A SERS detection platform based on nanoparticle assemblies and a pH detection method

Abstract

The present invention provides a SERS detection platform based on nanoparticle assemblies and a pH detection method, belonging to the technical field of analytical detection. The pH detection method of the present invention utilizes the characteristic that the intensity ratio of Raman characteristic peaks of pH-responsive cyano compounds changes regularly in different acidic environments, so that the corresponding relationship between the intensity ratio of Raman peaks and the pH value can be accurately analyzed, realizing the micro-detection of H + and can be widely applied to real-time pH monitoring in the fields of biology, chemical engineering, environmental monitoring, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical detection, and specifically relates to a SERS detection platform based on nanoparticle assemblies and a pH detection method. Background Art

[0002] In the technical field of analytical detection, the accurate detection of pH value is of great importance and is widely used in many fields such as biology, chemical engineering, and environmental monitoring. At present, common pH detection methods mainly include acid-base indicator method, glass electrode method, and chemiluminescence-based detection method, etc. The acid-base indicator method uses the color change of the indicator at different pH values to roughly judge the pH value. However, this method can only give a general pH range with low accuracy and cannot meet the scenarios with precise pH value requirements, such as the monitoring of cell culture environment in biological experiments. The glass electrode method is a widely used detection method at present. Its principle is to measure the pH value based on the potential difference generated by different hydrogen ion concentrations on both sides of the glass membrane. But this method has many limitations: on the one hand, the operation of the glass electrode is relatively complex, requiring a series of pretreatment steps such as calibration and cleaning, and has high requirements for the professional skills of the operator; on the other hand, the glass electrode has unstable performance in some special environments. For example, in high-temperature, high-salinity or strongly corrosive environments, the glass membrane is easily damaged, resulting in inaccurate detection results or even unable to detect. The chemiluminescence-based detection method detects pH through the relationship between the intensity of the light signal generated by a chemical reaction and the pH value. Although this method improves the detection sensitivity to a certain extent, it often requires adding a variety of chemical reagents for complex chemical reactions, which not only has a slow detection speed, but also the use of chemical reagents increases the detection cost and environmental pollution risk, and at the same time has high requirements for the detection equipment, limiting its application in some on-site rapid detection scenarios.

[0003] In summary, the existing pH detection methods have obvious deficiencies in terms of operation complexity, detection speed, and adaptability to special environments. Therefore, it is of great practical significance to develop a new type of pH detection method with simple operation, fast detection, and adaptability to complex environments. Based on such a background, the present invention proposes a method for detecting the environmental pH based on the displacement change of the Raman peak of cyanide groups under acidic conditions. Summary of the Invention

[0004] In order to solve the problems of complex detection method, long detection time, and high cost, the present invention provides a SERS (Surface Enhanced Raman Scattering) detection platform based on nanoparticle assemblies and a pH detection method.

[0005] Technical solution: An SERS detection platform based on a nanoparticle assembly, the SERS detection platform includes a sensing substrate for placing a sample to be measured, and a nanoparticle assembly containing a cyano group is assembled on the sensing substrate. The SERS detection platform detects the pH value of the sample to be measured based on the change in the intensity ratio of the Raman characteristic peaks of the nanoparticle assembly containing a cyano group in different acidic environments.

[0006] Further, in addition to the sensing substrate, the SERS detection platform further includes: a white light source, a Raman light source, a dark field condenser, an optical microscope magnifying objective lens, a reflector, a color CCD image sensor, and a spectral CCD image sensor;

[0007] The laser emitted by the white light source is irradiated on the sensing substrate through the dark field condenser. The nanoparticle assembly on the sensing substrate scatters after being excited; after the scattered light passes through the optical microscope magnifying objective lens, it enters the spectral CCD image sensor to form an LSPR image, and at the same time is reflected by the reflector to the color CCD image sensor to form a dark field scattering image;

[0008] The laser emitted by the Raman light source is irradiated on the nanoparticle assembly through the optical microscope magnifying objective lens. The nanoparticle assembly scatters after being excited, and the scattered light then passes through the optical microscope magnifying objective lens and enters the spectral CCD image sensor to form a Raman scattering spectrum image.

[0009] Further, the assembly method of the nanoparticle assembly is as follows:

[0010] S1. Modify the sensing substrate with (3-mercaptopropyl) trimethylsilane reagent to prepare a chemical modification layer;

[0011] S2. Drop the aqueous solution of spindle-shaped gold nanoparticles on the sensing substrate, keep it for a period of time and then remove it;

[0012] S3. Drop the ethanol solution of cyano-rhodamine derivative on the sensing substrate, keep it for a period of time and then remove it;

[0013] S4. Drop the aqueous solution of FeCl3 on the sensing substrate, keep it for a period of time and then remove it;

[0014] S5. Mix the aqueous solution of spherical gold nanoparticles and the ethanol solution of 6-mercaptonicotinonitrile for a period of time, then drop it on the sensing substrate, keep it for a period of time and then dry it.

[0015] Further, the cyano-rhodamine derivative in S3 is RBH-CN, and its structural formula is as follows:

[0016] 。

[0017] Further, the assembly method of the nanoparticle assembly is specifically as follows:

[0018] S1. Modify the sensing substrate with (3-mercaptopropyl) trimethylsilane reagent to prepare a chemical modification layer:

[0019] Mix an ethanol solution of (3-mercaptopropyl) trimethylsilane with a mass fraction of 95% and an absolute ethanol solution with a mass fraction of 97% according to a volume ratio of 1:2 - 1:10 to obtain a mixed solution. Immerse the sensing substrate in the mixed solution for 2 - 3 h, then take it out, rinse it with absolute ethanol, and place it in an oven at 60 - 80 °C for 15 - 20 min;

[0020] S2. Take 100 μL of an aqueous solution of spindle-shaped gold nanoparticles with a concentration of 10 −8 mol / L and drop it on the sensing substrate, and remove it after 10 - 15 min; The length of the spindle-shaped gold nanoparticles is 80 - 150 nm, and the width is 20 - 40 nm;

[0021] S3. Take 100 μL of an ethanol solution of RBH-CN with a concentration of 10 −7 mol / L and drop it on the sensing substrate obtained in S2, and remove it after 10 - 15 min;

[0022] S4. Take 200 μL of an aqueous solution of FeCl3 with a concentration of 10 −6 mol / L and drop it on the sensing substrate obtained in S3, and blow it dry after 30 - 40 min;

[0023] S5. Mix 1 mL of an aqueous solution of spherical gold nanoparticles with a concentration of 10 −8 mol / L and 200 μL of an ethanol solution of 6-mercaptonicotinonitrile with a concentration of 10 -7 mol / L for 15 - 20 min, then take 200 μL and drop it on the sensing substrate, and blow it dry after 20 min; The diameter of the spherical gold nanoparticles is 25 - 35 nm.

[0024] The present invention provides a pH detection method based on the SERS detection platform described above. This method realizes the detection of pH based on the intensity ratio of Raman characteristic peaks at 1705 cm −1 and 2344 cm −1 .

[0025] Further, the pH detection method includes the following steps:

[0026] Step 1: Turn on the Raman light source, select a laser with a wavelength of 633 nm, and irradiate it on the sensing substrate containing the nanoparticle assembly for 15 s. Obtain the Raman scattering spectrum image through a spectral CCD, and then obtain the Raman signal intensity of the nanoparticle assembly.

[0027] Step 2: Drop buffer solutions with different pH values on the sensing substrate containing the nanoparticle assembly, repeat the operations in Step 1, and obtain the intensity ratio of the Raman characteristic peaks at 1705 cm −1 and 2344 cm −1 so as to plot the standard curve of pH-Raman characteristic peak intensity ratio.

[0028] Step 3: Take the sample solution to be measured, drop it on the sensing substrate containing the nanoparticle assembly, turn on the Raman laser for Raman intensity measurement, and obtain the Raman peak intensity ratio at 1705 cm -1 and 2344 cm -1 ; Through the standard curve of pH-Raman characteristic peak intensity ratio plotted in Step 2, obtain the pH value corresponding to the sample solution to be measured.

[0029] Furthermore, the standard curve of pH-Raman characteristic peak intensity ratio is y = 0.18618x + 0.57857, where R 2 = 0.9717, y represents the Raman peak intensity ratio at 1705 cm -1 and 2344 cm -1 , and x represents the pH value.

[0030] Advantages

[0031] 1) The present invention uses a SERS detection platform based on a nanoparticle assembly to detect the cyano Raman peak, realizing the micro-detection of H + .

[0032] 2) The present invention provides a method for pH detection of a sample to be measured using a SERS detection platform based on a nanoparticle assembly, mainly based on a cyano-rich Raman labeling molecule. This method utilizes the characteristic that the intensity ratio of the Raman characteristic peaks of a pH-responsive cyano compound changes regularly in different acidic environments, thereby accurately analyzing the corresponding relationship between the Raman peak intensity ratio and the pH value. This method does not require complex chemical reagent pretreatment, and its Raman characteristic peak is located in the biological silent zone, which can effectively shield biological signal interference and can be used for in-situ micro-area rapid detection, and can be widely applied to real-time pH monitoring in fields such as biology, chemical engineering, and environmental monitoring.

[0033] 3) Compared with traditional detection methods, the method of the present invention has the advantages of high detection efficiency, strong adaptability, simple operation, low cost, etc., solves the problems of complex existing detection methods, long detection time and high cost, and has important practical value and broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the dark-field scattering imaging optical system constructed for Example 1 of the present invention.

[0035] Figure 2 Method for constructing nanoparticle assemblies on a sensing substrate.

[0036] Figure 3 Schematic diagram of the nanoparticle assembly in Example 2 of the present invention.

[0037] Figure 4 Organic molecules involved in Example 2 of the present invention, wherein A is RBH-CN and B is 6-mercaptonicotinonitrile.

[0038] Figure 5 TEM images of two kinds of nanoparticles in Example 2 of the present invention, wherein A is spindle-shaped gold nanoparticles and B is spherical gold nanoparticles.

[0039] Figure 6 SEM image of the nanoparticle assembly in Example 2 of the present invention.

[0040] Figure 7 Curve graph of the change of LSPR during the assembly of the nanoparticle assembly in Example 2 of the present invention.

[0041] Figure 8 Graph showing the relationship between the pH-Raman peak of the assembly in Example 3 of the present invention; wherein A is the response change of the cyano Raman peak to pH, and B is the fitting curve graph of the intensity ratio of the Raman peaks at 1705 cm -1 and 2344 cm -1 at the buffer solutions of pH = 3, 4, 5, 6, 7. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0042] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Here, it should also be noted that in order to avoid obscuring the present invention due to unnecessary details, only the structures and / or processing steps closely related to the solution of the present invention are shown in the drawings, and other details less related to the present invention are omitted.

[0043] The raw materials for constructing the nanoparticle assembly in the following embodiments are mainly purchased from Aladdin, and RBH-CN is the product after further synthesis of the purchased rhodamine B-hydrazide.

[0044] The synthesis method of RBH-CN is as follows:

[0045] The first step: Prepare the raw material rhodamine B-hydrazide, and the structural formula of rhodamine B-hydrazide is as follows:

[0046]

[0047] The second step: Dropwise add a solution of 3,5-dibromobenzoyl chloride (0.77 g, 2.6 mmol) in THF (5 mL) to a solution containing rhodamine B-hydrazide (200 mg, 0.44 mmol) and triethylamine (0.4 mL, 2.9 mmol) (the solvent is a mixed solvent of 50 mL of THF and 1 mL of water), and stir at 0 - 5 °C. Then, stir the mixture overnight at room temperature. Remove the solvent under reduced pressure to obtain an orange oil. Dissolve the oil in CH2Cl2 (20 mL), and then wash the solution successively with dilute NaOH solution and brine. The organic phase is dried over anhydrous Na2SO4, and concentrated and purified by silica gel column chromatography using petroleum ether (60–90 °C) / EtOAc (3:1, v / v) as the eluent to obtain white solid A (190 mg, yield 60%). The structural formula of white solid A is as follows:

[0048]

[0049] The third step: Dissolve 4-cyanophenylboronic acid (0.15 g, 1 mmol), the product obtained in the second step (0.72 g, 1 mmol), potassium carbonate (0.14 g, 1 mmol), and Pd(dppf)Cl2 (0.037 g, 0.05 mmol) in a mixed solvent of THF / water (30 mL / 20 mL), react at 100 °C for 12 h under nitrogen protection, then evaporate the solvent, and perform silica gel column chromatography with a petroleum ether / ethyl acetate system to obtain white solid B (0.36 g, yield 49%). The structural formula of white solid B is as follows:

[0050]

[0051] The fourth step: Dissolve 4-(hydroxymethyl)phenylboronic acid (0.15 g, 1 mmol), the product obtained in the third step (0.74 g, 1 mmol), potassium carbonate (0.14 g, 1 mmol), and Pd(dppf)Cl2 (0.037 g, 0.05 mmol) in a mixed solvent of THF / water (30 mL / 20 mL), react at 100 °C for 12 h under nitrogen protection, then evaporate the solvent, and perform silica gel column chromatography with a petroleum ether / ethyl acetate system to obtain white solid C (0.52 g, yield 68%). The structural formula of white solid C is as follows.

[0052]

[0053] Step 5: Add the product obtained in Step 4 (0.77 g, 1 mmol) and the carboxylic acid (5-(1,2-dithiolan-3-yl)pentanoic acid) (0.2 g, 1 mmol) into a reaction flask, dissolve them with dry CH2Cl2 (20 mL), stir for 15 min, then add DCC (N,N'-dicyclohexylcarbodiimide, 0.4 g, 2 mmol) and DMAP (4-dimethylaminopyridine 0.005 g, 0.004 mmol), protect with N2, and stir at room temperature for 24 h. After filtering out the precipitate, then rotary evaporate the solvent, and perform silica gel column chromatography with a petroleum ether / ethyl acetate system to obtain 0.86 g of an oily substance with a yield of 88%. The obtained product is RBH-CN. The structural formula of RBH-CN is as follows:

[0054]

[0055] Example 1: Construction of a SERS detection platform based on nanoparticle assemblies

[0056] Figure 1 It is a schematic diagram of a dark-field imaging optical system. Among them, the blue arrow represents the white light path, and the purple arrow represents the path of Raman light. According to the established dark-field imaging optical system in this example, a SERS detection platform based on nanoparticle assemblies was constructed. This detection platform includes a single-particle imaging module, a sample reaction module, and a single-particle image recording module.

[0057] (I) Single-particle imaging module: It includes a white light source, a Raman light source, a dark-field condenser, and an optical microscope magnifying objective lens;

[0058] (1) White light source: It is used to generate white incident laser light to excite a single noble metal nanoparticle to produce a local surface plasmon resonance effect (LSPR effect).

[0059] (2) Raman light source: It is used to generate laser light in the 633 nm band to excite a single noble metal nanoparticle to produce a Raman scattering effect.

[0060] (3) Dark-field condenser: The dark-field condenser is used to converge the white incident laser light onto the sample. The dark-field condenser is a dry condenser with a numerical aperture of 0.80 - 0.95.

[0061] (4) Optical microscopy magnifying objective lens: It is used to magnify the optical path signal so that the nanoparticle assembly on the surface of the sensing substrate meets the requirements for Raman testing. The magnification of the optical microscopy magnifying objective lens is 60, and the numerical aperture is 0.70.

[0062] (II) Sample reaction module: It includes a sensing substrate, and a chemical modification layer is provided on the sensing substrate for fixing the nanoparticle assembly.

[0063] In the present invention, on the surface of the sensing substrate, a plasmonic nanoparticle assembly is constructed by using a cyanide-rich Raman labeling molecule, so as to realize the surface-enhanced Raman spectroscopy for rapid in-situ pH testing.

[0064] (III) Single-particle image recording module: It includes a mirror, a color CCD image sensor, and a spectral CCD image sensor. The mirror is used to convert the optical path direction. The color CCD image sensor is used to obtain dark-field scattering imaging. The spectral CCD image sensor is used to obtain an LSPR image, a single-particle LSPR spectral image, and a Raman scattering spectral image when performing Raman scattering testing. Specifically:

[0065] (1) The spectral CCD image sensor records the signal generated by the local surface plasmon resonance effect excited by the white laser, and obtains an LSPR image;

[0066] (2) The spectral CCD image sensor records the signal generated by the Raman scattering effect excited by the red laser in the 633 nm band, and obtains a Raman scattering spectral image.

[0067] In the SERS detection platform, the white light source, the dark-field condenser, the sensing substrate, the optical microscopy magnifying objective lens, and the mirror are installed in sequence from top to bottom; the Raman light source is installed behind the optical microscopy magnifying objective lens, the spectral CCD is on the left side of the optical microscopy magnifying objective lens, and the color CCD is on the right side of the optical microscopy magnifying objective lens.

[0068] The laser direction emitted by the white light source is perpendicular to the plane where the dark-field condenser, the sensing substrate, and the optical microscopy magnifying objective lens are located, and the included angle between the laser direction emitted by the white light source and the mirror is 30 - 60°. The laser emitted by the Raman light source is refracted and then irradiates the sensing substrate from below the optical microscopy magnifying objective lens, and the refracted laser direction is opposite to the laser direction emitted by the white light source.

[0069] The laser emitted by the white light source is irradiated on the sensing substrate through a dark field condenser. After being excited, the nanoparticle assembly on the sensing substrate scatters. After the scattered light passes through an optical microscopic magnifying objective lens, it enters a spectral CCD image sensor to obtain an LSPR image, and at the same time is reflected by a mirror to a color CCD image sensor to form a dark field scattering image.

[0070] The laser emitted by the Raman light source is irradiated on the nanoparticle assembly through an optical microscopic magnifying objective lens. After being excited by the laser, the nanoparticle assembly scatters, and the scattered light passes through the optical microscopic magnifying objective lens again and enters a spectral CCD image sensor to obtain a Raman scattering spectrum image.

[0071] Example 2: Constructing a nanoparticle assembly on a sensing substrate

[0072] Figure 2 Shows a method for constructing a nanoparticle assembly on a sensing substrate in a SERS detection platform;

[0073] In this example, the ITO glass sheet used as the sensing substrate has a specification of 10 * 33 mm and a thickness of 5 mm. Before use, the ITO glass sheet is ultrasonically cleaned with dishwashing liquid, acetone, ethanol, and ultrapure water for 0.5 h each, and then dried with nitrogen for standby. The nanoparticle assembly is fixed to the surface of the substrate through gold-sulfur bonds, and the surface of the substrate is modified with (3-mercaptopropyl) trimethylsilane reagent. The method for constructing a nanoparticle assembly on a sensing substrate is as follows:

[0074] Step 1. Modify the ITO glass sheet with (3-mercaptopropyl) trimethylsilane reagent:

[0075] Take 1 ml of an ethanol solution of 95% mass fraction of mercapto (3-mercaptopropyl) trimethylsilane and 4 ml of an ethanol solution of 97% mass fraction of absolute ethanol. After mixing the two, immerse the ITO glass sheet in the mixed solution for 2 h, then take out the ITO glass sheet, rinse it with absolute ethanol, and then place it in an oven at 60 °C for 15 min.

[0076] Step 2. Assembly of the nanoparticle assembly:

[0077] Step 2.1. Take 100 μL of an aqueous solution of spindle-shaped gold nanoparticles with a concentration of 10 −8 mol / L and drop it on the ITO glass sheet in Step 1, and remove it after 10 minutes. The spindle-shaped gold nanoparticles are 80 nm long and 40 nm wide.

[0078] Step 2.2. Take 100 μL of an ethanol solution of cyanine-rhodamine derivative (RBH-CN) with a concentration of 10 −7 mol / L and drop it on the ITO glass sheet in Step 2.1, and remove it after 10 min.

[0079] In the structure of the cyano-rhodamine derivative, at 1705 cm −1 and 2344 cm −1 The intensity ratio of the Raman characteristic peaks is correspondingly enhanced in the range of pH 3 - 7, and the standard curve of the pH-Raman peak intensity ratio is obtained to realize the trace detection of pH in the environment.

[0080] Step 2.3: Take 200 μL of an aqueous solution of FeCl3 with a concentration of 10 −6 mol / L and drop it on the ITO glass sheet in Step 3, and blow it dry after 30 min. 200 μL of the aqueous solution of FeCl3 with a concentration of 10

[0081] Step 2.4: Take 1 mL of an aqueous solution of spherical gold nanoparticles with a concentration of 10 −8 mol / L and 30 nm in diameter and mix it with 200 μL of an ethanol solution of 6-mercaptonicotinonitrile with a concentration of 10 -7 mol / L for 10 min. Then take 200 μL and drop it on the ITO glass sheet in Step 2.3, and blow it dry after 20 min.

[0082] In the nanoparticle assembly, it is connected through gold-sulfur bonds and iron coordination bonds. The spindle-shaped gold nanoparticles are fixed on the surface of the ITO glass sheet by gold-sulfur bonds, the RBH-CN is connected to the gold cone by gold-sulfur bonds, and then the RBH-CN and 6-mercaptonicotinonitrile are connected by the coordination bond between Fe 3+ and the cyano group, and 6-mercaptonicotinonitrile is connected to the 30 nm spherical gold nanoparticles by gold-sulfur bonds, so that the spindle-shaped gold nanoparticles and the spherical gold nanoparticles are connected to construct a nanoparticle assembly.

[0083] The ITO glass sheet nanoparticle assembly formed on the ITO glass sheet is as Figure 3 shown. Figure 3 In it, the red balls represent spherical gold nanoparticles with a diameter of 30 nm, the yellow rhombuses represent spindle-shaped gold nanoparticles, the green layer represents the chemical modification layer, and the blue layer represents the ITO glass sheet.

[0084] The structural formula of the cyano-rhodamine derivative is as shown in A in Figure 4 , and the structural formula of 6-mercaptonicotinonitrile is as shown in B in Figure 4 .

[0085] Step 3: Collect the dark-field scattering images of the nanoparticle assemblies after each step in Step 2 is completed, and perform LSPR optical intensity tests on the same particles. Determine whether the assembly is successfully assembled according to the change in LSPR optical intensity.

[0086] The specific steps of Step 3 are as follows:

[0087] Step 3.1, Dark-field scattering imaging and target particle localization:

[0088] Only turn on the white light source. The white laser passes vertically through the dark-field condenser and hits the ITO glass slide. Then, the scattered light passing through the optical microscopic magnifying objective lens enters the spectral CCD to form an LSPR image, and at the same time, after being reflected by the mirror, it is collected and imaged by the color CCD to obtain a dark-field scattering image. In the dark-field scattering image, select a spindle-shaped metal nanoparticle as the target nanoparticle and record its position for subsequent tracking.

[0089] Step 3.2, Collect the LSPR spectrum of the target particle:

[0090] In the case of only turning on the white light source, combine the LSPR image obtained by the spectral CCD and the dark-field scattering image displayed by the color CCD, and adjust the incident slit of the spectral CCD so that the scattered light of the selected target nanoparticle enters the spectral CCD to obtain the LSPR spectrum of a single particle. Through data processing of the spectrum, LSPR optical intensity data is obtained.

[0091] In this step, both the color CCD and the spectral CCD are images obtained by LSPR scattering. The positions where they are taken are the same, but one is color and the other is black and white, and only by passing through the incident slit of the spectral CCD can the LSPR spectrum be obtained.

[0092] In the process of constructing the nanoparticle assembly, after each assembly step is completed, repeat the operations of Step 3.1 and Step 3.2, and draw a curve graph based on the obtained LSPR optical intensity data.

[0093] The curve graph of the change of LSPR during the assembly of the nanoparticle assembly is as Figure 7 shown. According to the dark-field imaging image of the nanoparticle obtained by the color CCD, track the same nanoparticle, obtain the LSPR spectrum at each step in the process of constructing the nanoparticle assembly, and then draw the change graph of the LSPR optical intensity during the process of constructing the nanoparticle assembly. If the displacement trend of the LSPR peak occurs as in Figure 7 each step, the nanoparticle assembly is successfully constructed.

[0094] Example 3: pH detection method

[0095] Based on the SERS detection platform constructed in Example 1 and the successfully assembled nanoparticle assembly in Example 2, perform Raman intensity detection. According to the intensity ratio of the Raman characteristic peaks of the nanoparticle assembly at 1705 cm −1 and 2344 cm −1 carry out micro-detection of pH.

[0096] Step 1: Turn off the white light source and turn on the Raman source. Select Raman laser with a laser length of 633 nm and irradiate it on the ITO glass slide on which the nanoparticle assembly is successfully assembled. The exposure time is 15 s. Obtain the Raman scattering spectrum image through the spectral CCD, and then obtain the Raman signal intensity (800 - 2600 cm -1 Raman scattering intensity at each position), repeat 3 times, and take the average value.

[0097] Step 2: Subsequently, repeat the above operation when 100 μL of pH buffer solutions with pH values of 3, 4, 5, 6, and 7 are dropped on the ITO glass slide, and obtain the intensity ratio of the Raman characteristic peaks at 1705 cm −1 and 2344 cm −1 Thereby, draw a standard curve of pH-Raman peak intensity ratio.

[0098] Result: As the concentration of H + ions in the solution changes, the intensity ratio of the Raman characteristic peaks of the cyanine-rhodamine derivative (RBH-CN) at 1705 cm −1 and 2344 cm −1 increases correspondingly within the pH range of 3 - 7. Drawing a standard curve of pH-concentration can achieve highly sensitive detection of pH in trace solutions. The formula for drawing the standard curve of pH-concentration is y = 0.18618x + 0.57857, where R 2 = 0.9717. In the formula, y represents the intensity ratio of the Raman peaks at 1705 cm -1 and 2344 cm -1 , and x represents the pH value.

[0099] Figure 8 is the change relationship diagram of the pH-Raman peak intensity ratio of the assembly; A is the Raman signal intensity of the nanoparticle assembly at different pH values, B is the displacement response fitting curve diagram, and the fitting curve of the intensity ratio of the Raman peaks at 1705 cm -1 and 2344 cm -1 under the pH = 3 - 7 buffer solution.

[0100] Step 3: Take 100 μL of the sample solution to be measured, drop it on the ITO glass slide containing the nanoparticle assembly, turn on the Raman laser to perform Raman intensity measurement on it, and obtain the intensity ratio of the Raman peaks at 1705 cm -1 and 2344 cm -1 . Through the drawn standard curve of pH-Raman peak intensity ratio, the corresponding pH value can be obtained. Based on the standard curve of pH-Raman peak intensity ratio, the specific value of pH in the environment can be clearly identified.

[0101] The pH detection method of the present invention utilizes the characteristic that the intensity ratio of Raman characteristic peaks of pH-responsive cyanide compounds changes regularly in different acidic environments, so that the corresponding relationship between the intensity ratio of Raman peaks and pH value can be accurately analyzed. This method does not require complex chemical reagent pretreatment, and its Raman characteristic peaks are located in the biological silent region, which can effectively shield biological signal interference. It can be used for in-situ micro-region rapid detection and can be widely applied to real-time pH monitoring in the fields of biology, chemical engineering, environmental monitoring, etc.

[0102] In summary, the present invention realizes the micro-detection of pH by using the SERS detection platform. In addition, this method has a low detection limit, easy availability of detection instruments, simple, rapid, sensitive detection method, good repeatability, and does not require professional training, solving the problems of complex existing detection methods, long detection time, and high cost.

[0103] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A SERS detection platform based on nanoparticle assemblies, characterized in that, The SERS detection platform includes a sensing substrate for placing a sample to be detected, on which an assembly of nanoparticles containing cyano groups is assembled. The SERS detection platform detects the pH value of the sample to be detected based on the change in the intensity ratio of Raman characteristic peaks of the assembly of nanoparticles containing cyano groups in different acidic environments. The assembly method of the nanoparticle assembly is as follows: S1. Modify the sensing substrate with (3-mercaptopropyl) trimethylsilane reagent to prepare a chemical modification layer. S2. Drop the aqueous solution of spindle-shaped gold nanoparticles on the sensing substrate, keep it for a period of time and then remove it. S3. Drop the ethanol solution of cyano-rhodamine derivative on the sensing substrate, keep it for a period of time and then remove it. S4. Drop the aqueous solution of FeCl3 on the sensing substrate, keep it for a period of time and then remove it. S5. Mix the aqueous solution of spherical gold nanoparticles and the ethanol solution of 6-mercaptonicotinonitrile for a period of time, then drop it on the sensing substrate, keep it for a period of time and then dry it. The cyano-rhodamine derivative in S3 is RBH-CN, and its structural formula is as follows: 。 2. The SERS detection platform according to claim 1, wherein In addition to the sensing substrate, the SERS detection platform further includes: a white light source, a Raman light source, a dark field condenser, an optical microscope magnifying objective lens, a reflector, a color CCD image sensor and a spectral CCD image sensor. The laser emitted by the white light source irradiates the sensing substrate through the dark field condenser. The nanoparticle assembly on the sensing substrate is scattered after being excited. After the scattered light passes through the optical microscope magnifying objective lens, it enters the spectral CCD image sensor to form an LSPR image, and at the same time is reflected by the reflector to the color CCD image sensor to form a dark field scattering image. The laser emitted by the Raman light source irradiates the nanoparticle assembly through the optical microscope magnifying objective lens. The nanoparticle assembly is scattered after being excited, and the scattered light passes through the optical microscope magnifying objective lens again and enters the spectral CCD image sensor to form a Raman scattering spectrum image.

3. The SERS detection platform according to claim 2, wherein The specific assembly method of the nanoparticle assembly is as follows: S1. Modify the sensing substrate with (3-mercaptopropyl) trimethylsilane reagent to prepare a chemical modification layer: Mix the ethanol solution of (3-mercaptopropyl) trimethylsilane with a mass fraction of 95% and the absolute ethanol solution with a mass fraction of 97% according to a volume ratio of 1:2 - 1:10 to obtain a mixed solution. Immerse the sensing substrate in the mixed solution for 2 - 3 h, then take it out, rinse it with absolute ethanol and place it in an oven at 60 - 80 °C for 15 - 20 min. S2. Take 100 μL of an aqueous solution of spindle-shaped gold nanoparticles with a concentration of 10 -8 mol / L and drop it onto the sensing substrate, and remove it after 10 - 15 minutes; the length of the spindle-shaped gold nanoparticles is 80 - 150 nm, and the width is 20 - 40 nm; S3. Take 100 μL of an ethanol solution of RBH-CN with a concentration of 10 -7 mol / L and drop it onto the sensing substrate obtained in S2, and remove it after 10 - 15 minutes; S4. Take 200 μL of an aqueous solution of FeCl3 with a concentration of 10 -6 mol / L and drop it onto the sensing substrate obtained in S3, and then dry it after 30 - 40 minutes. S5. Mix 1 mL of an aqueous solution of spherical gold nanoparticles with a concentration of 10 -8 mol / L with 200 μL of an ethanol solution of 6-mercaptonicotinonitrile with a concentration of 10 -7 mol / L for 15 - 20 minutes. Then, take 200 μL and drop it onto the sensing substrate, and blow it dry after 20 minutes. The diameter of the spherical gold nanoparticles is 25 - 35 nm.

4. The pH detection method of the SERS detection platform according to claim 2, characterized in that, Based on the intensity ratio of the Raman characteristic peaks of the nanoparticle assemblies at 1705 cm -1 and 2344 cm -1 to achieve the detection of pH.

5. The pH detection method according to claim 4, wherein It includes the following steps: Step 1. Turn on the Raman light source, select a laser with a length of 633 nm, irradiate it on the sensing substrate containing the nanoparticle assembly, obtain a Raman scattering spectrum image through the spectral CCD, and further obtain the Raman signal intensity of the nanoparticle assembly. Step 2: Drop buffer solutions with different pH values onto the sensing substrate containing the nanoparticle assembly, and repeat the operations in Step 1 above to obtain the intensity ratio of the Raman characteristic peaks at 1705 cm -1 and 2344 cm -1 , and thus plot a standard curve of pH-Raman characteristic peak intensity ratio; Step 3: Drop the test sample solution onto the sensing substrate containing the nanoparticle assembly, turn on the Raman laser, perform Raman intensity measurement on it, and obtain the Raman peak intensity ratio at 1705 cm -1 and 2344 cm -1 ; Obtain the pH value corresponding to the test sample solution through the standard curve of the pH-Raman characteristic peak intensity ratio drawn in Step 2.

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