAuCl 4溶液的体积比为4:1； The volume ratio of the added amount of the seed solution in step 5) to the HAuCl 4 solution in step 1) is 4:1;
    步骤5)中所述搅拌为磁力搅拌，速度为1500rpm，持续时间为30s；The stirring in step 5) is magnetic stirring, the speed is 1500rpm, and the duration is 30s;
    步骤5)中所述静置的温度为25-30℃，时间为8-12h；In step 5), the standing temperature is 25-30°C, and the time is 8-12h;
    步骤5)中所述离心的转速为8000r/min，持续时间为10minThe speed of centrifugation mentioned in step 5) is 8000r/min, and the duration is 10min
    步骤5)中所述CTAC溶液的浓度为80mM，所述CTAC溶液与所述种子溶液的体积比为1:2；In step 5), the concentration of the CTAC solution is 80 mM, and the volume ratio of the CTAC solution to the seed solution is 1:2;
    步骤6)中所述CTAC溶液的浓度为80mM，所述CTAC溶液与所述种子溶液的体积比为1:2；In step 6), the concentration of the CTAC solution is 80 mM, and the volume ratio of the CTAC solution to the seed solution is 1:2;
  7. 根据权利要求5所述的一种高效表面增强拉曼散射基底材料制备方法，其特征在于，步骤2中所述水为去离子水，所述稀释的倍数为8倍；The method for preparing a high-efficiency surface-enhanced Raman scattering base material according to claim 5, wherein the water in step 2 is deionized water, and the dilution factor is 8 times;
    所述硝酸溶液的浓度为10mM，所述硝酸银溶液与所述金纳米棒溶液的体积比为(1:0.4)-(1:5)；The concentration of the nitric acid solution is 10 mM, and the volume ratio of the silver nitrate solution to the gold nanorod solution is (1:0.4)-(1:5);
    所述抗坏血酸溶液的体积与所述硝酸银溶液的体积比为1:1；The volume ratio of the ascorbic acid solution to the silver nitrate solution is 1:1;
    所述超声波处理的频率为100kHz，处理时间为2min；The frequency of the ultrasonic treatment is 100kHz, and the treatment time is 2min;
    所述水浴保存的温度为63-68℃，时间为4h；The storage temperature in the water bath is 63-68°C, and the time is 4h;
    所述离心的转速为8000r/min，时间为10min；The rotation speed of the centrifugation is 8000r/min, and the time is 10min;
    所述分散所用的溶液为去离子水，所述去离子水与所述金纳米棒溶液体积比为1:2.The solution used for the dispersion is deionized water, and the volume ratio of the deionized water to the gold nanorod solution is 1:2.
  8. 根据权利要求5所述的一种高效表面增强拉曼散射基底材料制备方法，其特征在于，步骤3中所述维生素K4乙醇溶液与所述金核银壳纳米棒悬浊液的体积比为1:500；The method for preparing a high-efficiency surface-enhanced Raman scattering substrate material according to claim 5, wherein the volume ratio of the vitamin K4 ethanol solution to the gold core silver shell nanorod suspension in step 3 is 1 :500;
    所述温和摇动的时间为2h。The gentle shaking time is 2h.
  9. 权利要求1-4任一项所述的高效表面增强拉曼散射基底材料在制备检测肾损伤因子的表面增强拉曼散射基底材料中的用途。Use of the high-efficiency surface-enhanced Raman scattering substrate material of any one of claims 1 to 4 in preparing a surface-enhanced Raman scattering substrate material for detecting renal damage factors.
  10. 根据权利要求9所述的用途，其特征在于，所述肾损伤因子为糖蛋白类免疫物质。The use according to claim 9, wherein the kidney injury factor is a glycoprotein immune substance.
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