SERS (Surface Enhanced Raman Scattering) detection platform based on nanoparticle assembly and pH (Potential of Hydrogen) detection method

Abstract

The invention provides an SERS (Surface Enhanced Raman Scattering) detection platform based on a nanoparticle assembly and a pH (Potential of Hydrogen) detection method, and belongs to the technical field of analysis and detection.According to the pH detection method, the characteristic that the Raman characteristic peak intensity ratio of a pH responsive cyano compound in different acid environments can change regularly is utilized; therefore, the corresponding relation between the Raman peak intensity ratio and the pH value can be accurately analyzed, trace detection of H < + > is realized, and the method can be widely applied to real-time pH monitoring in the fields of biology, chemical engineering, environmental monitoring and the like.

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 crucial importance and is widely applied in multiple fields such as biology, chemical engineering, and environmental monitoring. Currently, common pH detection methods mainly include the acid-base indicator method, the glass electrode method, and the detection method based on chemiluminescence, 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 that require precise pH values, such as the monitoring of the cell culture environment in biological experiments. The glass electrode method is a currently widely used detection method. 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. However, 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 relatively high requirements for the professional skills of the operator; on the other hand, the performance of the glass electrode is unstable 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 inability to detect. The detection method based on chemiluminescence 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 the addition of multiple chemical reagents for complex chemical reactions, not only with a slow detection speed, but also the use of chemical reagents increases the detection cost and the risk of environmental pollution. At the same time, it has relatively high requirements for the detection equipment, restricting 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 the ability to adapt 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 methods, 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: A SERS detection platform based on a nanoparticle assembly. The SERS detection platform includes a sensing substrate for placing a sample to be tested, 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 tested 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 microscopic magnifying objective lens, a reflector, a color CCD image sensor, and a spectral CCD image sensor; 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 is scattered after being excited; after the scattered light passes through the optical microscopic 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 is irradiated on the nanoparticle assembly through the optical microscopic magnifying objective lens. The nanoparticle assembly is scattered after being excited, and the scattered light then passes through the optical microscopic magnifying objective lens and enters the spectral CCD image sensor to form a Raman scattering spectrum image.

[0007] Further, 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 FeCl 3 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-mercaptobenzonitrile for a period of time, then drop it on the sensing substrate, keep it for a period of time and then dry it.

[0008] Further, the cyano-rhodamine derivative in S3 is RBH-CN, and its structural formula is as follows: .

[0009] Further, 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: An ethanol solution of (3-mercaptopropyl)trimethylsilane with a mass fraction of 95% and an absolute ethanol solution with a mass fraction of 97% are mixed at a volume ratio of 1:2 - 1:10 to obtain a mixed solution, which is placed in the mixed solution for soaking treatment for 2 - 3 h, then taken out, rinsed with absolute ethanol and placed 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 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; 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; S4. Take 200 μL of an aqueous solution of −6 FeCl 3 with a concentration of 10 mol / L and drop it on the sensing substrate obtained in S3, and blow it dry after 30 - 40 min; 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 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.

[0010] 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 of the nanoparticle assembly.

[0011] Furthermore, the pH detection method 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, the exposure time is 15 s, obtain the 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 on the sensing substrate containing the nanoparticle assembly, repeat the operation in Step 1 above, obtain the intensity ratio of Raman characteristic peaks at 1705 cm −1 and 2344 cm −1 , and thus draw 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 for Raman intensity measurement, and obtain the Raman peak intensity ratio at 1705 cm -1 and 2344 cm -1 ; Obtain the pH value of the test sample solution through the standard curve of the pH-Raman characteristic peak intensity ratio drawn in Step 2.

[0012] Furthermore, the standard curve of the 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.

[0013] Beneficial Effects 1) The present invention uses a SERS detection platform based on nanoparticle assembly to detect the cyanide Raman peak, realizing the micro-detection of H + .

[0014] 2) The present invention provides a method for pH detection of a test sample using a SERS detection platform based on nanoparticle assembly, mainly based on cyanide-rich Raman labeling molecules. This method utilizes the characteristic that the Raman characteristic peak intensity ratio of pH-responsive cyanide compounds 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, 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.

[0015] 3) Compared with traditional detection methods, the method of the present invention has the advantages of high detection efficiency, strong adaptability, simple operation, and low cost, solving the problems of complex existing detection methods, long detection time, and high cost, and having important practical value and broad market application prospects. Description of the Drawings

[0016] Figure 1 is a schematic diagram of the dark-field scattering imaging optical system constructed in Example 1 of the present invention.

[0017] Figure 2 is a method for constructing a nanoparticle assembly on a sensing substrate.

[0018] Figure 3 is a schematic diagram of the nanoparticle assembly in Example 2 of the present invention.

[0019] Figure 4The organic molecule involved in Example 2 of the present invention, wherein A is RBH-CN and B is 6-mercaptonicotinonitrile.

[0020] 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.

[0021] Figure 6 SEM image of the nanoparticle assembly in Example 2 of the present invention.

[0022] Figure 7 Curve graph of the change of LSPR during the assembly of the nanoparticle assembly in Example 2 of the present invention.

[0023] Figure 8 Graph showing the relationship between pH and 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 Raman peak intensity ratio at 1705 cm -1 and 2344 cm -1 at the fitting curve of the Raman peak intensity ratio. Detailed implementation manners

[0024] 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, while other details less related to the present invention are omitted.

[0025] 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.

[0026] The synthesis method of RBH-CN is as follows: The first step: Prepare the raw material rhodamine B-hydrazide, and the structural formula of rhodamine B-hydrazide is as follows:

[0027] The second step: Drop the THF (5 mL) solution containing 3,5-dibromobenzoyl chloride (0.77 g, 2.6 mmol) into the 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 CH 2 Cl2 (20 mL), and then the solution was washed successively with dilute NaOH solution and brine. The organic phase was dried over anhydrous Na 2 SO 4 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:

[0028] Step 3: p-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), Pd(dppf)Cl 2 (0.037 g, 0.05 mmol) were dissolved in a mixed solvent of THF / water (30 mL / 20 mL), and the reaction was carried out at 100 °C for 12 h under nitrogen protection. Then the solvent was evaporated, and silica gel column chromatography was carried out using 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:

[0029] Step 4: p-Hydroxymethylphenylboronic 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), Pd(dppf)Cl 2 (0.037 g, 0.05 mmol) were in a mixed solvent of THF / water (30 mL / 20 mL), and the reaction was carried out at 100 °C for 12 h under nitrogen protection. Then the solvent was evaporated, and silica gel column chromatography was carried out using 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.

[0030]

[0031] Step 5: The product obtained in the fourth step (0.77 g, 1 mmol) and carboxylic acid (5-(1,2-dithiolan-3-yl)pentanoic acid) (0.2 g, 1 mmol) were added to the reaction flask and dissolved in dry CH 2 Cl 2 (20 mL), stirred for 15 min, and then DCC (N,N'-dicyclohexylcarbodiimide, 0.4 g, 2 mmol) and DMAP (4-dimethylaminopyridine 0.005 g, 0.004 mmol) were added. N2 Protect, stir at room temperature for 24 h. After filtering out the precipitate, the solvent was then rotary evaporated, and silica gel column chromatography was carried out with a petroleum ether / ethyl acetate system to obtain 0.86 g of an oily substance with a yield of 88%. The product obtained was RBH-CN. The structural formula of RBH-CN is as follows:

[0032] Example 1: Construction of a SERS detection platform based on nanoparticle assemblies 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.

[0033] (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; (1) White light source: It is used to generate white incident laser to excite single noble metal nanoparticles to produce local surface plasmon resonance effect (LSPR effect).

[0034] (2) Raman light source: It is used to generate a laser in the 633 nm band to excite single noble metal nanoparticles to produce Raman scattering effect.

[0035] (3) Dark-field condenser: The dark-field condenser is used to converge the white incident laser onto the sample. The dark-field condenser is a dry condenser with a numerical aperture of 0.80 - 0.95.

[0036] (4) Optical microscope magnifying objective lens: It is used to amplify the optical path signal so that the nanoparticle assemblies on the surface of the sensing substrate meet the requirements for Raman testing. The magnification of the optical microscope magnifying objective lens is 60, and the numerical aperture is 0.70.

[0037] (II) Sample reaction module: It includes a sensing substrate, and a chemical modification layer is provided on the sensing substrate for fixing nanoparticle assemblies.

[0038] 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 surface-enhanced Raman spectroscopy for in-situ rapid pH testing.

[0039] (3) 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 LSPR images, single-particle LSPR spectral images, and Raman scattering spectral images during Raman scattering tests. Specifically: (1) The spectral CCD image sensor records the signal generated by the local surface plasmon resonance effect excited by white laser, and obtains an LSPR image; (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.

[0040] In the SERS detection platform, the white light source, dark-field condenser, sensing substrate, optical microscope magnifying objective lens, and mirror are installed in sequence from top to bottom; the Raman light source is installed behind the optical microscope magnifying objective lens, the spectral CCD is on the left side of the optical microscope magnifying objective lens, and the color CCD is on the right side of the optical microscope magnifying objective lens.

[0041] The laser direction emitted by the white light source is perpendicular to the plane where the dark-field condenser, sensing substrate, and optical microscope 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 microscope magnifying objective lens, and the refracted direction is opposite to the laser direction emitted by the white light source.

[0042] The laser emitted by the white light source irradiates the sensing substrate through the dark-field condenser. After the nanoparticle assembly on the sensing substrate is excited, scattering occurs; the scattered light passes through the optical microscope magnifying objective lens and then enters the spectral CCD image sensor to obtain an LSPR image, and at the same time is reflected by the mirror to the color CCD image sensor to form a dark-field scattering image.

[0043] The laser emitted by the Raman light source passes through the optical microscope magnifying objective lens and irradiates the nanoparticle assembly. After the nanoparticle assembly is excited by the laser, scattering occurs, and the scattered light passes through the optical microscope magnifying objective lens again and enters the spectral CCD image sensor to obtain a Raman scattering spectral image.

[0044] Example 2: Construction of nanoparticle assembly on sensing substrate Figure 2 Shows the method of constructing a nanoparticle assembly on a sensing substrate in the SERS detection platform; In this embodiment, the sensing substrate uses an ITO glass sheet with 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 the nanoparticle assembly on the sensing substrate is as follows: Step 1. Modify the ITO glass sheet with (3-mercaptopropyl) trimethylsilane reagent: Take 1 ml of an ethanol solution of 95% mass fraction of mercapto (3-mercaptopropyl) trimethylsilane and 4 ml of an anhydrous ethanol solution of 97% mass fraction. 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 anhydrous ethanol, and then place it in an oven at 60 °C for 15 min.

[0045] Step 2. Assembly of the nanoparticle assembly: 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.

[0046] Step 2.2. Take 100 μL of an ethanol solution of cyano-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.

[0047] In the structure of the cyano-rhodamine derivative, the intensity ratio of the Raman characteristic peaks at 1705 cm −1 and 2344 cm −1 correspondingly increases in the pH range of 3 - 7, and a standard curve of pH-Raman peak intensity ratio is obtained to realize the trace detection of pH in the environment.

[0048] Step 2.3. Take 200 μL of an aqueous solution of FeCl −6 with a concentration of 10 3 , drop it on the ITO glass sheet in Step 3, and dry it after 30 min.

[0049] Step 2.4. Take 1 mL of an aqueous solution of spherical gold nanoparticles with a diameter of 30 nm with a concentration of 10 −8 mol / L and 200 μL of an aqueous solution with a concentration of 10 -7After mixing with an ethanol solution of 6-mercaptonicotinonitrile at a concentration of mol / L for 10 min, 200 μL was taken and dropped onto the ITO glass sheet in step 2.3, and then dried after 20 min.

[0050] In the nanoparticle assembly, connections are made through gold-sulfur bonds and iron coordination bonds. The spindle-shaped gold nanoparticles are fixed on the surface of the ITO glass sheet using gold-sulfur bonds, the RBH-CN is connected to the gold cone using gold-sulfur bonds, and then 3+ the RBH-CN and 6-mercaptonicotinonitrile are connected through the coordination bond between Fe and the cyano group, and then 6-mercaptonicotinonitrile is connected to the 30 nm spherical gold nanoparticles through gold-sulfur bonds, so that the spindle-shaped gold nanoparticles and the spherical gold nanoparticles are connected to construct a nanoparticle assembly.

[0051] The ITO glass sheet nanoparticle assembly formed on the ITO glass sheet is as Figure 3 shown. Figure 3 In it, the red spheres 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.

[0052] 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 shown.

[0053] Step 3: Collect the dark-field scattering images of the nanoparticle assemblies after each step in step 2, and perform LSPR optical intensity tests on the same particle. Determine whether the assembly is successfully assembled according to the change in LSPR optical intensity.

[0054] The specific steps of step 3 are as follows: Step 3.1: Dark-field scattering imaging and target particle positioning: Only turn on the white light source. The white laser passes vertically through the dark-field condenser and hits the ITO glass sheet. Then, the scattered light passing through the optical microscope magnifying objective lens enters the spectral CCD to form an LSPR image, and at the same time, it is reflected by the mirror 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.

[0055] Step 3.2: Collect the LSPR spectrum of the target particle: In the case of only turning on the white light source, combining the LSPR image obtained by the spectral CCD and the dark-field scattering image displayed by the color CCD, 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, and through data processing of the spectrum, obtain the LSPR optical intensity data.

[0056] In this step, both the color CCD and the spectral CCD are images obtained through LSPR scattering. They are taken at the same position, 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.

[0057] Step 3.3: During the process of constructing the nanoparticle assembly, after each assembly step, the operations of Step 3.1 and Step 3.2 are repeated, and a curve graph is plotted based on the obtained LSPR optical intensity data.

[0058] 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 under the color CCD, the same nanoparticle is traced, and the LSPR spectrum is obtained at each step during the process of constructing the nanoparticle assembly, and then the change graph of the LSPR optical intensity during the process of constructing the nanoparticle assembly is plotted. If the displacement trend of the LSPR peak occurs at each step as in Figure 7 the figure, the nanoparticle assembly is successfully constructed.

[0059] Example 3: pH detection method Based on the SERS detection platform constructed in Example 1 and the successfully assembled nanoparticle assembly in Example 2, Raman intensity detection is carried out. According to the intensity ratio of the Raman characteristic peaks of the nanoparticle assembly at 1705 cm −1 and 2344 cm −1 trace detection of pH is realized.

[0060] Step 1: Turn off the white light source, turn on the Raman source, select Raman laser with a laser length of 633 nm, and make it irradiate on the ITO glass sheet on which the nanoparticle assembly is successfully assembled. The exposure time is 15 s. The Raman scattering spectrum image is obtained through the spectral CCD, and then the Raman signal intensity (Raman scattering intensity at each position in the range of 800 - 2600 cm -1 of the nanoparticle assembly is obtained. Repeat 3 times and take the average value.

[0061] 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 sheet, 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 peak intensity ratio.

[0062] Result: As the concentration of H + ions in the solution changes, the cyano-rhodamine derivative (RBH-CN) at 1705 cm−1 and 2344 cm −1 The intensity ratio of Raman characteristic peaks increases correspondingly within the pH range of 3 - 7. By plotting the standard curve of pH-concentration, highly sensitive detection of pH in trace solutions can be achieved. The formula for plotting the pH-concentration standard curve is y = 0.18618x + 0.57857, where R 2 = 0.9717. In the formula, y represents the intensity ratio of Raman peaks at 1705 cm -1 and 2344 cm -1 , and x represents the pH value.

[0063] Figure 8 is the variation 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 Raman peaks at 1705 cm -1 and 2344 cm -1 under the pH = 3 - 7 buffer solution.

[0064] 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 Raman peaks at 1705 cm -1 and 2344 cm -1 . By using the standard curve of the pH-Raman peak intensity ratio plotted, the corresponding pH value can be obtained. Based on the standard curve of the pH-Raman peak intensity ratio, the specific value of pH in the environment can be clearly identified.

[0065] 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. This method does not require complex chemical reagent pretreatment, and its Raman characteristic peaks are located in the biological silent area, 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.

[0066] In summary, the present invention realizes the trace detection of pH by using the SERS detection platform. In addition, this method has a low detection limit, the detection instrument is easy to obtain, the detection method is simple, fast, sensitive, has good repeatability, and does not require professional training, solving the problems of complex existing detection methods, long detection time, and high cost.

[0067] 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 assembly, characterized in that: The SERS detection platform comprises a sensing substrate for placing a sample to be tested, on which a cyano-containing nanoparticle assembly is assembled. The SERS detection platform detects the pH value of the sample to be tested based on the change in the Raman characteristic peak intensity ratio of the cyano-containing nanoparticle assembly in different acidic environments.

2. The SERS detection platform according to claim 1, characterized in that: In addition to the sensing substrate, the SERS detection platform also includes: a white light source, a Raman light source, a dark field condenser, an optical microscope magnifying lens, a reflector, a color CCD image sensor and a spectral CCD image sensor; The laser emitted by the white light source is irradiated onto the sensing substrate through a dark field condenser, and the nanoparticle assembly on the sensing substrate is excited and scattered; the scattered light passes through the optical microscope magnification objective lens, enters the spectral CCD image sensor to form an LSPR image, and is reflected by the reflector to the color CCD image sensor to form a dark field scattering image; The laser light emitted by the Raman light source is irradiated on the nanoparticle assembly through the optical microscope magnification objective lens. The nanoparticle assembly is excited and scattered. The scattered light then passes through the optical microscope magnification objective lens and enters the spectrum CCD image sensor to form a Raman scattering spectrum image.

3. The SERS detection platform according to claim 1, characterized in that: The assembly method of the nanoparticle assembly is as follows: S1, modifying the sensing substrate with (3-mercaptopropyl)trimethylsilane reagent to prepare a chemically modified layer; S2, dropping an aqueous solution of spindle-shaped gold nanoparticles onto the sensing substrate, retaining it for a period of time and then removing it; S3, dropping an ethanol solution of a cyano-rhodamine derivative onto the sensing substrate, leaving it for a period of time and then removing it; S4, dropping FeCl3 aqueous solution on the sensing substrate, leaving it for a while and then removing it; S5. Take an aqueous solution of spherical gold nanoparticles and an ethanol solution of 6-mercaptonicotinonitrile, mix them for a period of time, drop them on the sensing substrate, keep them for a period of time, and then blow them dry.

4. The SERS detection platform according to claim 2, characterized in that: The cyano-rhodamine derivative in S3 is RBH-CN, and its structural formula is as follows: 。 5. The SERS detection platform according to claim 4, characterized in that: The assembly method of the nanoparticle assembly is specifically as follows: S1. Modify the sensing substrate with (3-mercaptopropyl)trimethylsilane reagent to prepare a chemically modified layer: A mixed solution is obtained by mixing a 95% (3-mercaptopropyl)trimethylsilane ethanol solution and a 97% anhydrous ethanol solution at a volume ratio of 1:2-1:10, and the mixture is immersed in the mixed solution for 2-3 hours, then taken out, rinsed with anhydrous ethanol, and placed in a 60-80°C oven for 15-20 minutes; S2, take the concentration of 10 −8 100 μL of an aqueous solution of mol / L spindle-shaped gold nanoparticles was dripped onto the sensing substrate and removed after 10-15 minutes; the spindle-shaped gold nanoparticles were 80-150 nm long and 20-40 nm wide; S3, take the concentration of 10 −7 100 μL of ethanol solution of RBH-CN at mol / L was added dropwise on the sensing substrate obtained in S2 and removed after 10-15 minutes; S4, take the concentration of 10 −6 200 μL of mol / L FeCl3 aqueous solution was dripped onto the sensing substrate obtained in S3 and blown dry after 30-40 minutes; S5, 1 mL of 10 −8 mol / L aqueous solution of spherical gold nanoparticles and 200 μL of 10 -7 After mixing the ethanol solution of 6-mercaptonicotinonitrile (6-Mercaptonicotinonitrile) at 1 mol / L for 15-20 minutes, 200 μL was added dropwise to the sensing substrate and blown dry after 20 minutes. The diameter of the spherical gold nanoparticles was 25-35 nm.

6. The pH detection method based on the SERS detection platform of claim 2, characterized in that: Based on the nanoparticle assembly at 1705 cm −1 and 2344 cm −1 The intensity ratio of Raman characteristic peaks can be used to detect pH.

7. The pH detection method according to claim 6, characterized in that: The steps include: Step 1: Turn on the Raman light source, select a laser with a length of 633 nm, and irradiate it on the sensing substrate containing the nanoparticle assembly, obtain the Raman scattering spectrum image through the spectral CCD, and then obtain the Raman signal intensity of the nanoparticle assembly; Step 2: Add buffer solutions of different pH values ​​onto the sensing substrate containing the nanoparticle assembly and repeat the above step 1 to obtain the −1 and 2344 cm −1 Raman characteristic peak intensity ratio, so as to draw a standard curve of pH-Raman characteristic peak intensity ratio; Step 3: Take the sample solution to be tested and drop it on the sensor substrate containing the nanoparticle assembly, turn on the Raman laser, and perform Raman intensity test to obtain its 1705cm -1 and 2344cm -1 The pH value corresponding to the sample solution to be tested is obtained by the standard curve of pH-Raman characteristic peak intensity ratio drawn in step 2.

Images