A SERS quantitative working curve method based on intensity ratio

Abstract

The embodiment of the present invention discloses a SERS quantitative working curve method based on intensity ratio, comprising: step 1, obtaining a quantitative standard curve. Prepare a first batch of SERS substrates, detect the SERS spectra of target molecules of different concentrations on the first batch of SERS substrates, and select the SERS intensity of any concentration as a reference to obtain the ratio between the characteristic peak intensity of the target molecules of different concentrations and the reference intensity, and fit the corresponding relationship between the ratio and the logarithm of the target molecule concentration as a quantitative standard curve; step 2, quantitative detection of molecular concentration. Prepare a second batch of SERS substrates, detect the SERS spectra of the target molecules of unknown concentration, and detect the SERS spectra of the target molecules of known concentration as an intensity reference, and determine the concentration value of the target molecules of unknown concentration according to the ratio between the characteristic peak intensities and the corresponding relationship obtained in step 1.

Description

Technical Field

[0001] The invention relates to the technical field of surface enhanced Raman spectroscopy (SERS) detection and quantitative analysis, in particular to a SERS quantitative working curve method based on intensity ratio. Background Art

[0002] Surface enhanced Raman scattering is an efficient molecular spectroscopy detection technology. By observing the molecular vibration, the chemical composition of the molecular system can be obtained. Therefore, Raman spectroscopy is also called "molecular fingerprint spectroscopy". With the development of nanotechnology, the improvement of Raman instruments, and the further optimization of SERS active particles, it is no longer a problem to use SERS spectroscopy to identify and detect the substances to be tested. Therefore, people have begun to apply SERS technology to the quantitative detection of substances. The linear relationship between SERS intensity and the concentration of the molecule to be tested is the key to achieve SERS quantitative detection. However, SERS measurement is susceptible to laser power fluctuations, focusing, optical alignment drift, oxidation and contamination. These external factors make the reproducibility of the measured SERS intensity poor between batches. On the other hand, the random binding of molecules to "hot spots" and the thermal diffusion of molecules inside or outside the "hot spots" can also bring uncertainty. These intrinsic factors can also cause fluctuations in SERS intensity, even one to two orders of magnitude. Therefore, even if the experimental parameters in different batches remain unchanged, it is difficult to obtain a robust SERS standard curve.

[0003] In order to improve the accuracy and reliability of SERS in quantitative detection, a variety of methods have been reported to solve the above problems, such as the internal standard method and the external standard method. The external standard method usually uses silicon peaks, standard solutions, etc. as reference signals to eliminate the influence of external conditions such as instruments and environments. However, this method cannot eliminate the influence of substrate heterogeneity on SERS signals. For the internal standard method that adds a certain amount of additional samples as reference signals, it can remove the signal fluctuations caused by substrate heterogeneity to a certain extent. However, the internal standard method still has the following disadvantages, such as: the SERS intensity of the internal standard substance is unstable, it interferes with the adsorption of analyte molecules, and it is insufficient to enhance ultrasensitive detection.

[0004] Therefore, it is urgent to develop a new SERS quantitative detection method. Summary of the invention

[0005] The purpose of the embodiments of the present invention is to provide a SERS quantitative working curve method based on intensity ratio to solve at least one of the above technical problems.

[0006] To achieve the above object, an embodiment of the present invention provides a SERS quantitative working curve method based on intensity ratio, comprising:

[0007] Step 1, obtaining a quantitative standard curve. Prepare the first batch of SERS substrates, detect the SERS spectra of target molecules of different concentrations on the first batch of SERS substrates, and select the SERS intensity of any concentration as a reference to obtain the ratio between the characteristic peak intensity of the target molecules of different concentrations and the reference intensity, and fit the corresponding relationship between the ratio and the logarithm of the target molecule concentration as a quantitative standard curve;

[0008] Step 2, quantitative detection of molecular concentration. Prepare a second batch of SERS substrates, detect the SERS spectra of the target molecules of unknown concentration on the second batch of SERS substrates, and detect the SERS spectra of the target molecules of known concentration on the second batch of SERS substrates as an intensity reference, calculate the ratio between the characteristic peak intensity of the target molecules of unknown concentration and the reference intensity, and determine the concentration value of the target molecules of unknown concentration based on the ratio between the characteristic peak intensities and the corresponding relationship obtained in step 1.

[0009] Preferably, the ratio between the characteristic peak intensity of the target molecule at different concentrations and the reference intensity obtained in step 1 comprises:

[0010] Select any one of the concentrations as the standard concentration, and use the characteristic peak intensity of the target molecule at the standard concentration as the standard to calculate the ratio between the characteristic peak intensity corresponding to other concentrations and the characteristic peak intensity corresponding to the standard concentration;

[0011] The known concentration in step 2 at least includes the standard concentration.

[0012] Preferably, the standard concentration is selected in the following manner:

[0013] preparing a third batch of SERS substrates, detecting the SERS spectra of the target molecules of different concentrations on the third batch of SERS substrates again, obtaining a second ratio based on the intensity of the characteristic peak of the target molecules at one concentration, and fitting a second corresponding relationship between the second ratio and the logarithm of the target molecule concentration;

[0014] The corresponding relationship is compared with the second corresponding relationship, and if the two are consistent, one of the concentrations is determined to be the standard concentration.

[0015] Preferably, the corresponding relationship between the ratio fitted in step 1 and the logarithm of the target molecule concentration is a linear relationship.

[0016] Preferably, a SERS substrate is prepared using a microfluidic chip and SERS detection is performed; wherein the microfluidic chip includes four injection holes: the first injection hole and the second injection hole form a Y-shaped structure, the third injection hole and the fourth injection hole form a Y-shaped structure, and the first injection hole and the second injection hole as a whole and the third injection hole and the fourth injection hole as a whole form a Y-shaped structure; the microfluidic chip also includes a mixing area for mixing different solutions, an illumination area for laser irradiation, and a drainage hole.

[0017] Preferably, preparing a SERS substrate using a microfluidic chip comprises:

[0018] Use a micro-syringe to connect the interface of the microfluidic chip injection hole;

[0019] Controlling the first and second syringes to introduce silver nitrate and sodium citrate, and using a laser to irradiate the mixed reagents to form silver nanoparticle aggregates;

[0020] After controlling the third syringe to pass deionized water to rinse the silver nitrate and sodium citrate, laser irradiation is performed again to further form silver nanoparticles on the silver nanoparticle aggregates using residual reagents;

[0021] The silver nanoparticle aggregate is used as the SERS substrate.

[0022] Preferably, performing SERS detection using a microfluidic chip includes:

[0023] The target molecule is injected through a fourth syringe, and the SERS spectrum of the target molecule is detected using the silver nanoparticle aggregate as the SERS substrate.

[0024] Compared with the prior art, the present invention has at least the following advantages:

[0025] By using the SERS intensity of the test substance in the same batch as a reference signal to calibrate the SERS intensity of different concentrations, a robust SERS working curve can be obtained, thereby achieving accurate prediction of the concentration of the test substance in terms of order of magnitude. The selected reference intensity also reflects the fluctuations of conditions such as the SERS substrate heterogeneity, molecular adsorption, and laser focusing of the measuring instrument.

[0026] Moreover, the present invention avoids the introduction of other substances as reference signals, and further avoids the competitive adsorption of the reference substance and the substance to be tested on the "hot spot", so it can realize the quantitative analysis of substances such as R6G at the single molecule level, and has an extremely wide concentration monitoring range (10 -7 mol / L~10 -13mol / L). The present invention is universal and applicable to all liquid substances with Raman signals. In addition, the experiment also has the advantages of high integration, semi-automation, fast response, and sample saving. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic diagram of the process of the SERS quantitative detection method provided by an embodiment of the present invention.

[0028] Figure 2 It is a schematic diagram of the process of the SERS quantitative detection method provided by an example of the present invention.

[0029] Figure 3 It is a schematic diagram of the structure of the microfluidic chip provided in an embodiment of the present invention.

[0030] Figure 4 A and B are the SERS intensity and concentration logarithm of R6G obtained in two batches of experiments provided in the present embodiment (10 -7 mol / L~10 -10 mol / L), where (I) is the SERS intensity before calibration, and (II) is the SERS intensity after calibration.

[0031] Figure 5 A and B are the SERS intensity and concentration logarithm of R6G obtained in two batches of experiments provided in the present embodiment (10 -10 mol / L~10 -13 mol / L), where (I) is the SERS intensity before calibration, and (II) is the SERS intensity after calibration.

[0032] Figure 6 It is a schematic diagram of the prediction result of R6G concentration according to the method provided in an embodiment of the present invention.

[0033] Figure 7 A is a schematic diagram of the Raman scattering spectra of thiram of different concentrations detected by the method provided in an embodiment of the present invention.

[0034] Figure 7 B is a schematic diagram of the prediction results of thiram concentration according to the method provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0035] In the drawings, the same or similar reference numerals are used to represent the same or similar elements or elements with the same or similar functions. The embodiments of the present invention are described in detail below in conjunction with the drawings.

[0036] In the description of the present invention, the terms "center", "longitudinal", "lateral", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate directions or positional relationships based on the directions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore should not be understood as limiting the scope of protection of the present invention.

[0037] In the absence of conflict, the technical features in the various embodiments and implementations of the present invention may be combined with each other and are not limited to the embodiments or implementations to which the technical features are applied.

[0038] Surface-enhanced Raman Scattering (SERS) technology has been proven to be a very effective analytical tool due to its high sensitivity, high selectivity and fluorescence quenching characteristics. Since the advent of SERS more than 50 years ago, nano-processing technology, laser manufacturing technology and corresponding equipment theory have developed rapidly and improved. It can be said that using SERS to achieve ultra-low concentration analyte recognition and detection in complex environments is no longer a problem. Most of the research and inventions on SERS are also mainly focused on the manufacture and discovery of new SERS substrates with high sensitivity, high stability or screening functions. However, there is a problem with many SERS substrates, that is, the molecules located at the hot spot position are less than 1%, but they contribute about 70% of the SERS intensity. This huge difference in SERS contribution inevitably leads to poor quantitative ability. At present, SERS technology is mostly used for qualitative and semi-quantitative analysis of samples in practice. It is still a major difficulty to use SERS technology to quantitatively analyze trace targets in actual samples.

[0039] In the embodiments of the present invention, the SERS intensity of the same substance in the same batch is used as the intensity calibration reference, which effectively eliminates the influence of substrate heterogeneity and instrument fluctuation. This method of using the SERS intensity of the substance to be tested as a reference completely avoids the introduction of other substances, effectively prevents the competitive adsorption of other substances with the substance to be tested, and realizes rapid, wide-range, and order-of-magnitude quantitative analysis of the analyte.

[0040] The embodiment of the present invention provides a SERS quantitative working curve method based on intensity ratio, such as Figure 1 As shown, the method includes:

[0041] Step 1, obtaining a quantitative standard curve. Prepare the first batch of SERS substrates, detect the SERS spectra of target molecules of different concentrations on the first batch of SERS substrates, and select the SERS intensity of any concentration as a reference to obtain the ratio between the characteristic peak intensity of the target molecules of different concentrations and the reference intensity, and fit the corresponding relationship between the ratio and the logarithm of the target molecule concentration as a quantitative standard curve;

[0042] Step 2, quantitative detection of molecular concentration. Prepare a second batch of SERS substrates, detect the SERS spectra of the target molecules of unknown concentration on the second batch of SERS substrates, and detect the SERS spectra of the target molecules of known concentration on the second batch of SERS substrates as an intensity reference, calculate the ratio between the characteristic peak intensity of the target molecules of unknown concentration and the reference intensity, and determine the concentration value of the target molecules of unknown concentration based on the ratio between the characteristic peak intensities and the corresponding relationship obtained in step 1.

[0043] Wherein, the ratio between the characteristic peak intensity of the target molecule at different concentrations and the reference intensity obtained in step 1 includes:

[0044] Select any one of the concentrations as the standard concentration, and use the characteristic peak intensity of the target molecule at the standard concentration as the standard to calculate the ratio between the characteristic peak intensity corresponding to other concentrations and the characteristic peak intensity corresponding to the standard concentration;

[0045] The known concentration in step 2 at least includes the standard concentration.

[0046] Optionally, a concentration is randomly selected as the standard concentration.

[0047] Alternatively, you can select the standard concentration by:

[0048] preparing a third batch of SERS substrates, detecting the SERS spectra of the target molecules of different concentrations on the third batch of SERS substrates again, obtaining a second ratio based on the intensity of the characteristic peak of the target molecules at one concentration, and fitting a second corresponding relationship between the second ratio and the logarithm of the target molecule concentration;

[0049] The corresponding relationship is compared with the second corresponding relationship, and if the two are consistent, one of the concentrations is determined to be the standard concentration.

[0050] Optionally, the corresponding relationship between the ratio fitted in step 1 and the logarithm of the target molecule concentration is a linear relationship.

[0051] Optionally, a SERS substrate is prepared using a microfluidic chip and SERS detection is performed; wherein the microfluidic chip includes four injection holes: the first injection hole and the second injection hole form a Y-shaped structure, the third injection hole and the fourth injection hole form a Y-shaped structure, and the first injection hole and the second injection hole as a whole and the third injection hole and the fourth injection hole as a whole form a Y-shaped structure; the microfluidic chip also includes a mixing area for mixing different solutions, an illumination area for laser irradiation, and a drainage hole.

[0052] The process of preparing a SERS substrate using a microfluidic chip may include:

[0053] Use a micro-syringe to connect the interface of the microfluidic chip injection hole;

[0054] Controlling the first and second syringes to introduce silver nitrate and sodium citrate, and using a laser to irradiate the mixed reagents to form silver nanoparticle aggregates;

[0055] After controlling the third syringe to pass deionized water to rinse the silver nitrate and sodium citrate, laser irradiation is performed again to further form silver nanoparticles on the silver nanoparticle aggregates using residual reagents;

[0056] The silver nanoparticle aggregate is used as the SERS substrate.

[0057] SERS detection using microfluidic chips can include:

[0058] The target molecule is injected through a fourth syringe, and the SERS spectrum of the target molecule is detected using the silver nanoparticle aggregate as the SERS substrate.

[0059] It should be understood that the specific parameters in this example are preferred results, but not the only results. Without violating the technical ideas of the present invention, those skilled in the art can adjust the parameters in the following examples to obtain various examples, which should all fall within the scope of protection of the present invention.

[0060] The following takes the target molecule as R6G molecule and the SERS substrate prepared by using a microfluidic chip as an example to introduce the SERS quantitative working curve method based on intensity ratio provided by the embodiment of the present invention. Figure 2 As shown, the method includes:

[0061] Step 21, using silver nanoparticle aggregates prepared in the channel of the microfluidic chip by double laser induced reduction technology as a SERS substrate.

[0062] Figure 3The schematic diagram of the structure of the microfluidic chip is shown, which includes four injection holes 21: the first injection hole 1 and the second injection hole 2 form a Y-shaped structure, the third injection hole 3 and the fourth injection hole 4 form a Y-shaped structure, and the first injection hole and the second injection hole as a whole and the third injection hole and the fourth injection hole as a whole form a Y-shaped structure. The microfluidic chip also includes a mixing area 22 for mixing different solutions, an illumination area 23 for laser irradiation, and a drainage hole 24.

[0063] Take four clean micro-syringes and inhale the corresponding solutions respectively, and fix them on the syringe pump. Take the customized microfluidic chip and fix it on the sample stage of the micro-Raman spectrometer; take the centrifuge tube and place it in the waste liquid recovery area to collect the waste liquid; connect the needle of the micro-syringe to the interface of the four injection holes of the microfluidic chip respectively; use wires to connect the control panel to the four syringe pumps respectively; connect the control panel to the power supply; control syringes 2 and 3, and simultaneously pass silver nitrate and sodium citrate at a flow rate of 400nL / s to make them fully mixed in the microfluidic chip, and use a laser with a wavelength of 532nm and a power of 0.17mW to irradiate the mixed reagents. After 90s, a silver nanoparticle aggregate will be initially formed; control syringe 1, pass deionized water at a flow rate of 400nL / s to rinse off the silver nitrate and sodium citrate in the channel, and then use a laser with a wavelength of 532nm and a power of 1.2mW to irradiate the silver nanoparticle aggregate again, and use the residual reagents to form finer silver nanoparticles.

[0064] It is easy to understand that a SERS substrate can be obtained by irradiating light only once, and a second irradiation can make the silver nanoparticles on the surface of the SERS substrate more uniform and delicate.

[0065] Step 22, detecting the SERS spectrum of the R6G aqueous solution on the SERS substrate, and fitting the relationship between the ratio of the characteristic peak intensities of R6G at different concentrations and the logarithm of the concentration.

[0066] The R6G aqueous solution (10 -7 mol / L~10 -10 mol / L) and plotted the SERS spectrum of R6G at 612 cm -1 The SERS intensity (I SERS ) and the logarithm of the concentration. Repeated experiments found that the slope and intercept of the linear relationship between the two batches were very different. It is difficult to achieve quantitative detection of R6G using the linear relationship of absolute intensity as the standard working curve.

[0067] Choose R6G in 10 -10 mol / L SERS intensity (I -10 ) was used as a reference to calibrate the above intensity (I SERS / I -10 , IR ), that is, the concentration of R6G is 10 -10 mol / L is the standard concentration, calculate the SERS intensity of R6G at other concentrations and SERS intensity (I -10 ), i.e., the relative intensity I R . Plot the relative intensity I R The relationship between the calibration curves of the two batches is shown in Table 1. The slope and intercept of the calibration curves of the two batches are consistent with each other. The specific process includes:

[0068] Control syringe 4 to adjust the concentration to 10 -7 mol / L、10 -8 mol / L、10 -9 mol / L、10 -10 mol / L R6G aqueous solution was passed into the microfluidic chip at a flow rate of 400 nL / s. After it was fully adsorbed, SERS signal acquisition was performed. For each concentration, multiple measurements were performed to read the R6G molecule at 612 cm -1 The absolute SERS intensity (I SERS ), with the logarithm of R6G concentration as the horizontal axis, I SERS The linear function relationship is obtained by fitting the ordinate; repeat the experiment to obtain the linear function relationship of the absolute intensity of the second batch. Due to the heterogeneity of the SERS substrate and the difference in experimental conditions, the linear relationships obtained from different batches are not consistent. Select 10 -10 mol / L is the standard concentration, R6G is at 10 -10 mol / L SERS intensity (I -10 ) was used as a reference to calibrate the above intensity (I SERS / I -10 , I R ), plot the relative intensity I R The results showed that the slope and intercept of the calibration curves of the two batches showed good consistency.

[0069] Therefore, the SERS intensity was calibrated by selecting the standard concentration (I SERS / I -10, I R ), which can effectively eliminate the influence of SERS substrate heterogeneity or experimental conditions, thereby obtaining a reproducible SERS calibration curve, which provides a reliable guarantee for quantitative analysis.

[0070] Step 23, measuring the SERS spectrum of R6G at a lower concentration, and fitting the relationship between the ratio of the characteristic peak intensities of R6G at different concentrations and the logarithm of the concentration.

[0071] Continue to measure lower concentrations of R6G (10 -10mol / L~10 -13 mol / L) and plotted the SERS spectrum of R6G at 612 cm -1 The SERS intensity (I SERS ) and the logarithm of concentration. Repeated experiments revealed that the slope and intercept of the linear relationship between the two batches also had a large difference.

[0072] Choose R6G in 10 -10 mol / L SERS intensity (I -10 ) was used as a reference to calibrate the above intensity (I SERS / I -10 , I R ). Plot the relative intensity I R The relationship between the calibration curves of the two batches is shown in Table 1. The slope and intercept of the calibration curves of the two batches are consistent with each other. The specific process includes:

[0073] Control syringe 4 to adjust the concentration to 10 -10 mol / L、10 -11 mol / L、10 -12 mol / L、10 -13 mol / L R6G aqueous solution was passed into the microfluidic chip at a flow rate of 400 nL / s. After it was fully adsorbed, SERS signal acquisition was performed. For each concentration, multiple measurements were performed to read the R6G molecule at 612 cm -1 The absolute SERS intensity (I SERS ), with the logarithm of R6G concentration as the horizontal axis, I SERS The linear function relationship is obtained by fitting the ordinate; the experiment is repeated to obtain the linear function relationship of the absolute intensity of the second batch. Due to the heterogeneity of the SERS substrate and the difference in experimental conditions, the linear relationships obtained from different batches are not consistent. -10 mol / L SERS intensity (I -10 ) was used as a reference to calibrate the above intensity (I SERS / I -10 , I R ). Plot the relative intensity I R The results showed that the slope and intercept of the calibration curves of the two batches showed good consistency.

[0074] Preferably, due to the ultra-sensitive characteristics of the SERS substrate, R6G can be detected at ultra-low concentrations (10 -10 mol / L~10 -13 mol / L) SERS detection, thus expanding the detection range of concentration.

[0075] Step 24, using the calibration curve of the first batch as the standard working curve, and using the calibration intensity of the second batch to reversely predict the concentration of R6G, the results show that quantitative analysis of the concentration of R6G can be achieved in terms of order of magnitude.

[0076] For concentration 10 -7 mol / L~10 -10 mol / L, the linear relationship of the first batch is Y=7+0.6lgC R6G , the calibration intensity obtained from the second batch is substituted into the above linear function, the measured concentration is calculated and compared with the true concentration. -10 mol / L~10 -13 mol / L, the linear relationship of the first batch is Y=4.39+0.34lgC R6G， The calibration intensity obtained from the second batch was substituted into the above linear function, and the measured concentration was calculated and compared with the true concentration.

[0077] Preferably, the linear relationship described in step 4 is used as a quantitative curve, and accurate prediction of the order of magnitude of R6G can be achieved through calibration of the SERS intensity.

[0078] Step 25, detecting the SERS spectrum of the thiram aqueous solution on the SERS substrate, and fitting the relative intensity I R The relationship between the concentration and the logarithm of the concentration is obtained, and the concentration of the thiram aqueous solution is reversely deduced using this relationship.

[0079] Detection of Thiram aqueous solution (10 -3 mol / L~10 -7 mol / L) SERS spectrum, and selected Thiram at 10 -3 mol / L SERS intensity (I -3 ) was used as the intensity reference to calibrate the above intensity (I SERS / I -3 , I R ). Plot the relative intensity I R The relationship between the concentration and the logarithm. The experiment was repeated to obtain two batches of experimental data. The calibration curve of the first batch was used as the standard working curve, and the calibration intensity of the second batch was used to reversely predict the concentration of thiram. The results showed that the quantitative analysis of the concentration of thiram in terms of order of magnitude could be achieved.

[0080] Preferably, in 10 -3 mol / L~10 -7 mol / L concentration range, the I R The logarithm of the concentration of thiram has good repeatability and can achieve accurate prediction of thiram in terms of order of magnitude.

[0081] In order to make the SERS quantitative detection method based on the microfluidic detection chip provided by the present invention more clear, another specific example is described in detail below. The example is introduced through the following aspects: 1. Sample weighing; 2. Instruments and equipment; 3. Preparation of SERS substrate; 4. Detection and analysis of R6G; 5. Detection and analysis of thiram.

[0082] 1. Sample weighing:

[0083] (1) Use a balance to weigh 0.01 mol (18.5 mg is optimal) of silver nitrate and 0.008 mol (22.5 mg is optimal) of sodium citrate, and dissolve each of them in 10 mL of deionized water (deionized water with a resistivity of 18.5 MΩ·cm).

[0084] (2) Weigh 4.79 mg of R6G powder and dissolve it in 10 mL of deionized water to obtain 10 -3 mol / L R6G aqueous solution, and take 1mL and add 9mL of deionized water to dilute tenfold to obtain a concentration of 10 -4 mol / L R6G aqueous solution. Repeat this step to obtain a concentration of 10 -7 mol / L~10 -13 mol / L R6G aqueous solution.

[0085] (3) Weigh 24 mg of thiram powder and dissolve it in 10 mL of deionized water to obtain 10 -2 mol / L thiram aqueous solution, and take 1mL and add 9mL of deionized water to dilute tenfold to obtain a concentration of 10 -3 mol / L aqueous solution of thiram. Repeat this step to obtain a concentration of 10 -3 mol / L~10 -7 mol / L aqueous solution of thiram.

[0086] 2. Instruments and equipment:

[0087] Customized microfluidic chip, 500 μL microinjector, syringe pump (TS-2A / L0107-2A), semi-micro balance (such as METTLER TOLEDO semi-micro balance), Raman spectrometer (such as Renishaw micro Raman spectrometer RH13325 (R-2000)), and a laser with a wavelength of 532 nm.

[0088] 3. Preparation of SERS substrate: including:

[0089] 1) Take four clean micro-syringes and inhale the corresponding solutions respectively, and fix them on the syringe pump; take the customized microfluidic chip and fix it on the sample stage of the micro-Raman spectrometer; take the centrifuge tube and place it in the waste liquid recovery area to collect the waste liquid;

[0090] 2) Connect the needles of the micro-injectors to the interfaces of the four injection holes of the microfluidic chip respectively; connect the control panel to the four injection pumps respectively using wires; connect the control panel to the power supply; control syringes 2 and 3 to simultaneously introduce silver nitrate and sodium citrate at a flow rate of 400 nL / s to fully mix them in the microfluidic chip, and irradiate the mixed reagents with a laser with a wavelength of 532 nm and a power of 0.17 mW, and a silver aggregate will be initially formed after 90 seconds;

[0091] 3) Control the syringe 1 to pass deionized water at a flow rate of 400 nL / s to flush out the silver nitrate and sodium citrate in the channel, and then use a laser with a wavelength of 532 nm and a power of 1.2 mW to irradiate the silver aggregates again, and use the residual reagents to form finer silver nanoparticles.

[0092] 4. Detection and analysis of R6G:

[0093] 1) Control the syringe 4 to adjust the concentration to 10 -7 mol / L~10 -10 mol / L and 10 -10 mol / L~10 -13 mol / L R6G aqueous solution was passed into the microfluidic chip at a flow rate of 400nL / s. After it was fully adsorbed, a laser with a wavelength of 532nm and a power of 0.17mW was used to collect SERS signals. The experiment was repeated to obtain two batches of experimental data.

[0094] 2) For 10 -7 mol / L~10 -10 mol / L, statistical R6G molecules at 612cm -1 The SERS intensity of the two batches was calculated and the linear relationship between the SERS intensity and the logarithm of the concentration was fitted, as shown in Figure 4 As shown in Figure (I) of A and B, the horizontal axis is lg[C R6G (M)], which is the logarithm of the concentration of R6G molecules, and the ordinate is the absolute intensity of the SERS intensity. Due to the heterogeneity of the SERS substrate and the differences in experimental conditions, the slope and intercept of the linear relationship between different batches are significantly different. -10 The linear relationship between the two batches was calibrated by the SERS intensity of mol / L. Figure 4Figure (II) in A and B is the result of calibration of two batches, the horizontal axis remains unchanged, and the vertical axis is the relative intensity after calibration. It can be seen that the similarity of its slope and intercept is greatly improved, which provides a basis for the quantitative detection of R6G.

[0095] 3) For 10 -10 mol / L~10 -13 mol / L, statistical R6G molecules at 612cm -1 The SERS intensity of the two batches was calculated and the linear relationship between the SERS intensity and the logarithm of the concentration was fitted, as shown in Figure 5 As shown in Figure (I) of A and B, the horizontal axis is lg[C R6G (M)], which is the logarithm of the R6G molecular concentration, and the ordinate is the absolute intensity of the SERS intensity. The slope and intercept of the linear relationship between different batches are still significantly different. -10 The linear relationship between the two batches was calibrated by the SERS intensity of mol / L. Figure 5 Figure (II) in A and B is the result after calibration of the two batches. It can be seen that the similarity of their slopes and intercepts is greatly improved.

[0096] 4) Figure 4 The calibration curves in Figures A and (II) in 5A are standard quantitative curves. Figure 4 The calibration intensity of Figure (II) in Figure 5B was used as the observed value to predict the concentration of R6G. The results are shown in Figure 6 As shown, the horizontal axis is the actual concentration and the vertical axis is the predicted concentration. In order to facilitate the observation of the relationship between the two, the oblique line segment of the function "y=x" is drawn. The closer the data point is to the line segment, the closer the predicted concentration is to the actual concentration. It can be seen that this method can achieve accurate prediction of the concentration of R6G in terms of order of magnitude.

[0097] 5. Detection and analysis of Thiram

[0098] Detection of Thiram aqueous solution (10 -3 mol / L~10 -7 mol / L) SERS spectrum, and selected Thiram at 10 -3 mol / L SERS intensity (I -3 ) was used as a reference to calibrate the above intensity (I SERS / I -3 , I R ). Plot the relative intensity I R The relationship between the concentration and the logarithm. Repeat the experiment to obtain two batches of experimental data. The calibration curve of the first batch was used as the standard working curve, and the calibration intensity of the second batch was used to reversely predict the concentration of thiram. The results showed that the quantitative analysis of the concentration of thiram in terms of magnitude can be achieved. Including:

[0099] Control syringe 4 to adjust the concentration to 10 -3 mol / L、10 -4 mol / L、10 -5 mol / L、10 -6 mol / L、10 -7 mol / L thiram aqueous solution was passed into the microfluidic chip at a flow rate of 400 nL / s. After it was fully adsorbed, SERS signal acquisition was performed. For each concentration, multiple measurements were performed to read the thiram 1387 cm -1 The absolute SERS intensity (I SERS ), choose Thiram in 10 -3 mol / L SERS intensity (I -3 ) was used as the intensity reference to calibrate the above intensity (I SERS / I -3 , I R ). The relative intensity I R Repeat the experiment to obtain the calibration intensity of the second batch, bring it into the working curve of the first batch to predict the concentration of thiram, and draw a data image between the predicted concentration and the actual concentration. Figure 7 A is a schematic diagram of the Raman scattering spectra of thiram of different concentrations detected by the method provided in an embodiment of the present invention. Figure 7 B is the result of predicting the concentration of thiram according to the method provided in an embodiment of the present invention. It can be seen that the method can achieve accurate prediction of the concentration of thiram in terms of order of magnitude.

[0100] In an embodiment of the present invention, a robust SERS working curve can be obtained by calibrating the SERS intensities of different concentrations using the SERS intensity of the test substance in the same batch as a reference, thereby achieving an accurate prediction of the concentration of the test substance in terms of order of magnitude. The selected reference intensity also reflects the fluctuations of conditions such as the SERS substrate heterogeneity, molecular adsorption, and laser focusing of the measuring instrument.

[0101] Moreover, the present invention avoids the introduction of other substances as reference signals, and further avoids the competitive adsorption of the reference substance and the substance to be tested on the "hot spot", so it can realize the quantitative analysis of substances such as R6G at the single molecule level, and has an extremely wide concentration monitoring range (10 -7 mol / L~10 -13 mol / L). The present invention is universal and applicable to all liquid substances with Raman signals. In addition, the experiment also has the advantages of high integration, semi-automation, fast response, and sample saving.

[0102] Finally, it should be pointed out that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some of the technical features thereof may be replaced by equivalents; these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A SERS quantitative working curve method based on intensity ratio, It is characterized in that include: Step 1, obtaining a quantitative standard curve; comprising: preparing a first batch of SERS substrates, detecting SERS spectra of target molecules of different concentrations on the first batch of SERS substrates, and selecting the SERS intensity of any concentration as a reference, obtaining the ratio between the characteristic peak intensity of the target molecules of different concentrations and the reference intensity, and fitting the corresponding relationship between the ratio and the logarithm of the target molecule concentration as a quantitative standard curve; Step 2, performing quantitative detection of molecular concentration; including: preparing a second batch of SERS substrates, detecting the SERS spectra of the target molecules of unknown concentration on the second batch of SERS substrates, and detecting the SERS spectra of the target molecules of known concentration on the second batch of SERS substrates as an intensity reference, calculating the ratio between the characteristic peak intensity of the target molecules of unknown concentration and the reference intensity, and determining the concentration value of the target molecules of unknown concentration based on the ratio between the characteristic peak intensities and the corresponding relationship obtained in step 1.

2. The method according to claim 1, It is characterized in that The ratio between the characteristic peak intensity of the target molecule at different concentrations and the reference intensity obtained in step 1 includes: Select any one of the concentrations as the standard concentration, and use the characteristic peak intensity of the target molecule at the standard concentration as the standard to calculate the ratio between the characteristic peak intensity corresponding to other concentrations and the characteristic peak intensity corresponding to the standard concentration; The known concentration in step 2 at least includes the standard concentration.

3. The method according to claim 2, It is characterized in that The standard concentrations were selected as follows: preparing a third batch of SERS substrates, detecting the SERS spectra of the target molecules of different concentrations on the third batch of SERS substrates again, obtaining a second ratio based on the intensity of the characteristic peak of the target molecules at one concentration, and fitting a second corresponding relationship between the second ratio and the logarithm of the target molecule concentration; The corresponding relationship is compared with the second corresponding relationship, and if the two are consistent, one of the concentrations is determined to be the standard concentration.

4. The method according to claim 1, It is characterized in that The corresponding relationship between the ratio fitted in step 1 and the logarithm of the concentration of the target molecule is a linear relationship.

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