CN109001176A - A kind of preparation method of the SERS substrate of Au@Ag nanoparticle and method using substrate detection glucose - Google Patents

Abstract

The invention discloses a kind of preparation methods of the SERS substrate of Au@Ag nanoparticle.Nano Au particle is made using reduction of sodium citrate gold chloride in this method, and 4- mercaptoaniline connects silver ion in modification, adds a certain proportion of ascorbic acid and sodium hydroxide restores silver-colored shell.Meanwhile the morphology of core-shell structure size is controllable, plasma resonance is realized in structure height coupling, so-called " hot spot " is formed inside core-shell structure, so that the 4- mercaptoaniline as internal standard molecule has very strong Raman signal.4- mercaptophenyl boronic acid is modified on this Au Ag core-shell structure, to capture glucose molecule, to realize the qualitative and quantitative detection of glucose.The present invention has the advantages that doing signal contrast using internal standard substance, detection sensitivity will not be improved by the interference of complex material in sample (such as urine), preparation cost is low, really realizes the qualitative and quantitative detection of glucose.

Description

A preparation method of a SERS substrate of Au@Ag nanoparticles and the use of the substrate to detect How to measure glucose

technical field

The invention belongs to the technical field of detection, and in particular relates to a method for preparing a SERS substrate of Au@Ag nanoparticles and a method for detecting glucose by using the substrate.

Background technique

Diabetes (diabetes) is an endocrine and metabolic disease mainly manifested by glucose metabolism disorders. In recent decades, the number of diabetics worldwide has grown at an astonishing rate. It has become a major public health problem that seriously affects the physical and mental health of Chinese people. There is currently no effective treatment for it, and it can produce many complications. Therefore, early prevention appears to be particularly important. Plasma glucose is currently the only reliable diagnostic index for diabetes, and it is also the main basis for judging the condition and control of diabetes.

Currently commonly used detection methods for blood glucose detection include: fasting plasma glucose detection, 2-hour postprandial plasma glucose detection, venous plasma glucose detection, etc., but there are some shortcomings, such as fasting blood glucose is limited by time, low sensitivity, and easy to miss diagnosis; Blood glucose is unstable and has poor reproducibility; and blood glucose detection is painful, so we need a glucose detection method that can achieve non-destructive testing and is less affected by the environment, with strong reproducibility and high sensitivity.

The Surface Enhanced Raman Scattering (SERS) technology has the advantages of high sensitivity, high resolution and fast response because it can reflect the structural information of molecules very well. In particular, the development of medical sensor detection is very rapid. However, it is still a difficult point in current research to precisely control and prepare SERS active hotspots, and to achieve large-area, low-cost and high-efficiency preparation of highly enhanced SERS active substrates.

A patented glucose detection method based on surface-enhanced Raman scattering and bimolecular probes (CN2016104501037), which specifically discloses a method using gold-silver-shell core nanorods as SERS active substrates, and using primary glucose acceptor molecules (with 4-mercapto Phenylboronic acid) modified on the SERS active substrate, then soak the above SERS active substrate in glucose solution to capture glucose on the SERS active substrate; then add secondary glucose acceptor molecules (using 4-cyano Phenylboronic acid), the secondary glucose receptor molecule selectively covalently binds to the glucose molecule on the substrate, and finally performs the SERS spectrum test. Although the above method improves the sensitivity of glucose detection to a certain extent, the detection of glucose using the substrate is complicated and requires the addition of glucose molecular probes twice. And because the Raman signal of the cyano group it uses is weak, the glucose concentration range that can be detected is even smaller, only reaching 10-2 mol/L, which will not be suitable for the detection of trace glucose.

Contents of the invention

In order to solve the above problems, the present invention provides a SERS substrate of Au@Ag nanoparticles, a preparation method and a method for detecting glucose using the substrate. The glucose SERS detection substrate based on Au@Ag nanoparticles is realized by using a double metal core-shell structure Plasmon resonance forms a SERS active hotspot, so that the internal standard substance 4-mercaptoaniline wrapped in the middle not only enhances the characteristic SERS signal, but also avoids the interference of the detection environment. At the same time, the 4-mercaptophenylboronic acid modified outside the core-shell structure can specifically recognize glucose molecules, which effectively guarantees the sensitivity, specificity and signal uniformity of the SERS substrate, and on the other hand simplifies the detection steps of glucose.

In order to achieve the purpose of the present invention, the present invention uses Au@Ag nanoparticles with an internal standard substance --- 4-mercaptoaniline as a substrate, and modifies the glucose capture molecule 4-mercaptophenylboronic acid to achieve specific detection of glucose Effect.

The aforementioned method also includes selecting five glucose standard solutions with different concentrations for reaction before detection, and using SERS for detection; at the same time, the method of adding a trace amount of glucose is used to detect the glucose in urine. And the obtained characteristic SERS spectrum is internally standardized to obtain a working curve for detecting glucose, which is used for detecting the concentration of glucose in urine.

The bimetallic nano-core-shell structure involved in this invention, wherein the diameter of the nano-gold core is 23-27nm, the diameter of the nano-silver shell is 5-10nm, and the middle is 4-mercaptoaniline, wherein the concentration is 5×10 ^-4 mol/L Grooming at its best.

A method for detecting glucose in urine comprises the following steps:

1) Add 90mL distilled water to 10mL chloroauric acid solution with a concentration of 2.4mmol/L, heat to boiling, add 1% sodium citrate solution to react for 15min, and then centrifuge, wash the precipitate with distilled water and absolute ethanol, at 80°C Dried to get nano gold.

2) Add 15 μL-40 μL of nano-gold to 0.05 mmol/L-5 mmol/L 4-mercaptoaniline solution to react for 12-20 hours, then centrifuge and wash.

3) Add 100mmol/L silver nitrate solution, 100mmol/L ascorbic acid and 100mmol/L sodium hydroxide to 20mL of the nanogold prepared in step (2) according to the volume ratio of 0.5-0.8:20-35:40-55 Solution, react at room temperature for 4-6 hours, thereby reducing nano-silver shells on the surface of nano-gold, centrifuging and washing, and taking the lower layer solution is Au@Ag nano-core-shell structure colloid;

4) Take 20mL of the prepared Au@Ag nano core-shell structure colloid, add 5μL-30μL of 4-mercaptophenylboronic acid solution with a concentration of 1mmol/L, and react for 2-3 hours under stirring conditions, and the reaction temperature is 60℃-80℃ ℃, centrifugal washing and drying to obtain the SERS substrate of Au@Ag nanoparticles.

5) Mix the prepared glucose SERS detection substrate with 5-6 prepared glucose standard solutions with different concentration gradients and stir for 0.5-1 hour, drop it on the cover glass, and use the Renishaw Raman spectrometer to analyze the Raman signal The detection of the corresponding concentration of the SERS spectrum.

6) Process the spectrum, take the peak at 1388cm- ¹ for normalization, and then obtain the corresponding working curve according to the relationship between the intensity of the three characteristic peaks at 1077, 1177, and 1584cm ^-1 and the glucose concentration.

7) Detection of unknown sample concentration: Mix the glucose SERS detection substrate prepared with the measured sample of unknown concentration, then use a laser Raman spectrometer to test, and perform spectral peak intensity normalization processing to obtain an unknown concentration of glucose surface-enhanced Raman Spectrogram; Calculate the concentration of glucose by formula.

Select the working curve at 1177cm ^-1 as the calculation template, take the corresponding amount of glucose solution and add it to the urine of untreated healthy people, and react with the glucose SERS substrate for 0.5-1 hour in the same way, measure the SERS spectrum, and draw it The curves are compared; using the present invention to detect, it is found that the linearity is basically consistent, indicating that the detection method is less interfered by complex substances in urine, and it can be seen that the glucose SERS substrate of the invention has high sensitivity, good specificity, strong reproducibility, etc. features.

Description of drawings

Figure 1 shows the preparation process of Au@Ag nano core-shell structure glucose SERS detection substrate.

FIG. 2 is a transmission electron microscope image of the Au@Ag nano core-shell structure of Example 4.

FIG. 3 is a transmission electron microscope image of the Au@Ag nano-core-shell colloid of Example 1. FIG.

FIG. 4 is a transmission electron microscope image of the Au@Ag nano core-shell structure colloid of Example 2.

Fig. 5 is the transmission electron microscope picture of the Au@Ag nano core-shell structure colloid of embodiment 3

Fig. 6 is the SERS spectra of different concentrations of glucose standard solutions detected by using the Au@Ag nano core-shell glucose SERS detection substrate prepared in Example 4.

Fig. 7 is a working curve diagram of the peak intensity at 1177 cm ^-1 as a function of glucose concentration, made using the SERS spectrum in Fig. 4 .

Fig. 8 is the SERS spectrum of detecting different concentrations of glucose in urine using the Au@Ag nano core-shell glucose SERS detection substrate prepared in Example 4.

Fig. 9 is a working curve diagram of the peak intensity at 1177 cm ^-1 as a function of glucose concentration made by using the SERS spectrum in Fig. 8 .

Detailed ways

In order to better understand the present invention, the present invention will be further described by the following examples, which are only used to explain the present invention and do not constitute any limitation to the present invention.

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

Example 1

A method for detecting glucose in urine, comprising the following steps:

1) Add 90mL distilled water to 10mL, 2.4mmol/L chloroauric acid solution, heat to boiling, add 1% sodium citrate solution and react for 15min to obtain gold nanoparticles.

2) The gold nanoparticles were reacted with 4-mercaptoaniline solutions of different concentrations (0.05mmol/L, 20μL) for at least 12 hours, and then washed by centrifugation.

3) Add 100mmol/L silver nitrate solution, 100mmol/L ascorbic acid and 100mmol/L sodium hydroxide solution in sequence by volume ratio (0.6:25:50) to 20mL of gold nanoparticles prepared in step (2), and react at room temperature for 4 hours , to restore the nano-silver shell to obtain the Au@Ag nano-core-shell structure particles we need, centrifuge and wash, and take the lower layer solution to become the Au@Ag nano-core-shell structure colloid.

4) Take 20mL of the prepared Au@Ag nano core-shell structure colloid, add (1mmol/L, 10μL) 4-mercaptophenylboronic acid solution, react for 2 hours, control the temperature at about 60°C, and wash by centrifugation.

5) React the glucose SERS detection substrate prepared above with the configured glucose standard solutions of different concentrations (0.1, 1, 2, 4, 6mmol/L) for 0.5 hours, detect the SERS signal, and obtain the SERS spectrum of the corresponding concentration .

6) Process the spectrogram, take the peak at 1388cm ^-1 for normalization, and then obtain the corresponding working curve according to the relationship between the intensity of the three characteristic peaks at 1177cm ^-1 and the glucose concentration.

Example 2

1) Add 90mL distilled water to 10mL, 2.4mmol/L chloroauric acid solution, heat to boiling, add 1% sodium citrate solution and react for 15min to obtain gold nanoparticles.

2) The gold nanoparticles were reacted with 4-mercaptoaniline solutions of different concentrations (0.5mmol/L, 30μL) for at least 12 hours, and then washed by centrifugation.

3) Add silver nitrate solution, ascorbic acid, and sodium hydroxide solution to 2) in proportion (0.6:25:50) in turn, react at room temperature for 4 hours, and restore the nano-silver shell to obtain the Au@Ag nano-core we need The particles with the shell structure are washed by centrifugation, and the solution of the lower layer is Au@Ag nano core-shell structure colloid.

4) Take the prepared Au@Ag nano core-shell structure colloid, add (1mmol/L, 10μL) 4-mercaptophenylboronic acid solution, react for 2 hours, control the temperature at about 60°C, and centrifuge and wash.

5) React the glucose SERS detection substrate prepared above with the configured glucose standard solutions of different concentrations (0.1, 1, 2, 4, 6mmol/L) for 0.5 hours, detect the SERS signal, and obtain the SERS spectrum of the corresponding concentration .

6) Process the spectrogram, take the peak at 1388cm ^-1 for normalization, and then obtain the corresponding working curve according to the relationship between the intensity of the three characteristic peaks at 1177cm ^-1 and the glucose concentration.

Example 3

1) Add 90mL distilled water to 10mL, 2.4mmol/L chloroauric acid solution, heat to boiling, add 1% sodium citrate solution and react for 15min to obtain gold nanoparticles.

2) The gold nanoparticles were reacted with 4-mercaptoaniline solutions of different concentrations (5 mmol/L, 30 μL) for at least 12 hours, and then washed by centrifugation.

3) Add silver nitrate solution, ascorbic acid, and sodium hydroxide solution to 2) in proportion (0.6:25:50) in turn, react at room temperature for 4 hours, and restore the nano-silver shell to obtain the Au@Ag nano-core we need The particles with the shell structure are washed by centrifugation, and the solution of the lower layer is Au@Ag nano core-shell structure colloid.

4) Take the prepared Au@Ag nano core-shell structure colloid, add (1mmol/L, 20μL) 4-mercaptophenylboronic acid solution, react for 2 hours, control the temperature at about 60°C, and wash by centrifugation.

5) React the glucose SERS detection substrate prepared above with the configured glucose standard solutions of different concentrations (0.1, 1, 2, 4, 6mmol/L) for 0.5 hours, detect the SERS signal, and obtain the SERS spectrum of the corresponding concentration .

6) Process the spectrogram, take the peak at 1388cm ^-1 for normalization, and then obtain the corresponding working curve according to the relationship between the intensity of the three characteristic peaks at 1177cm ^-1 and the glucose concentration.

Example 4

1) Add 90mL distilled water to 10mL, 2.4mmol/L chloroauric acid solution, heat to boiling, add 1% sodium citrate solution and react for 15min to obtain gold nanoparticles.

2) The gold nanoparticles were reacted with 4-mercaptoaniline solutions of different concentrations (0.5mmol/L, 30μL) for at least 12 hours, and then washed by centrifugation.

3) Add silver nitrate solution, ascorbic acid, and sodium hydroxide solution to 2) in proportion (0.6:25:50) in turn, react at room temperature for 4 hours, and restore the nano-silver shell to obtain the Au@Ag nano-core we need Shell-structured particles, washed by centrifugation.

4) Take the prepared Au@Ag nano core-shell structure colloid, add (1mmol/L, 30μL) 4-mercaptophenylboronic acid solution, react for 2 hours, control the temperature at about 60°C, and wash by centrifugation.

5) React the glucose SERS detection substrate prepared above with the configured glucose standard solutions of different concentrations (0.1, 1, 2, 4, 6mmol/L) for 0.5 hours, detect the SERS signal, and obtain the SERS spectrum of the corresponding concentration .

6) Process the spectrum, take the peak at 1388cm- ¹ for normalization, and then obtain the corresponding working curve according to the relationship between the intensity of the three characteristic peaks at 1077, 1177, and 1584cm ^-1 and the glucose concentration.

Performance testing:

A corresponding amount of glucose solution was added to the urine of untreated healthy people, and the glucose SERS substrate prepared by the same method as in Example 4 was reacted for 0.5 hours, and the SERS spectrum was measured. The above spectrum was processed, and the peak at 1388cm ^-1 was taken for normalization, and then the corresponding working curve was obtained according to the relationship between the intensity of the three characteristic peaks at 1177cm ^-1 and the glucose concentration (Figure 9).

From Figure 2 (Example 4), Figure 3 (Example 1), Figure 4 (Example 2) and Figure 5 (Example 3), it can be seen that the SERS substrate of Au@Ag nanoparticles has been successfully synthesized; from Figure 7 It can be seen that, using the SERS spectrum detected by the SERS detection substrate prepared in Example 4, the working curve of the peak intensity at 1177 cm ^-1 changing with the glucose concentration has a high linear correlation. It can be seen from Figure 9 that the composite material prepared in Example 4 is used as the base, and the urine of different glucose solutions added to untreated healthy people is detected by SERS, and the peak intensity at 1177 cm ^-1 is obtained by using the measured SERS spectrum The working curve with the glucose concentration changes, and compared with Figure 7, the linearity is basically consistent. It can be seen that the detection method is less interfered by complex substances in the urine. It can be seen that the glucose SERS substrate of the invention has high sensitivity and specificity. Good performance and strong reproducibility.

The above is only an embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process conversion made by using the content of the description of the present invention, or directly or indirectly used in other related technical fields, shall be The same reasoning is included in the patent protection scope of the present invention.

Claims (9)

1. A SERS substrate of Au@Ag nanoparticles, characterized in that: the SERS substrate is a kind of Au@Ag nanoparticles with 4-mercaptoaniline as a substrate, and glucose is modified on the surface of Au@Ag nanoparticles A composite material that captures the molecule 4-mercaptophenylboronic acid; the Au@Ag nanoparticles use nano-gold as the core and nano-silver as the shell, 4-mercaptoaniline is between the nano-gold core and nano-silver shell, and the nano-gold The diameter of the core is 23-27nm, and the thickness of the nano-silver shell is 5-10nm. 2. A method for preparing a SERS substrate of Au@Ag nanoparticles, comprising the following steps: (1) Reducing chloroauric acid by adding sodium citrate to obtain gold nanoparticles; (2) Loading 4-mercaptoaniline on gold nanoparticles; (3) The 4-mercaptoaniline on the carrier is used to capture the silver ions in the silver nitrate solution as a precursor, and then add ascorbic acid and sodium hydroxide to reduce the nano-silver shell to complete the preparation of the Au@Ag nano-core-shell structure; (4) Modification of 4-mercaptophenylboronic acid on the synthesized Au@Ag nano-core-shell structure was used to specifically recognize glucose molecules. 3. The method for preparing a SERS substrate of Au@Ag nanoparticles according to claim 2, characterized in that, the specific steps of step (1) are: adding Heat 90mL of distilled water to boiling, add 1% sodium citrate solution to react for 15min, and then centrifuge. The precipitate is washed with distilled water and absolute ethanol, and dried at 80°C to obtain gold nanoparticles. 4. The method for preparing a SERS substrate of Au@Ag nanoparticles according to claim 2, characterized in that, the specific step of step (2) is: adding 15 μL-40 μL of nano-gold to 0.05mmol/L-5mmol /L of 4-mercaptoaniline solution reacted for 12-20 hours, then centrifuged and washed. 5. The method for preparing a SERS substrate of Au@Ag nanoparticles according to claim 2, characterized in that, the specific step of step (3) is: adding 20mL of gold nanoparticles prepared in step (2) to a volume ratio of 0.5-0.8: 20-35: 40-55 Add 100mmol/L silver nitrate solution, 100mmol/L ascorbic acid and 100mmol/L sodium hydroxide solution in turn, react at room temperature for 4-6 hours, thereby reducing the The nano-silver shell is washed by centrifugation, and the solution of the lower layer is Au@Ag nano-core-shell structure colloid. 6. A method for preparing a SERS substrate of Au@Ag nanoparticles according to claim 2, characterized in that, the specific step of step (4) is: take 20 mL of the prepared Au@Ag nano core-shell structure colloid, add 5 μL-30 μL of 4-mercaptophenylboronic acid solution with a concentration of 1 mmol/L was reacted for 2-3 hours under stirring conditions at a reaction temperature of 60°C-80°C, washed and dried by centrifugation to obtain a SERS substrate of Au@Ag nanoparticles. 7. A method for detecting glucose in urine, comprising the following steps: S1: Mix the glucose SERS substrate prepared according to any one of claims 2-6 with the prepared 5-6 glucose standard solutions of different concentration gradients and mix and stir for 0.5-1 hour, drop it on the cover glass, and use Raney The Raman spectrometer detects the Raman signal and obtains the SERS spectrum corresponding to the concentration; S2: Take the corresponding SERS substrate and conduct Raman spectrum test separately to obtain the Raman signal of the substrate; S3: Using the background Raman signal as an internal standard to normalize the surface-enhanced Raman spectrum of the glucose standard solution; S4: Establish the relative intensity-concentration standard control working curve of the Raman spectral line of the glucose standard solution; S5: Detect unknown sample concentration: Mix the measured sample of unknown concentration with the glucose SERS substrate prepared in any one of claims 2-6, then use a laser Raman spectrometer to test, and perform spectral peak intensity normalization processing to obtain The surface-enhanced Raman spectrum of glucose at an unknown concentration; the concentration of glucose was calculated by the formula. 8. A method for detecting glucose in urine according to claim 7, characterized in that: the concentration change value of the glucose standard solution samples of the 5-6 concentration gradients is between 0.1-6 mmol/L. 9. A method for detecting glucose in urine according to claim 7, characterized in that: the unknown sample in step S5 is human urine.