CN109001176B - Preparation method of SERS substrate of Au @ Ag nanoparticles and method for detecting glucose by using substrate - Google Patents
Preparation method of SERS substrate of Au @ Ag nanoparticles and method for detecting glucose by using substrate Download PDFInfo
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Abstract
The invention discloses a preparation method of an SERS substrate of Au @ Ag nanoparticles. The method comprises the steps of reducing chloroauric acid by using sodium citrate to prepare gold nanoparticles, modifying 4-mercaptoaniline to connect silver ions, and adding a certain proportion of ascorbic acid and sodium hydroxide to reduce silver shells. Meanwhile, the core-shell structure is controllable in shape and size, plasma resonance is realized through high coupling of the structure, so-called 'hot spots' are formed inside the core-shell structure, and 4-mercaptoaniline serving as an internal standard molecule has a strong Raman signal. 4-mercaptophenylboronic acid is modified on the Au @ Ag core-shell structure to capture glucose molecules, so that qualitative and quantitative detection of glucose is realized. The invention has the advantages that: the internal standard substance is used for signal comparison, so that the interference of complex substances in a sample (such as urine) is avoided, the detection sensitivity is improved, the preparation cost is low, and the qualitative and quantitative detection of the glucose is really realized.
Description
Technical Field
The invention belongs to the technical field of detection, and particularly relates to a preparation method of an SERS substrate of Au @ Ag nanoparticles and a method for detecting glucose by using the SERS substrate.
Background
Diabetes (diabetes) is an endocrine metabolic disease mainly manifested by disorders of carbohydrate metabolism. In recent decades, the rapid increase of the number of diabetes patients in the world at an alarming speed has become a major public health problem which seriously affects the physical and mental health of people in China, and the treatment thereof has no specific method at present and can generate a plurality of complications, so that the early prevention is very important. Plasma glucose is the only reliable diagnostic index of diabetes at present, and is also the main basis for judging the state of illness and controlling condition of diabetes.
The current commonly used detection methods for blood glucose detection are as follows: fasting plasma glucose detection, 2-hour postprandial plasma glucose detection, venous plasma glucose detection and the like, but all have some defects, such as fasting blood glucose is limited by time, the sensitivity is low, and missed diagnosis is easy; the postprandial blood sugar is unstable and has poor reproducibility, and the blood sugar detection has pain, so a glucose detection method which can realize nondestructive detection and has small influence on the environment, strong reproducibility and high sensitivity is needed.
The Surface Enhanced Raman Scattering (SERS) technology can well reflect the structural information of molecules, has the advantages of high sensitivity, high resolution, rapid reaction and the like, and is very rapidly developed in the aspects of chemical, environmental and biological sensing and detection, particularly medical sensing and detection in recent years. However, the accurate regulation and preparation of the SERS active hotspot can still realize the preparation of the high-enhanced SERS active substrate with large area, low cost and high efficiency.
The patent relates to a glucose detection method (CN 2016501037) based on surface enhanced Raman scattering and a bimolecular probe, and particularly discloses a method which comprises the steps of utilizing a gold-silver shell core nanorod as a SERS active substrate, modifying the SERS active substrate with a primary glucose receptor molecule (4-mercaptophenylboronic acid), soaking the SERS active substrate in a glucose solution, and capturing glucose on the SERS active substrate; and then adding a secondary glucose receptor molecule (4-cyanobenzene boronic acid) into the SERS active substrate, and selectively and covalently combining the secondary glucose receptor molecule with the glucose molecule on the substrate, and finally performing SERS spectrum test. Although the method improves the sensitivity of glucose detection to a certain extent, the method for detecting glucose by using the substrate is complex, and 2 times of adding glucose molecular probes are required. And because the Raman signal of the used cyano group is weaker, the concentration range of the glucose which can be detected is smaller and can only reach 10 < -2 > mol/L, so that the method is not suitable for detecting trace glucose.
Disclosure of Invention
In order to solve the problems, the invention provides an Au @ Ag nanoparticle SERS substrate, a preparation method and a method for detecting glucose by using the substrate. Meanwhile, the core-shell structure externally modified 4-mercaptophenylboronic acid can specifically recognize glucose molecules, so that the sensitivity, specificity and signal uniformity of the SERS substrate are effectively guaranteed, and the detection step of glucose is simplified.
In order to realize the purpose of the invention, the invention uses Au @ Ag nano particles with an internal standard substance of 4-mercaptoaniline as a substrate to modify a glucose capture molecule of 4-mercaptophenylboronic acid so as to achieve the effect of specifically detecting glucose.
The method also comprises the steps of selecting five glucose standard solutions with different concentrations for reaction before detection, and detecting by using SERS; meanwhile, a method of adding trace glucose is adopted to detect glucose in urine. And performing internal standard treatment on the obtained characteristic SERS spectrum to obtain a working curve for detecting glucose, wherein the working curve is used for detecting the concentration of glucose in urine.
The bimetal nanometer nuclear shell structure related in the invention, wherein the diameter of the nanometer gold core is 23-27nm, 5-10nm of nano silver shell, and 4-mercaptoaniline in the middle of the silver shell, wherein 5 is multiplied by 10-4The concentration modification of mol/L is the best.
A method for detecting glucose in urine comprises the following steps:
1) adding 90mL of distilled water into 10mL of chloroauric acid solution with the concentration of 2.4mmol/L, heating to boil, adding 1% of sodium citrate solution, reacting for 15min, centrifuging, washing precipitate with distilled water and absolute ethyl alcohol, and drying at 80 ℃ to obtain the nano-gold.
2) Adding 15-40 mul of nano gold into 0.05-5 mmol/L4-mercaptoaniline solution for reaction for 12-20 h, and centrifuging and washing.
3) Adding 20mL of nano gold prepared in the step (2) into the mixed solution according to the volume ratio of 0.5-0.8: 20-35: 40-55, sequentially adding 100mmol/L silver nitrate solution, 100mmol/L ascorbic acid and 100mmol/L sodium hydroxide solution, reacting for 4-6 hours at normal temperature, so as to reduce a nano silver shell on the surface of the nano gold, and centrifugally washing to obtain a lower layer solution, namely Au @ Ag nano core-shell structure colloid;
4) and (2) adding 5-30 mul of 4-mercaptophenylboronic acid solution with the concentration of 1mmol/L into 20mL of prepared Au @ Ag nano core-shell structure colloid, reacting for 2-3 hours under the stirring condition, wherein the reaction temperature is 60-80 ℃, and centrifugally washing and drying to obtain the SERS substrate of the Au @ Ag nano particles.
5) Mixing and stirring the prepared glucose SERS detection substrate and 5-6 prepared glucose standard solutions with different concentration gradients for 0.5-1 hour, dripping the glucose SERS detection substrate on a cover glass, and detecting a Raman signal by using a Ranishao Raman spectrometer to obtain an SERS spectrogram with corresponding concentration.
6) Processing the spectrogram to obtain 1388cm-1Normalizing at peak, and normalizing according to 1077, 1177 and 1584cm-1And obtaining a corresponding working curve according to the relation between the intensities of the three characteristic peaks and the glucose concentration.
7) Detecting the concentration of an unknown sample: mixing a glucose SERS detection substrate prepared from a sample with unknown concentration and measurement, testing by using a laser Raman spectrometer, and performing spectral peak intensity normalization to obtain a glucose surface enhanced Raman spectrum with unknown concentration; the glucose concentration was calculated by the formula.
1177cm was selected-1Taking the working curve as a calculation template, adding a corresponding amount of glucose solution into untreated urine of a healthy person, reacting with a glucose SERS substrate for 0.5-1 hour in the same way, and measuring an SERS spectrum, namely drawing a curve for comparison; the detection is carried out by using the method, the linearity is basically consistent, and the detection method is less interfered by complex substances in urine, so that the glucose SERS substrate has the characteristics of high sensitivity, good specificity, strong reproducibility and the like.
Drawings
FIG. 1 shows a preparation process of a glucose SERS detection substrate with an Au @ Ag nano core-shell structure.
FIG. 2 is a transmission electron micrograph 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 structure colloid of example 1.
FIG. 4 is a transmission electron micrograph of the Au @ Ag nano core-shell structure colloid of example 2.
FIG. 5 is a transmission electron micrograph of the colloid of Au @ Ag nano core-shell structure of example 3
FIG. 6 shows SERS spectra of glucose standard solutions with different concentrations detected by using the Au @ Ag nano core-shell structured glucose SERS detection substrate prepared in example 4.
FIG. 7 is a graph of 1177cm made using the SERS spectrum of FIG. 4-1Working curve of peak intensity as a function of glucose concentration.
FIG. 8 shows SERS spectra of different concentrations of glucose in urine measured using the Au @ Ag nano core-shell structured glucose SERS detection substrate prepared in example 4.
FIG. 9 is a graph of 1177cm made using the SERS spectrum of FIG. 8-1Working curve of peak intensity as a function of glucose concentration.
Detailed Description
In order to better understand the present invention, the following examples are further described, which are only used to explain the present invention and do not limit the present invention.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the 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 commercially available products.
Example 1
A method for detecting glucose in urine comprises the following steps:
1) and adding 90mL of distilled water into 10mL of 2.4mmol/L chloroauric acid solution, heating to boil, adding 1% sodium citrate solution, and reacting for 15min to obtain the nanogold.
2) The nano-gold is respectively reacted with 4-mercaptoaniline solutions with different concentrations (0.05 mmol/L, 20 mu L) for at least 12 hours, and then centrifugally washed.
3) Adding the gold nanoparticles into 20mL of the nanogold prepared in the step (2) according to the volume ratio (0.6: 25: 50) and sequentially adding 100mmol/L silver nitrate solution, 100mmol/L ascorbic acid and 100mmol/L sodium hydroxide solution, reacting for 4 hours at normal temperature, reducing a nano silver shell to obtain the required particles of the Au @ Ag nano core-shell structure, and centrifugally washing to obtain a lower layer solution, namely the Au @ Ag nano core-shell structure colloid.
4) And (3) adding 20mL of prepared Au @ Ag nano core-shell structure colloid into a (1mmol/L,10 mu L) 4-mercaptophenylboronic acid solution, reacting for 2 hours, controlling the temperature to be about 60 ℃, and centrifuging and washing.
5) And (3) reacting the prepared glucose SERS detection substrate with prepared glucose standard solutions with different concentrations (0.1, 1, 2, 4 and 6 mmol/L) for 0.5 hour, and detecting an SERS signal to obtain an SERS spectrogram with corresponding concentration.
6) Processing the spectrogram to obtain 1388cm-1Normalizing at peak, and normalizing according to 1177cm-1And obtaining a corresponding working curve according to the relation between the intensities of the three characteristic peaks and the glucose concentration.
Example 2
1) And adding 90mL of distilled water into 10mL of 2.4mmol/L chloroauric acid solution, heating to boil, adding 1% sodium citrate solution, and reacting for 15min to obtain the nanogold.
2) The nano-gold is respectively reacted with 4-mercaptoaniline solutions with different concentrations (0.5 mmol/L, 30 mu L) for at least 12 hours, and then centrifugally washed.
3) To 2) (0.6: 25: 50) and sequentially adding a silver nitrate solution, ascorbic acid and a sodium hydroxide solution, reacting for 4 hours at normal temperature, reducing a nano silver shell to obtain the required particles of the Au @ Ag nano core-shell structure, centrifugally washing, and taking a lower layer solution to obtain the Au @ Ag nano core-shell structure colloid.
4) Adding the prepared Au @ Ag nano core-shell structure colloid into a (1mmol/L,10 mu L) 4-mercaptophenylboronic acid solution, reacting for 2 hours, controlling the temperature to be about 60 ℃, and centrifuging and washing.
5) And (3) reacting the prepared glucose SERS detection substrate with prepared glucose standard solutions with different concentrations (0.1, 1, 2, 4 and 6 mmol/L) for 0.5 hour, and detecting an SERS signal to obtain an SERS spectrogram with corresponding concentration.
6) Processing the spectrogram to obtain 1388cm-1Normalizing at peak, and normalizing according to 1177cm-1And obtaining a corresponding working curve according to the relation between the intensities of the three characteristic peaks and the glucose concentration.
Example 3
1) And adding 90mL of distilled water into 10mL of 2.4mmol/L chloroauric acid solution, heating to boil, adding 1% sodium citrate solution, and reacting for 15min to obtain the nanogold.
2) The nano-gold is respectively reacted with 4-mercaptoaniline solutions with different concentrations (5 mmol/L, 30 mu L) for at least 12 hours, and then centrifugally washed.
3) To 2) (0.6: 25: 50) and sequentially adding a silver nitrate solution, ascorbic acid and a sodium hydroxide solution, reacting for 4 hours at normal temperature, reducing a nano silver shell to obtain the required particles of the Au @ Ag nano core-shell structure, centrifugally washing, and taking a lower layer solution to obtain the Au @ Ag nano core-shell structure colloid.
4) Adding the prepared Au @ Ag nano core-shell structure colloid into a (1mmol/L, 20 mu L) 4-mercaptophenylboronic acid solution, reacting for 2 hours, controlling the temperature to be about 60 ℃, and centrifuging and washing.
5) And (3) reacting the prepared glucose SERS detection substrate with prepared glucose standard solutions with different concentrations (0.1, 1, 2, 4 and 6 mmol/L) for 0.5 hour, and detecting an SERS signal to obtain an SERS spectrogram with corresponding concentration.
6) Processing the spectrogram to obtain 1388cm-1Normalizing at peak, and normalizing according to 1177cm-1And obtaining a corresponding working curve according to the relation between the intensities of the three characteristic peaks and the glucose concentration.
Example 4
1) And adding 90mL of distilled water into 10mL of 2.4mmol/L chloroauric acid solution, heating to boil, adding 1% sodium citrate solution, and reacting for 15min to obtain the nanogold.
2) The nano-gold is respectively reacted with 4-mercaptoaniline solutions with different concentrations (0.5 mmol/L, 30 mu L) for at least 12 hours, and then centrifugally washed.
3) To 2) (0.6: 25: 50) sequentially adding silver nitrate solution, ascorbic acid and sodium hydroxide solution, reacting for 4 hours at normal temperature, reducing a nano silver shell to obtain the required particles with the Au @ Ag nano core-shell structure, and centrifuging and washing.
4) Adding the prepared Au @ Ag nano core-shell structure colloid into a (1mmol/L, 30 mu L) 4-mercaptophenylboronic acid solution, reacting for 2 hours, controlling the temperature to be about 60 ℃, and centrifuging and washing.
5) And (3) reacting the prepared glucose SERS detection substrate with prepared glucose standard solutions with different concentrations (0.1, 1, 2, 4 and 6 mmol/L) for 0.5 hour, and detecting an SERS signal to obtain an SERS spectrogram with corresponding concentration.
6) Processing the spectrogram to obtain 1388cm-1Normalizing at peak, and normalizing according to 1077, 1177 and 1584cm-1And obtaining a corresponding working curve according to the relation between the intensities of the three characteristic peaks and the glucose concentration.
And (3) performance detection:
a corresponding amount of glucose solution was added to the urine of an untreated healthy person, and the glucose SERS substrate prepared in the same manner as in example 4 was reacted for 0.5 hour to measure the SERS spectrum. Processing the spectrogram to obtain 1388cm-1Wave crest treatmentNormalized according to 1177cm-1The relationship between the intensities of the three characteristic peaks and the glucose concentration yields the corresponding working curve (fig. 9).
As can be seen from fig. 2 (example 4), fig. 3 (example 1), fig. 4 (example 2) and fig. 5 (example 3), SERS substrates of Au @ Ag nanoparticles have been successfully synthesized; as can be seen from FIG. 7, 1177cm of SERS spectrum was obtained from the SERS spectrum detected by the SERS detection substrate prepared in example 4-1The linear correlation of the working curve graph of the peak intensity along with the change of the glucose concentration is high. As can be seen from FIG. 9, with the composite material prepared in example 4 as a substrate, SERS was used to detect urine of untreated healthy persons from different glucose solutions, and 1177cm was obtained from the measured SERS spectrum-1The working curve graph of the peak intensity along with the change of the glucose concentration is compared with the working curve graph shown in the figure 7, and the linearity is basically consistent, so that the detection method is less interfered by complex substances in urine, and the glucose SERS substrate disclosed by the invention has the characteristics of high sensitivity, good specificity, strong repeatability and the like.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (6)
1. An Au @ Ag nanoparticle SERS substrate is characterized in that: the SERS substrate is a composite material which takes Au @ Ag nano particles with 4-mercaptoaniline as a substrate and modifies glucose capture molecules 4-mercaptophenylboronic acid on the surfaces of the Au @ Ag nano particles; the Au @ Ag nano particle takes nano gold as a core and nano silver as a shell, 4-mercaptoaniline is arranged between the nano gold core and the nano silver shell, the diameter of the nano gold core is 23-27nm, and the thickness of the nano silver shell is 5-10 nm;
the preparation method comprises the following steps:
(1) reducing chloroauric acid by adding sodium citrate to obtain nanogold;
(2) adding 15-40 mu L of the nanogold prepared in the step (1) into 0.05-5 mmol/L of 4-mercaptoaniline solution for reaction for 12-20 hours, and then centrifuging and washing;
(3) adding 20mL of nano gold prepared in the step (2) into the mixed solution according to the volume ratio of 0.5-0.8: 20-35: 40-55, sequentially adding 100mmol/L silver nitrate solution, 100mmol/L ascorbic acid and 100mmol/L sodium hydroxide solution, reacting for 4-6 hours at normal temperature, so as to reduce a nano silver shell on the surface of the nano gold, and centrifugally washing to obtain a lower layer solution, namely Au @ Ag nano core-shell structure colloid;
(4) the specific steps of the step (4) are as follows: and (2) adding 5-30 mul of 4-mercaptophenylboronic acid solution with the concentration of 1mmol/L into 20mL of prepared Au @ Ag nano core-shell structure colloid, reacting for 2-3 hours under the stirring condition, wherein the reaction temperature is 60-80 ℃, and centrifugally washing and drying to obtain the SERS substrate of the Au @ Ag nano particles.
2. A preparation method of an Au @ Ag nanoparticle SERS substrate is characterized by comprising the following steps:
(1) reducing chloroauric acid by adding sodium citrate to obtain nanogold;
(2) adding 15-40 mu L of the nanogold prepared in the step (1) into 0.05-5 mmol/L of 4-mercaptoaniline solution for reaction for 12-20 hours, and then centrifuging and washing;
(3) adding 20mL of nano gold prepared in the step (2) into the mixed solution according to the volume ratio of 0.5-0.8: 20-35: 40-55, sequentially adding 100mmol/L silver nitrate solution, 100mmol/L ascorbic acid and 100mmol/L sodium hydroxide solution, reacting for 4-6 hours at normal temperature, so as to reduce a nano silver shell on the surface of the nano gold, and centrifugally washing to obtain a lower layer solution, namely Au @ Ag nano core-shell structure colloid;
(4) the specific steps of the step (4) are as follows: and (2) adding 5-30 mu L of 1 mmol/L4-mercaptophenylboronic acid solution for capturing glucose into 20mL of prepared Au @ Ag nano core-shell structure colloid, reacting for 2-3 hours under the stirring condition, wherein the reaction temperature is 60-80 ℃, and centrifugally washing and drying to obtain the SERS substrate of the Au @ Ag nano particles.
3. The method for preparing the SERS substrate of Au @ Ag nano particles according to claim 2, wherein the step (1) comprises the following steps: adding 90mL of distilled water into 10mL of chloroauric acid solution with the concentration of 2.4mmol/L, heating to boil, adding 1% of sodium citrate solution, reacting for 15min, centrifuging, washing precipitate with distilled water and absolute ethyl alcohol, and drying at 80 ℃ to obtain the nano-gold.
4. A method for detecting glucose in urine is characterized by comprising the following steps:
s1: mixing and stirring the glucose SERS substrate prepared according to any one of claims 2 to 3 with 5 to 6 prepared glucose standard solutions with different concentration gradients for 0.5 to 1 hour, dripping the mixture on a cover glass, and detecting a Raman signal by using a Renilsha Raman spectrometer to obtain an SERS spectrogram with corresponding concentration;
s2: taking the corresponding SERS substrate to independently perform Raman spectrum test to obtain a substrate background Raman signal;
s3: normalizing the surface enhanced Raman spectrum of the glucose standard solution by using the substrate background Raman signal as an internal standard;
s4: establishing a relative intensity-concentration standard comparison working curve of a Raman spectrum line of the glucose standard solution;
s5: detecting the concentration of an unknown sample: mixing a sample with a determination of unknown concentration with the glucose SERS substrate prepared according to any one of claims 2 to 3, then testing by using a laser Raman spectrometer, and carrying out spectrum peak intensity normalization processing to obtain a glucose surface enhanced Raman spectrum with unknown concentration; the glucose concentration was calculated by the formula.
5. The method for detecting glucose in urine according to claim 4, wherein: the concentration change value of the glucose standard solution samples with the 5-6 concentration gradients is between 0.1 and 6 mmol/L.
6. The method for detecting glucose in urine according to claim 4, wherein: the unknown sample of step S5 is human urine.
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