Efficient surface-enhanced Raman scattering substrate material and preparation method thereof
阅读说明：本技术 一种高效表面增强拉曼散射基底材料及制备方法 (Efficient surface-enhanced Raman scattering substrate material and preparation method thereof ) 是由 熊良钟 张茂峰 熊清爵 陈敏 阮志燕 王振 于 2019-09-11 设计创作，主要内容包括：本发明公开了一种基于表面增强拉曼技术的基底材料及其制备方法。其中,表面增强拉曼技术的基底材料包括：纳米内核,纳米内核是由金纳米棒构成；纳米外壳,纳米外壳是由纳米银材料构成,纳米外壳均匀完全的包裹于纳米内核表面,纳米外壳外表面结合有维生素K4。该基底材料用作表面增强拉曼检测的探针可以显著的增强检测的灵敏度,实现尿液中KIM-1含量的高敏感度检测,从而达到大动态范围的KIM-1含量测定。(The invention discloses a substrate material based on a surface enhanced Raman technology and a preparation method thereof. The substrate material of the surface enhanced Raman technology comprises: the nano inner core is composed of gold nanorods; the nano-shell is made of nano-silver materials, the nano-shell is uniformly and completely wrapped on the surface of the nano-core, and vitamin K4 is combined on the outer surface of the nano-shell. The substrate material used as a probe for surface enhanced Raman detection can significantly enhance the detection sensitivity and realize high-sensitivity detection of the KIM-1 content in urine, thereby achieving the KIM-1 content determination in a large dynamic range.)
1. A high efficiency surface enhanced raman scattering substrate material comprising:
the nano inner core is composed of gold nanorods; and
the nano shell is made of nano silver materials, the nano shell is uniformly and completely wrapped on the surface of the nano core, and Raman detection molecules are combined on the outer surface of the nano shell.
2. The surface-enhanced raman scattering substrate material of claim 1, wherein said raman detecting molecule is vitamin K4, said vitamin K4 being introduced into a thiol group by diazotization sulfhydrylation.
3. The surface-enhanced raman scattering substrate material according to claim 1, wherein said nanoinner core has a diameter of 19-26nm and a length of 80-96 nm.
4. The surface-enhanced raman scattering substrate material according to claim 1, wherein the thickness of said nano-shell is 2-18 nm.
5. A method for preparing the high-efficiency surface-enhanced Raman scattering substrate material according to claims 1-4, comprising the following steps:
(1) preparing gold nanorods;
1) by water pairing of HAuCl4Diluting the solution to obtain a diluent, and adding a CTAB solution into the diluent to obtain a first solution;
2) solution one is injected into NaBH quickly4Stirring the solution to obtain a seed solution, and standing the seed solution;
3) dissolving CTAB and sodium oleate in water, cooling, adding a silver nitrate solution, and preserving heat to obtain a second solution;
4) injecting HAuCl into the second solution while stirring4Continuously stirring the solution for the first time, changing the stirring speed for the second time, adding HCl solution while stirring, continuously stirring for the second time, finally adding ascorbic acid solution, and performing the first timeStirring for three times to obtain a growth solution;
5) injecting the seed solution into the growth solution to obtain a mixed solution, standing the mixed solution, centrifuging the standing mixed solution, collecting the precipitate, and dispersing the precipitate in a CTAC solution;
6) repeating the step 5) for three times, and storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution;
(2) preparing a gold-core silver-shell nanorod;
diluting the gold nanorod solution with water, adding a silver nitrate solution into the diluent, performing ultrasonic treatment, adding an ascorbic acid solution, and performing water bath preservation, centrifugation and dispersion to obtain a gold-core-silver-shell nanorod suspension;
(3) detecting the bonding of molecules by Raman;
and diazotizing and sulfhydryzing the vitamin K4 ethanol solution to introduce sulfhydryl, adding the mixture into the gold-core silver-shell nanorod suspension to obtain a mixture, and gently shaking the mixture to obtain the gold-core silver-shell nanorod marked with vitamin K4.
6. The method for preparing the high efficiency surface enhanced Raman scattering substrate according to claim 5, wherein the water in step 1) is deionized water;
the HAuCl4The concentration of the solution is 25mM, and the dilution factor is 50 times;
the concentration of the CTAB solution is 0.2M;
the HAuCl4The volume ratio of the solution to the CTAB solution is 1: 50;
the NaBH in the step 2)4The concentration of the solution is 0.01M, and the solution is prepared at present;
the NaBH4Solution with the HAuCl4The volume ratio of the solution is 1: 6;
the stirring in the 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 ℃;
in the step 3), the dissolving temperature is 50 ℃, and the cooling temperature is 28-30 ℃;
the water in the step 3) and the NaBH in the step 2)4The volume ratio of the solution is 1250: 3;
the mass of CTAB and sodium oleate and the volume of silver nitrate solution in the step 3) and the HAuCl in the step 1)4The volume ratio of the solution is 70 g: 12.34 g: 180 ml: 1 ml;
the concentration of the silver nitrate solution is 4.0M, and the heat preservation time is 1 min;
HAuCl described in step 4)4The concentration of the solution is 1.0mM, the concentration of the HCl solution is 37 wt%, and the concentration of the ascorbic acid solution is 0.064M;
HAuCl described in step 4)4The volume ratio of the solution, the HCl solution and the ascorbic acid solution to the water in the step 3) is 250:2.1:1.25: 250;
the first stirring in the step 4) is magnetic stirring, the speed is 700rpm, and the duration is 90 min;
the second stirring in the step 4) is magnetic stirring, the speed is 400rpm, and the duration is 15 min;
the third stirring in the step 4) is magnetic stirring, the speed is 1200rpm, and the duration is 30 s;
the addition amount of the seed solution in the step 5) and the HAuCl in the step 1)4The volume ratio of the solution is 4: 1;
in the step 5), the stirring is magnetic stirring, the speed is 1500rpm, and the duration is 30 s;
the standing temperature in the step 5) is 25-30 ℃, and the standing time is 8-12 h;
the rotating speed of the centrifugation in the step 5) is 8000r/min, and the duration time is 10min
The concentration of the CTAC solution in the step 5) is 80mM, and the volume ratio of the CTAC solution to the seed solution is 1: 2;
the concentration of the CTAC solution in the step 6) is 80mM, and the volume ratio of the CTAC solution to the seed solution is 1:2.
7. The method for preparing a high efficiency surface enhanced Raman scattering substrate according to claim 5, wherein the water in step 2 is deionized water, and the dilution factor is 8 times;
the concentration of the nitric acid solution is 10mM, 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 ultrasonic treatment is 100kHz, and the treatment time is 2 min;
the temperature for water bath preservation is 63-68 ℃, and the time is 4 h;
the rotating speed of the centrifugation is 8000r/min, and the time is 10 min;
the solution used for dispersing is deionized water, and the volume ratio of the deionized water to the gold nanorod solution is 1:2.
8. 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;
the time for the gentle shaking was 2 h.
9. Use of the high efficiency surface enhanced raman scattering substrate material of any one of claims 1 to 4 in the preparation of a surface enhanced raman scattering substrate material for the detection of a renal injury factor.
10. The use of claim 9, wherein the kidney injury factor is a glycoprotein based immune substance.
Technical Field
The invention relates to the technical field of Raman scattering, in particular to a high-efficiency surface-enhanced Raman scattering substrate material and a preparation method thereof.
Background
At present, acute and chronic kidney diseases characterized by kidney injury are one of challenging health problems worldwide, several indexes of urine routine tests, osmotic pressure, blood creatinine, urea nitrogen, endogenous creatinine clearance and the like are generally adopted for judging the kidney injury, but when the indexes are increased, a plurality of kidney injuries are very serious or irreversible damage is generated, so that a plurality of scholars indicate that the activity of urine N-acetyl-beta-D-glucosaminidase (NAG) can be used as an early index of the injury, but the index is generally shown when the kidney disease is in 8-16 hours, at present, researches prove that the content of kidney injury molecule 1(KIM-1) in urine has obvious specificity for early diagnosis of acute kidney injury, and the KIM-1 content can be changed when the kidney injury is 4-6 hours, can quickly, sensitively and specifically reflect the damage and recovery process of various kidney diseases, and can be a reliable biological marker for detecting early kidney damage, so that the high-precision detection of the KIM-1 content in urine has great significance for the diagnosis and treatment of acute and chronic kidney diseases characterized by kidney damage.
At present, the method for measuring the KIM-1 content in urine is mainly an ELISA method, and the sensitivity is pg/ml, so that the method cannot realize quantitative detection of the KIM-1 content in urine within a large dynamic range.
The Surface Enhanced Raman Scattering (SERS) technique has become an attractive and powerful analytical technique due to its high sensitivity, narrow linewidth and fingerprint effect, enabling multiple biosensing. The surface enhanced Raman scattering has excellent Raman enhancement efficiency which can reach 14 to 15 orders of magnitude, so that the surface enhanced Raman scattering can reliably and accurately detect ultra-trace or even single molecule level analytes. In addition, SERS detection can be done quickly within minutes. Therefore, surface-enhanced raman scattering has been widely applied in various fields such as chemical sensing, biological analysis, biological sensing and early cancer diagnosis, but at present, most of the surface-enhanced raman scattering substrate materials select smooth macroscopic glass, gold, silver or bimetallic films as substrate materials, and do not have sufficient plasma "hot spots", which results in limited sensitivity of the surface-enhanced raman technology.
In order to improve the sensitivity of SERS detection, currently, a Raman detection molecule is bonded on a substrate material for SERS detection, wherein the best effect is a P-ATP molecule, the sensitivity is ng/ml-pg/ml, and although the sensitivity of SERS detection is improved after the P-ATP molecule is bonded, the detection sensitivity and the detection range width required by many detected objects are higher
Disclosure of Invention
The invention aims to provide an efficient surface-enhanced Raman scattering substrate material and a preparation method thereof, which can realize high-sensitivity detection of the KIM-1 content in urine, and can reach fg/ml level, thereby achieving the KIM-1 content determination in a large dynamic range.
In one aspect of the present invention, a high efficiency surface enhanced raman scattering substrate includes:
the nano inner core is composed of gold nanorods; and
the nano-shell is made of a nano-silver material, the nano-shell is uniformly and completely wrapped on the surface of the nano-core, and the outer surface of the nano-shell is bonded with Raman detection molecule vitamin K4.
Further, the vitamin K4 is introduced into sulfhydryl groups through diazotization sulfhydrylation.
Furthermore, the diameter of the nanometer inner core is 19-26nm, and the length of the nanometer inner core is 80-96 nm.
Further, the thickness of the nano shell is 2-18 nm.
Another aspect of the present invention provides a method for preparing a high efficiency surface enhanced raman scattering substrate material, comprising the following steps:
(1) preparing gold nanorods;
1) by water pairing of HAuCl4Diluting the solution to obtain a diluent, and adding a CTAB solution into the diluent to obtain a first solution;
2) solution one is injected into NaBH quickly4Stirring the solution to obtain a seed solution, and standing the seed solution;
3) dissolving CTAB and sodium oleate in water, cooling, adding a silver nitrate solution, and preserving heat to obtain a second solution;
4) injecting HAuCl into the second solution while stirring4Continuously stirring the solution for the first time, changing the stirring speed for the second time, adding the HCl solution while stirring, continuously stirring for the second time, finally adding the ascorbic acid solution, and stirring for the third time to obtain a growth solution;
5) injecting the seed solution into the growth solution to obtain a mixed solution, standing the mixed solution, centrifuging the standing mixed solution, collecting the precipitate, and dispersing the precipitate in a CTAC solution;
6) repeating the step 5) for three times, and storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution;
(2) preparing a gold-core silver-shell nanorod;
diluting the gold nanorod solution with water, adding a silver nitrate solution into the diluent, performing ultrasonic treatment, adding an ascorbic acid solution, and performing water bath preservation, centrifugation and dispersion to obtain a gold-core-silver-shell nanorod suspension;
(3) detecting the bonding of molecules by Raman;
and diazotizing and sulfhydryzing the vitamin K4 ethanol solution to introduce sulfhydryl, adding the mixture into the gold-core silver-shell nanorod suspension to obtain a mixture, and gently shaking the mixture to obtain the gold-core silver-shell nanorod marked with vitamin K4.
Further, the water in the step 1) is deionized water;
the HAuCl4The concentration of the solution is 25mM, and the dilution factor is 50 times;
the concentration of the CTAB solution is 0.2M;
the HAuCl4The volume ratio of the solution to the CTAB solution is 1: 50;
the NaBH in the step 2)4The concentration of the solution is 0.01M, the temperature is 25-30 ℃, and the solution is prepared at present;
the NaBH4Solution with the HAuCl4The volume ratio of the solution is 1: 6;
the stirring in the 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 ℃;
in the step 3), the dissolving temperature is 50 ℃, and the cooling temperature is 28-30 ℃;
the water in the step 3) and the NaBH in the step 2)4The volume ratio of the solution is 1250: 3;
the volume ratio of the CTAB, the mass of sodium oleate and the volume of silver nitrate solution in the step 3) to the HAuCl4 solution in the step 1) is 70 g: 12.34 g: 180 ml: 1 ml;
the concentration of the silver nitrate solution is 4.0M, and the heat preservation time is 1 min;
HAuCl described in step 4)4The concentration of the solution is 1.0mM, the concentration of the HCl solution is 37 wt%, and the concentration of the ascorbic acid solution is 0.064M;
HAuCl described in step 4)4The volume ratio of the solution, the HCl solution and the ascorbic acid solution to the water in the step 3) is 250:2.1:1.25: 250;
the first stirring in the step 4) is magnetic stirring, the speed is 700rpm, and the duration is 90 min;
the second stirring in the step 4) is magnetic stirring, the speed is 400rpm, and the duration is 15 min;
the third stirring in the step 4) is magnetic stirring, the speed is 1200rpm, and the duration is 30 s;
the addition amount of the seed solution in the step 5) and the HAuCl in the step 1)4The volume ratio of the solution is 4: 1;
in the step 5), the stirring is magnetic stirring, the speed is 1500rpm, and the duration is 30 s;
the standing temperature in the step 5) is 28-30 ℃, and the standing time is 8-12 h;
the rotating speed of the centrifugation in the step 5) is 8000r/min, and the duration time is 10min
The concentration of the CTAC solution in the step 5) is 80mM, and the volume ratio of the CTAC solution to the seed solution is 1: 2;
the concentration of the CTAC solution in the step 6) is 80mM, and the volume ratio of the CTAC solution to the seed solution is 1: 2;
further, the water in the step 2 is deionized water, and the dilution multiple is 8 times;
the concentration of the nitric acid solution is 10mM, 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 ultrasonic treatment is 100KHz, and the treatment time is 2 min;
the temperature for water bath preservation is 63-68 ℃, and the time is 4 h;
the rotating speed of the centrifugation is 8000r/min, and the time is 10 min;
the solution used for dispersing is deionized water, and the volume ratio of the deionized water to the gold nanorod solution is 1:2.
Further, the volume ratio of the vitamin K4 ethanol solution to the gold-core silver-shell nanorod suspension in the step 3 is 1: 500;
the time for the gentle shaking was 2 h.
The invention also provides application of the high-efficiency surface-enhanced Raman scattering substrate material in preparing a surface-enhanced Raman scattering substrate material for detecting the kidney injury factor.
Further, the kidney injury factor is a glycoprotein immune substance.
The invention has the advantages that:
the high-efficiency surface-enhanced Raman scattering substrate material provided by the invention does not use macroscopic materials widely cited in the prior art, but selects the nanoscale metal particles, and the local electromagnetic field of the nanoscale metal particles can be remarkableEnhancement, compared with the traditional gold nanoparticles, the gold-core silver-shell nanoparticles can further enhance the electromagnetic signal, and vitamin K4 is bonded on the surface of the gold-core silver shell, so that the electromagnetic coupling between two metal nanoparticles at the interface of the two metal nanoparticles can generate 10 times of14The SERS Enhancement Factor (EF), and the enhancement of the signal can greatly enhance the detection sensitivity.
The preparation method can well adjust the thickness of the silver shell by controlling the concentrations of silver nitrate and ascorbic acid in the silver shell growth solution, and can prepare the silver shells with different thicknesses according to requirements.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
Drawings
Further objects, features and advantages of the present invention will become apparent from the following description of embodiments of the invention, with reference to the accompanying drawings, in which:
FIG. 1A is a transmission electron microscope image of gold nanorods; FIGS. 1B-1F are transmission electron microscope images of gold core silver shell nanorods with silver shells of different thicknesses prepared in 0.2, 0.5, 1.0, 2.0, and 2.5mL silver nitrate solutions;
FIG. 2 is a UV-NIR extinction spectrum of gold nanorods and gold core silver shell nanorods with different thickness silver shells prepared in 0.2, 0.5, 1.0, 2.0 and 2.5mL silver nitrate solutions;
FIG. 3 is a Raman spectrum of vitamin K4 absorbed on a film of a monolayer array of gold core silver shell nanorods and gold nanorods of examples 1-5;
FIG. 4 shows a spectrum at 1080cm-1Plot of raman intensity as a function of silver shell thickness;
FIG. 5 is a high-sensitivity recognition capability of a gold-core silver-shell nanorod monolayer array film on low-concentration KIM-1 in human urine;
FIG. 6 shows SERS intensity at 1145cm-1With biomarker concentrations, the insert shows its wide dynamic detection range.
Detailed Description
The objects and functions of the present invention and methods for accomplishing the same will be apparent by reference to the exemplary embodiments. However, the present invention is not limited to the exemplary embodiments disclosed below; it can be implemented in different forms. The nature of the description is merely to assist those skilled in the relevant art in a comprehensive understanding of the specific details of the invention.
Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the drawings, the same reference numerals denote the same or similar parts, or the same or similar steps.