CN111398246A - Rapid high-flux liquid interface enhanced Raman spectroscopy detection method - Google Patents

Abstract

The invention discloses a rapid high-flux liquid interface enhanced Raman spectroscopy detection method, which comprises the following steps: mixing the noble metal nano sol, the oil phase solvent and the object to be detected to obtain a mixed sample; placing the mixed sample in a container unit which can be spliced and irradiated by multi-surface laser, and assembling the noble metal nano particles in the mixed sample to a liquid-liquid interface to form a three-dimensional interface plasma array, namely obtaining a three-dimensional liquid sample to be tested; and after laser calibration is carried out once, the Raman instrument is used for detecting the sample group to be detected. In the measuring process, the method can detect a plurality of samples only by carrying out one-time accurate laser calibration on the sample group to be detected, and can obviously improve the detection flux; the method is simple to operate, good in stability, high in speed, high in flux and high in accuracy, and can be used for large sample analysis, such as food safety, environmental protection or clinical inspection.

Description

A fast and high-throughput liquid interface-enhanced Raman spectroscopy detection method

technical field

The invention belongs to the field of sensitive detection and analysis, in particular to a fast high-throughput liquid interface enhanced Raman spectroscopy detection method.

Background technique

The three-dimensional interfacial ionic array is formed by the emulsification and assembly of noble metal nanoparticles at the liquid-liquid interface to form a surface-enhanced Raman spectroscopy (SERS) substrate with controllable hot spots (Anal.Chem.2019,91,2288-2295), With stable physical properties and self-healing ability (Nanoscale 2016, 8, 7723-7737), the shell of the 3D interfacial ion array has a uniform SERS hotspot density, which can generate biphasic accessible hotspots, making them ideal for detecting nonadsorbed molecules 's model. Due to their excellent optical stability and laser alignment tolerance, they are commonly used in SERS sensors (ACSSensor. 2019, 4, 1798-1805). In addition, the 3D interfacial plasmon array can perform quantitative SERS analysis in the general analyte concentration range and can analyze individual analytes in multiplex detection, which is ideal for in situ microreaction monitoring. Here we refer to the three-dimensional interfacial ion array containing the test object as the three-dimensional liquid test sample.

However, the current sensing applications of 3D liquid samples to be tested are mostly completed by static measurement. Although static measurements make full use of the advantages of 3D liquid interface plasma arrays, laser calibration is required for each sample to be tested. , which limits the detection throughput of the SERS sensing platform. The present invention fully recognizes the superiority of the three-dimensional liquid interface plasma array, and hopes to combine it with a container unit and a Raman instrument that can be spliced and can be irradiated by multi-faceted lasers to develop a simpler, more accurate and faster high-pass Quantitative Raman detection method. Precise laser alignment of each 3D liquid test sample is not required before each measurement, which can significantly increase detection throughput and automate SERS multi-sample detection. As a miniaturized yet powerful platform for identification of small molecules or compounds, 3D liquid interface plasmonic arrays are particularly important for disease diagnosis, environmental safety, toxicant screening, and field detection, because these sites include large sample numbers, but small sample sizes and limited. However, a SERS substrate that can be applied to large sample analysis needs to have the following characteristics: simple fabrication; good stability; fast detection speed; high accuracy; and high sensitivity. The present invention fully complies with the above requirements, and opens up a new idea for large sample analysis.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the purpose of the present invention is to provide a fast and high-throughput liquid interface enhanced Raman spectroscopy detection method, which has fast detection speed, high accuracy and good stability. Can be used for large sample analysis.

To achieve the above object, the technical scheme adopted in the present invention is:

A fast high-throughput liquid interface enhanced Raman spectroscopy detection method, comprising the following steps:

a. Mix the precious metal nanosol, the oil phase solvent and the analyte to obtain a mixed sample;

b. The mixed sample is placed in a container unit that can be spliced and can be irradiated by multi-faceted lasers, and the precious metal nanoparticles in the mixed sample reach the liquid-liquid interface and assemble to form a three-dimensional interface plasma array, that is, a three-dimensional liquid sample to be tested is obtained;

c. Multiple three-dimensional liquid samples to be tested are spliced together between container units to form a sample group to be tested. After laser calibration once, the sample group to be tested is detected with a Raman instrument, that is, fast high-throughput SERS detection is realized.

Further scheme, in step a, the oil phase solvent is one or both of organic reagents with density greater than water such as chloroform, 1,2-dichloroethane, dichloromethane, etc.; or organic reagents with density less than water Such as one or both of hexane, cyclohexane, toluene, xylene and pentane.

In a further scheme, if the analyte is oil-soluble, it can be dissolved in the oil phase; if it is water-soluble, it can be dissolved in pure water, or in benign solvents such as ethanol and methanol.

In a further scheme, in step b, the container unit that can be spliced and can be irradiated by multi-faceted laser is a microporous container, and the volume of the microporous container is 10-1000 μL; the material can be a hydrophilic material or a hydrophobic material, for example, Glass, plexiglass, quartz, polyethylene or polypropylene, etc.; the shape of the container unit can be a cylinder, a cuboid or a hemisphere, etc. The container unit can receive vertical or horizontal laser irradiation.

In a further scheme, in step b, the three-dimensional interface plasma array is of an oil-in-water type or a water-in-oil type, and the volume is 1 μL to 1 mL.

In a further scheme, in step b, the assembly of noble metal nanoparticles may be promoted to form a three-dimensional interface plasmon array by physical means or chemical means. Further preferably, the physical method is ultrasonic, vibration or a combination of ultrasonic and vibration, and the time is 1 to 5 minutes; the chemical method is to add a modifier, accelerator or acid solution to the assembly system; the modifier It is 1-pentanethiol, pyridine, 1,10-phenanthroline, 1-heptanethiol, dodecanethiol, benzenethiol, tetra-n-octylammonium bromide, 5,10,15,20-tetra( One of 1,-methyl-4-pyridyl)-21H, 23H-porphine, the main purpose is to improve the hydrophobicity of particles by means of adsorption or coordination; the accelerator is a hydrophobic cation/anion salt , containing a hydrophobic ion carrying the opposite charge to the nanoparticle, including transition metal complexes, crown ethers, and organic salts such as tetrabutylammonium nitrate, tetraphenylarsenide trifluoromethanesulfonamide, tetraphenylboronate, Tetraphenyl arsenide triphenyl borate, etc.

In a further scheme, in step c, the method of using the Raman instrument to detect the sample group to be tested is as follows: keep the laser light source still, and detect by moving the sample group to be tested; or keep the sample group to be tested still, and move the laser light source. light source for detection. Further preferably, a microprocessor is equipped on the Raman instrument, and the sample group to be tested or the laser light source is automatically moved by controlling the mechanical drive mechanism of the microprocessor to move along the X direction and the Y direction.

Compared with the prior art, the present invention has the following beneficial effects:

The detection method provided by the present invention combines a three-dimensional liquid sample to be tested with a container unit that can be spliced and can be irradiated by multi-faceted lasers and a Raman meter. The detection of each sample overcomes the need for accurate laser calibration for each sample to be tested in the existing detection methods, and can significantly improve the detection throughput; the method is simple in operation, good in stability, fast in speed, and high in throughput. And with high accuracy, it can be used for large sample analysis, such as food safety, environmental protection or clinical testing.

Description of drawings

Fig. 1 is the flow chart of a kind of fast high-throughput liquid interface enhanced Raman spectroscopy detection method of the present invention;

Fig. 2 is the splicing and laser incident direction schematic diagram of the container unit in the present invention;

Fig. 3 is the SERS diagram of high-throughput detection of Rhodamine 6G (R6G) standard sample in Example 1;

Fig. 4 is the SERS diagram of high-throughput detection of food hazard fumetidine and melamine in Example 2;

FIG. 5 is a SERS image of high-throughput detection of fumedox on apple and orange peels in Example 3. FIG.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to specific embodiments. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention.

1 and 2, a detailed description is given of the fast high-throughput liquid interface-enhanced Raman spectroscopy detection method of the present invention. In Fig. 2, the small grid line box represents a container unit, in which a three-dimensional liquid test sample is placed, and the laser can be incident from a vertical or horizontal direction (the arrow indicates the incident direction of the laser). Extended example 1 shows that the container unit is spliced left and right to form a test group with 12 three-dimensional liquid test samples. Example 1 shows its front view. The extended example 2 indicates that the container unit is spliced at the front, back, left and right to form a test group with 96 three-dimensional liquid test samples. Example 2 shows its top view.

Example 1

In this embodiment, the Raman parameters are set as: excitation wavelength of 785 nm, laser power of 3%, integration time of 3 s, and accumulation times of 1 time.

The high-throughput SERS detection capability of this measurement technique. 80 μL of 1 ppm rhodamine 6G (R6G) dissolved in chloroform and 200 μL of gold nanosol were added to each 300 μL container unit, respectively. Among them, R6G, as the test substance, also has a positive charge. The chemical method of adding 10 μL HCl (V/V 5%) can promote the assembly of the three-dimensional liquid test sample, and perform high-throughput SERS detection. The three-dimensional liquid test sample is in the water-in-oil state in the hydrophobic container unit (polypropylene material) (Figure 3A). The inset shows that the three-dimensional liquid test sample is in the same state in the 1.5mL centrifuge tube; The container unit (quartz material) was in an oil-in-water state ( FIG. 3B ). This shows the ultra-stability and excellent container adaptability of the three-dimensional liquid test sample. Then, the same 12 three-dimensional liquid test samples were prepared for the study of SERS stability. 10 spectra were taken for each 3D liquid test sample. Since the three-dimensional liquid test sample is prepared off-line, the on-line measurement only refers to the in-situ acquisition of the spectrum with the laser focus fixed. Three characteristic peaks 1358, 1504, 1646 cm ^-1 from R6G were selected for statistical analysis. As shown in Figure 3C, the variation of the three characteristic peak intensities relative to the mean value in the 120 selected spectra yielded relative standard deviations of 8.4%, 8.1%, and 7.8%, respectively, further demonstrating the ultra-stability of the three-dimensional liquid test sample. SERS features. The inset in Fig. 3C shows all the time-resolved spectra of the 12 three-dimensional liquid test samples during continuous irradiation, without obvious fluctuations or the appearance of new Raman spectral bands. These results indicate that the 3D liquid test samples have good stability and good SERS uniformity.

Example 2

In this embodiment, the Raman parameters are set as: excitation wavelength of 785 nm, laser power of 10%, integration time of 8 s, and accumulation times of 1 time.

High-throughput detection of two food hazards using the measurement technique of the present invention. Fumei Shuang is a protective fungicide that can control downy mildew, fusarium wilt, anthracnose, head smut and yellow blight at the seedling stage. Melamine is a chemical raw material and is not allowed to be added to food, but some unscrupulous traders add melamine to dairy products to improve the detection index of protein content. Current detection methods are inefficient, so there is an urgent need to develop rapid detection methods to achieve accurate analysis of them. The specific process is as follows: (1) Dissolve Famex in chloroform to prepare Famex standard solution with concentration gradient of 0, 0.1, 1, 10, 20, 30, 40, 50ppb. Dissolve melamine in chloroform to prepare a melamine standard solution with a concentration gradient of 0, 1.7, 3.3, 6.7, 10.0, 13.3, 16.7ppb; (2) respectively take the above 80 μL standard solution and 200 μL gold nanosol and add it to the container unit made of quartz , and assembled into a three-dimensional liquid plasma array, that is, the sample to be tested; these two food hazards are not only the test substance, but also have a positive charge, which is opposite to that of the citrate-stabilized gold nanoparticles. Therefore, the test object itself can be used as an accelerator, that is, to promote the assembly of the three-dimensional liquid test sample by chemical means; (3) splicing the above-mentioned multiple container units to form a test sample group, after laser calibration once, use a Raman meter Test the group of samples to be tested. As shown in Figures 4A and 4B are the Raman spectra detected by this method for standard solutions of fumetidine and melamine with different concentrations. As the concentration of the analyte increases, the corresponding Raman intensity also increases, and the minimum detection limit reached 0.1ppb and 1.7ppb, respectively. Figures 4C and 4D are the fitted linear curves between the concentration and the relative Raman intensities of the standard solutions of Fumei and melamine, respectively, and the correlation coefficients R ² are 0.92 and 0.98, respectively, where 1377 cm ^-1 of Fumei and 710 cm ^-1 of melamine are selected The Raman peak at 665 cm -1 was used as the quantitative basis, and the characteristic peak of chloroform at 665 cm ^-1 was used as the internal standard. In order to test the reproducibility of SERS, 30 Raman spectra of the 50ppb dual standard solution and 55 Raman spectra of the melamine standard solution with a concentration of 16.7ppb were collected. As shown in Figures 4E and 4F, the relative standard deviations of the characteristic peak intensities at 1377 cm ^-1 and 710 cm ^-1 were 9.0% and 8.0%. The above results demonstrate that this measurement technique has good SERS quantification and high-throughput analysis capabilities for food contaminants.

Example 3

In this embodiment, the Raman parameters are set as: excitation wavelength of 785 nm, laser power of 3%, integration time of 8 s, and accumulation times of 1 time.

Non-extractive detection of pesticide residues on peels. The specific process is as follows: (1) Take a peel with a thickness of 0.3mm with a knife; (2) Take a uniform round peel with a diameter of 4mm with a punch; (3) Add drops of 0, 10, 50, 100, 200, 300, and 400ppb of 50 μL of each 50 μL of Fumei Shuang solution, dripped on orange peel with 50 μL of each of 0, 100, 200, 300, 400, 500 ppb Fumei Shuang solution, and dried at room temperature; (4) Directly The peels containing different concentrations of fomeshuang are added into the container unit made of quartz material to assemble a three-dimensional liquid sample to be tested; (5) the above-mentioned multiple container units are spliced to form a sample group to be tested. After laser calibration once, use Raman The instrument detects the sample group to be tested. This process combines the steps of extraction and assembly, which greatly saves experimental time. As shown in Figure 5, picture A is the detection of Fumeishuang on apple peels, and picture B is the detection of Fumeishuang on orange peels. The SERS intensity gradually increased with the increase of the concentration of fometaxel in the peel. The lowest concentrations of fometaxel detected in apples and oranges were 10 ppb and 100 ppb, respectively. It should be noted that the Raman signal intensities of different peels under the same pesticide dosage are different, indicating that the Raman signals of different contents in the peel and the interaction strength between the pesticide and the peel are different. The results show that the method has strong robustness to the analysis of actual complex samples.

The technical features of the above-described embodiments can be combined arbitrarily. To simplify the description, all possible combinations of the technical features of the above-described embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims (10)

1. a fast high-throughput liquid interface enhanced Raman spectroscopy detection method, is characterized in that: comprise the following steps: a. Mix the precious metal nanosol, the oil phase solvent and the analyte to obtain a mixed sample; b. The mixed sample is placed in a container unit that can be spliced and can be irradiated by multi-faceted lasers, and the precious metal nanoparticles in the mixed sample reach the liquid-liquid interface and assemble to form a three-dimensional interface plasma array, that is, a three-dimensional liquid sample to be tested is obtained; c. Multiple three-dimensional liquid samples to be tested are spliced together between container units to form a sample group to be tested. After laser calibration once, the sample group to be tested is detected with a Raman instrument, that is, fast high-throughput SERS detection is realized. 2. detection method according to claim 1 is characterized in that: in step a, described oil phase solvent is chloroform, 1,2-dichloroethane, methylene chloride, hexane, cyclohexane, toluene, At least one of xylene or pentane. 3. The detection method according to claim 1, characterized in that: in step b, the container unit that can be spliced and can be irradiated with multi-faceted laser light is a microporous container, and the volume of the microporous container is 10-1000 μL; its material is Glass, plexiglass, quartz, polyethylene or polypropylene. 4 . The detection method according to claim 3 , wherein the container unit that can be spliced and can be irradiated with multi-faceted laser can receive laser irradiation in a vertical direction or a horizontal direction. 5 . 5 . The detection method according to claim 1 , wherein in step b, the type of the three-dimensional interface plasma array is an oil-in-water type or a water-in-oil type. 6 . 6 . The detection method according to claim 5 , wherein the assembly of noble metal nanoparticles can be promoted by physical means or chemical means to form a three-dimensional liquid test sample. 7 . 7 . The detection method according to claim 6 , wherein the physical method is ultrasound, vibration or a combination of ultrasound and vibration. 8 . 8. detection method according to claim 6 is characterized in that: described chemical method is to add modifier, accelerator or acid solution in assembly system; Described modifier is 1-pentanethiol, pyridine, 1, 10-phenanthroline, 1-heptanethiol, dodecanethiol, benzenethiol, tetra-n-octylammonium bromide, 5,10,15,20-tetrakis(1,-methyl-4-pyridyl) One of -21H, 23H-porphine; the accelerator is a hydrophobic cation/anion salt, which contains a hydrophobic ion carrying an opposite charge to the nanoparticle, including transition metal complexes, crown ethers and organic salts. 9. detection method according to claim 1, is characterized in that: in step c, the method to be tested sample group is detected with Raman instrument is: keep laser light source still, detect by moving to be tested sample group; Or keep the sample group to be tested still, and perform detection by moving the laser light source. 10. The detection method according to claim 9 is characterized in that: a microprocessor is equipped on the Raman instrument, and the mechanical drive mechanism of the microprocessor is controlled to move along the X direction and the Y direction automatically to move the test to be measured. Sample group or laser light source.

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