Method for rapidly detecting liquid phase diffusion coefficient of analyte through surface enhanced Raman spectroscopy based on microchannel and application

Abstract

The invention discloses a method for rapidly detecting a liquid phase diffusion coefficient of an analyte based on surface enhanced Raman spectroscopy of a microchannel, which ingeniously combines the characteristics of nondestructive, non-contact, label-free, rapid and sensitive laser confocal Raman spectroscopy with the characteristics of trace, high-throughput analysis and self-injection of a microfluidic technology. Wide application prospects are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of physical chemistry and analytical chemistry, and specifically relates to a method and application of rapidly detecting liquid-phase diffusion coefficient of analytes by surface-enhanced Raman spectroscopy based on microchannels. Background Art

[0002] Diffusion is the phenomenon of the migration of particles such as atoms, molecules or ions in space. On a microscopic level, diffusion is caused by the random thermal motion of molecules; on a macroscopic level, diffusion is caused by the concentration gradient between two diffusion systems [doi:10.1002 / ange.19530651912]. Diffusion is usually divided into mass transfer, momentum transfer and heat transfer [doi:10.1038 / s41598-022-13890-y]. Among them, mass transfer is the process of transferring substances from high concentration areas to low concentration areas. This concentration difference drives the diffusion of substances [doi:10.1002 / cite.330620126] [doi:10.1146 / annurev-fluid-010518-040306]. Molecular diffusion in liquids is a key process in many systems [doi: 10.1016 / j.jconhyd.2010.05.002]. It is often a factor that influences the reaction rate in biological or chemical reactions [doi: 10.1021 / es403105b].

[0003] Generally speaking, the liquid phase diffusion coefficient is a function of the solution concentration, and changes with the concentration of the diffusion solution. It can usually be calculated through empirical correlation or obtained experimentally [doi:10.1002 / aic.690420403][doi:10.1016 / j.optlaseng.2012.03.006]. However, the values ​​calculated by the Einstein-Stokes relationship are usually not very accurate, and the deviation is greater in the case of high concentration or irregular molecular shape [doi:10.1103 / PhysRevE.103.L030103]. Therefore, experiment is the only way to accurately obtain the diffusion coefficient. There are currently a variety of techniques for measuring liquid phase diffusion coefficients, such as the membrane pool method [doi:10.1111 / j.2042-7158.1981.tb13730.x], holographic interferometry [doi:10.3390 / jimaging8070196], Taylor dispersion analysis [doi:10.1002 / elps.202200184], liquid core cylindrical lens method, etc. The membrane pool method is a diffusion coefficient determination method based on Fick's first law. It consists of two liquid pools separated by a permeable microporous membrane, allowing the analyte to diffuse from one liquid pool to the other. This method requires the detection of the initial and final concentrations of the analyte in the liquid pool [doi:10.1021 / cr60048a001][doi:10.1021 / JA01158A032]. However, this technique is very time-consuming (sometimes up to several days) and requires precise calibration with analytes of known diffusion coefficients in advance. The pore size and material of the microporous membrane will also have a certain impact on the experimental results [doi:10.1016 / s0043-1354(01)00356-6]. Holographic interferometry is an interference measurement method that uses holographic photography to observe and record the deformation or displacement of an object. It is necessary to record and reconstruct the interference pattern to determine the concentration distribution and thus the diffusion coefficient of the analyte [doi:10.1364 / AO.15.000729]. Analyzing interference-related data is usually complicated and requires cumbersome mathematical tools and algorithms [doi:10.1364 / AO.455775]. In addition, holographic interferometry has high requirements for wavelength stability, quality of optical components and installation and debugging accuracy. The experimental conditions are quite harsh, and small external disturbances (vibration, temperature and humidity changes) may have a large noise impact on the experimental results [doi:10.1364 / OE.459213][doi:10.1364 / AO.444678]. Taylor dispersion analysis is based on the analysis of solute dispersion in laminar flow. This method obtains a concentration distribution similar to a Gaussian distribution curve by detecting the change in the refractive index of the system [doi:10.1063 / 1.4818733].However, the implementation of Taylor dispersion experiments requires precise flow control and complex data analysis methods. At the same time, Taylor dispersion uses a spiral capillary that is several meters long, and the mobile phase needs to flow through a circular cross-section at a constant flow rate. The experimental conditions are very harsh and the accuracy is not high, and the experimental data must be subjected to complex mathematical processing [doi:10.1021 / acs.analchem.5b02053].

[0004] By using a transparent capillary or a liquid core cylindrical lens as a liquid diffusion cell to observe and record the spatial variation of the refractive index of the analyte, the diffusion coefficient of one liquid in another liquid can also be measured [doi:10.1016 / j.jcis.2012.06.053][doi:10.1364 / OE.25.005626]. However, this type of technology has high requirements for the stability and accuracy of the experimental device and the experimental environment, and the scope of application of this type of method is limited. It is powerless for opaque solutions or substances whose refractive index does not change significantly with concentration [doi:10.1364 / OE.388656].

[0005] It can be seen that the existing diffusion coefficient detection technologies mostly rely on refractive index changes or molecular labels, which have the disadvantages of large sample consumption, long detection time, and complicated and cumbersome calculation process. Summary of the invention

[0006] The purpose of the present invention is to provide a method for rapidly detecting the liquid phase diffusion coefficient of an analyte by using surface enhanced Raman spectroscopy based on a microchannel.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a method for rapidly detecting the liquid phase diffusion coefficient of an analyte by surface enhanced Raman spectroscopy based on a microchannel, comprising the following steps:

[0009] In the first step, a capillary (with an outer diameter of 1.0 mm, an inner diameter of 0.8 mm, and a length of 15 mm) is soaked in acetone, ethanol, and a pure water solution in turn and ultrasonically cleaned several times, the capillary is dried (at a temperature of 100-120° C., preferably 110° C.), and the dried capillary is immersed in a piranha solution (a volume ratio of concentrated sulfuric acid to 30% hydrogen peroxide of 1-10:1, preferably 3:1) for 0.5-2 h (preferably 1 h), and ultrasonically cleaned with pure water until there are no bubbles on the inner wall of the capillary, and then dried (at a temperature of 100-120° C., preferably 110° C.), and the dried capillary is immersed in a 3-aminopropyltrimethoxysilane ethanol solution with a volume fraction of 0.5-3% (preferably 1%) for 0.5-24 h (preferably 12 h), and repeatedly washed with ethanol and dried again to obtain a capillary with positively charged amino groups modified on the inner wall;

[0010] The Au TOH solution is sucked into the capillary tube, and allowed to stand (for 10 to 40 minutes, preferably 30 minutes), and repeatedly washed with pure water, and dried to obtain a treated capillary tube (i.e., SERS substrate, abbreviated as Au TOH / Cap);

[0011] Step 2: Chip production:

[0012] A first groove is made on a polydimethylsiloxane (PDMS) chip (75 mm*25 mm*3 mm), the first groove is parallel to the long side of the chip and is at least 4 mm (preferably 5 mm) away from the port, and a second groove is set at one end of the first groove to ensure that the edge of the first groove is exactly on the tangent line of the second groove;

[0013] The first groove is a semicircular groove with a depth of 0.5 mm and a length of 15 mm.

[0014] The second groove is a cylindrical groove with a diameter of 3 mm and a depth of 1.1 mm.

[0015] Step 3: Preparation of the injection device: bend the tip of the microinjection needle 0.8-1.3 cm (preferably 1 cm) at 90° toward the back of the needle, so that the tip of the microinjection needle is perpendicular to the second groove on the chip, and the microinjection needle is repeatedly ultrasonically cleaned with deionized water to remove all bubbles;

[0016] The needle tube length of the microinjection needle is 10 cm and the maximum volume is 50 uL.

[0017] Step 4: Combine the injection pump and chip

[0018] Place the capillary prepared in the first step into the first groove of the chip prepared in the second step, place the prepared injection device on the LongerPump micro-injection pump (TS-2A Syringe Pump Controller), and suspend the needle of the injection device in the second groove on the chip away from the first groove;

[0019] Step 5: Laser confocal Raman time series detection of analyte signal changes

[0020] Use the LongerPump micro-injection pump to draw the solution of the object to be tested into the injection device, turn on the LongerPump micro-injection pump, slowly and evenly inject the solution of the object to be tested into the second groove, use the confocal Raman detector to start time-series acquisition at the center point of the upper edge of the inner wall of the capillary in the first groove, set the integration time and the spectrum acquisition speed, and then obtain a series of spectrum information at the observation point within a certain period of time, and calculate the liquid phase diffusion coefficient of the analyte;

[0021] The setting parameters of the confocal Raman detector are: HORIBA XploRA PLUS, and the parameters are set as follows: Range: 300~1200 cm -1 ; Acq time(s): 1; Accumulation: 1; Objective: 10X; Grating: 1200(750 nm); Filter: 1%; Laser: 785nm; Slit: 100um; Hole: 300um.

[0022] Step 6: Derivation of the formula for calculating the diffusion coefficient of the physical-liquid phase

[0023] ① Experimental setup

[0024] The total length of the capillary is 1 cm, and the pore diameter is dm. The volume V1 of the analyte solution with a concentration of C0 is instantaneously injected into one end of the capillary, x = 0, and the analyte begins to diffuse unidirectionally in the aqueous solution medium with a volume V2, from one end of the capillary x = 0 to the other end x = l; let the volume unit be L, the concentration unit be M, and set the signal detection point of the analyte at a certain position x in the middle of the capillary, that is, When the diffusion time , the concentration of the analyte in the capillary will tend to be consistent everywhere. Let this equilibrium concentration be C e , calculated by the following formula:

[0025] (1)

[0026] ②Basic diffusion equation

[0027] Use Fick's second law to describe the diffusion process of molecules. The one-dimensional form of Fick's second law is:

[0028] (2)

[0029] in:

[0030] C(x,t) is the (local) concentration of the analyte at capillary position x with diffusion time t;

[0031] D is the diffusion coefficient of the analyte;

[0032] Note: It is assumed here that the capillary length l is much greater than x, that is, within the detection time, the analyte has not diffused to the capillary end point l;

[0033] ③ Initial conditions for molecular diffusion

[0034] Ⅰ Instantaneous point source release: When t = 0, at x = 0, the concentration of the analyte is C0, that is, C(0,0) = C0;

[0035] This means that all analyte molecules are initially concentrated at the origin of the capillary; in other words, it is assumed that the origin of the capillary is completely occupied by the analyte solution, thus forming an ideal instantaneous point source;

[0036] Ⅱ Initial spatial distribution: When t = 0, in the region of x > 0, the concentration of the analyte is zero at the initial moment, that is ; This means that the analyte molecules are initially concentrated only at the origin of the capillary (x = 0) and have not yet distributed to other areas;

[0037] ④Boundary conditions for molecular diffusion

[0038] ⅠC(x→∞,t) = 0, at time t, the infinite distance has not yet diffused;

[0039] Ⅱ The actual length of the capillary is much greater than x. Then, as time goes by, the system reaches a dynamic equilibrium state, at which the concentration of the analyte becomes uniform throughout the system and there is no longer any concentration gradient; this can be expressed as:

[0040] when , where Ce is the final equilibrium concentration;

[0041] ⑤Diffusion equation

[0042] According to the experimental setting conditions, the unidirectional diffusion of the analyte in the aqueous solution in the capillary is regarded as a one-dimensional non-steady state diffusion process starting from an instantaneous point source, that is, it is expressed as a one-dimensional diffusion problem in a semi-infinite medium; assuming that the initial conditions are C(x,0) = 0 (except x=0), C(0,t) = C0, and the boundary conditions are C(x→∞, t) = 0, solving the diffusion equation (2), the equation describing the diffusion of the analyte molecules is obtained as:

[0043] (3)

[0044] Where erf(·) is the error function, which is defined as:

[0045] .

[0046] In the method, a semiconductor temperature control platform is used to cool or heat the entire detection platform, the predetermined temperature is set at 25°C, and the temperature difference is controlled within + / -0.1°C.

[0047] The preparation method of the Au TOH solution comprises the following steps:

[0048] Dissolve hexadecyltrimethylammonium bromide (CTAB) in water and stir to dissolve at a temperature of 25-30°C (preferably 27°C); add HAuCl4 solution to the above solution, then quickly add NaBH4 solution under stirring, stir and stand for 3-5 hours to obtain a CTAB-coated gold nanocluster solution;

[0049] The hexadecyltrimethylammonium chloride solution and the CTAB-coated gold nanocluster solution are mixed, and then ascorbic acid solution is added. After the reaction is complete, HAuCl4 solution is added, and the reaction is carried out for 10 to 30 minutes (preferably 15 minutes), followed by centrifugation (centrifugation at 10000 rpm for 25 minutes), and the mixture is resuspended in the hexadecyltrimethylammonium chloride solution to obtain an Au NSs solution;

[0050] The Au NSs solution is added to the hexadecyltrimethylammonium chloride solution, and then the ascorbic acid solution is added to the mixed solution. After mixing, the HAuCl4 solution is added to the above solution, and the reaction is carried out at a temperature of 25-30°C (preferably 27°C) for 10-30min (preferably 15min), and centrifuged (centrifuged at 7000 rpm for 10min). Then, the precipitate is dispersed in a PVP ethanol solution with a mass fraction of 0.5-2% (preferably 1%), ultrasonicated (10min), centrifuged (centrifuged at 7000 rpm for 10min), the supernatant is removed, and the precipitate is dispersed in ethanol to obtain an Au TOH (nano-gold trioctahedron) solution.

[0051] The method of the present invention for measuring the liquid phase diffusion coefficient has a wide range of uses: first, physical and chemical research. The liquid phase diffusion coefficient is an important basic data for studying mass transfer processes, calculating mass transfer rates, and chemical design and development. By measuring the liquid phase diffusion coefficient, the transport mechanism and reaction kinetics of substances in liquids can be revealed. Second, material science research. Measuring the liquid phase diffusion coefficient helps to understand the stability and reactivity of materials in liquid environments. This is of great significance for developing new materials, optimizing material properties, and improving material utilization. Third, chemical design and development. In chemical production, the liquid phase diffusion coefficient is one of the key factors for optimizing process flow and improving production efficiency. By measuring the liquid phase diffusion coefficient, the reaction conditions can be optimized, and the reaction rate and yield can be improved. Fourth, environmental monitoring. In the field of environmental protection, measuring the liquid phase diffusion coefficient helps to evaluate the diffusion rate and impact range of pollutants in the environment. It is of great significance for formulating effective pollution control measures, protecting the ecological environment, and ensuring human health.

[0052] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0053] The present invention provides a method for rapidly detecting the liquid-phase diffusion coefficient of an analyte by surface-enhanced Raman spectroscopy based on a microchannel. The method skillfully combines the non-destructive, contactless, label-free, rapid and sensitive characteristics of laser confocal Raman spectroscopy with the micro-volume, high-throughput analysis and self-sampling characteristics of microfluidic technology, and has broad application prospects.

[0054] The present invention uses surface enhanced Raman technology to perform non-invasive and continuous monitoring (<2 min) of the diffusion state of local and time-dependent chemical components in the capillary, thereby quickly determining the diffusion coefficient of one liquid diffusing into another liquid. The present invention proves the effectiveness of this method by accurately measuring the concentration trend of urea solution (5M) at a certain location over time. It is worth noting that this method of determining the diffusion coefficient using surface enhanced Raman technology has great advantages over other existing technologies in terms of time resolution, convenience, and sample consumption. In addition, the present invention expands this method to low concentrations of methylene blue (1.0*10 -5 M) Diffusion coefficient determination in water, highlighting its potential use in studying liquid-liquid systems at different concentration levels.

[0055] The present invention innovatively establishes a reliable Raman spectroscopy method, which can obtain the liquid phase diffusion coefficient based on the concentration-time variation of the analyte. This technology, which combines Raman spectroscopy detection technology with the technology of dynamic monitoring of molecular diffusion through microchannels, is a technology with considerable application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Schematic diagram of nanoparticle electron microscope and SERS substrate.

[0057] Figure 2 Schematic diagram of the performance results of the microchannel SERS substrate.

[0058] Figure 3 This is a schematic diagram of the test results of the liquid phase diffusion coefficient of the urea solution.

[0059] Figure 4 This is a schematic diagram of the test results of the liquid phase diffusion coefficient of the methylene blue solution.

[0060] Figure 5 Schematic diagram of the detection platform. DETAILED DESCRIPTION

[0061] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0062] Reagents required for the present invention: urea (CH4N2O), methylene blue (MB), chloroauric acid (HAuCl4), sodium borohydride (NaBH4), polyvinylpyrrolidone (PVP), hexadecyltrimethylammonium chloride (CTAC), hexadecyltrimethylammonium bromide (CTAB), ascorbic acid (AA), sulfuric acid (H2SO4), hydrogen peroxide (H2O2), 3-aminopropyltrimethoxysilane (ATPMS, analytical grade, Shanghai Titan Technology Co., Ltd.); distilled water (Guangzhou Watsons Food and Beverage Co., Ltd.).

[0063] Example 1

[0064] First step: Preparation and characterization of nanoparticles and microchannels

[0065] Preparation of gold nanoparticles (Au NSs) using the seed-mediated growth method [doi:10.1002 / ppsc.201300256].

[0066] Dissolve 0.365 g CTAB in 5 mL water and stir to dissolve at 27°C. Add 5 mL HAuCl4 solution with a concentration of 0.5 mmol / L to the above solution, and then quickly add 0.6 mL NaBH4 solution with a concentration of 10 mmol / L under stirring, stir for 2 min and leave for 3-5 h to ensure complete reaction, to obtain CTAB-coated 3 nm gold nanocluster solution.

[0067] Mix 2 mL of 0.2 mol / L CTAC solution with 50 uL of gold nanocluster solution, then add 1.5 mL of 0.1 mol / L AA solution. After the reaction is complete, add 2 mL of 0.5 mmol / L HAuCl4 solution. After reacting for 15 min, centrifuge at 10000 rpm for 25 min, resuspend in 1 mL of 20 mmol / L CTAC solution to obtain an Au NSs solution with a particle size of 10 nm, which is stored at 4°C for later use.

[0068] Take 20 uL of Au NSs solution with a particle size of 10 nm and add it to 10 ml of CTAC solution with a concentration of 0.1 mol / L, then add 650 uL of AA solution with a concentration of 0.1 mol / L to the mixed solution. After mixing, add 10 mL of HAuCl4 solution with a concentration of 0.5 mmol / L to the above solution, react at 27°C for 15 min, centrifuge at 7000 rpm for 10 min, then disperse the precipitate into 0.5 mL of PVP ethanol solution with a mass fraction of 1%, ultrasonicate for 10 min, centrifuge at 7000 rpm for 10 min, remove the supernatant and disperse the precipitate in 0.5 mL of ethanol to obtain a nano-gold trioctahedron (Au TOH) solution with a particle size of 75 nm [doi:10.1021 / acs.analchem.2c00157]. The electron microscope image of the nanoparticle is shown in Figure 1 As shown in a. Figure 1 Figure 1 is a schematic diagram of the electron microscope of nanoparticles and SERS substrate. Figure 2 is a schematic diagram of the electron microscope of nanogold trioctahedron. It can be seen from the figure that the prepared nanoparticles are trioctahedral in shape with a diameter of about 75 nanometers.

[0069] In the second step, the quartz capillary (microchannel) was pretreated as follows: first, the quartz capillary (outer diameter 1.0 mm, inner diameter 0.8 mm, length 15 mm) was soaked in acetone solution and ultrasonically cleaned several times with ethanol and pure water alternately, and the capillary was placed in an oven at 110°C to dry. The dried capillary was immersed in piranha solution (concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 3:1) for 1 h, and ultrasonically cleaned with pure water until there were no bubbles on the inner wall of the capillary, and then dried at 110°C with air blast, and the dried capillary was immersed in 1% ATPMS ethanol solution for 12 h, and repeatedly cleaned with ethanol to remove excess ATPMS and dried again to obtain a capillary with positively charged amino groups modified on the inner wall [doi:10.1016 / j.talanta.2010.03.023][ doi:10.1016 / j.saa.2020.118193]. The Au TOH solution was sucked into the capillary tube by capillary action, so that the gold nanosol filled the entire capillary tube [doi:10.1021 / j150290a010]. After standing for 30 minutes, the excess Au TOH solution was sucked out and repeatedly washed with pure water. The treated capillary tube (i.e., SERS substrate, abbreviated as Au TOH / Cap) was dried and used for later use. The obtained SERS substrate is as follows Figure 1 As shown in b, b is a schematic diagram of capillary SERS optics. As can be seen from the figure, the modified SERS substrate is red, indicating that the gold nanoparticles have been successfully fixed to the inner wall of the capillary.

[0070] Investigation of the stability, reproducibility, uniformity and sensitivity of microchannel SERS substrate

[0071] In SERS substrate detection, the uniformity, reproducibility and stability of the substrate are important indicators for evaluating substrate performance. Therefore, in order to investigate the uniformity, reproducibility and stability of Au TOH / Cap, the present invention uses methylene blue (MB) as a probe molecule and selects 40 sites axially on the SERS substrate for SERS signal detection. When collecting signals, the capillary action is used to directly absorb a concentration of 1.0*10 -5 MB solution was prepared, and then a site was taken every 5 μm along the capillary. -1 、498 cm -1 Statistical analysis of the peak intensity at the position and calculation of the relative standard deviation. The results are as follows Figure 2 As shown in (a) and (b), Figure 2 Schematic diagram of the performance results of the microchannel SERS substrate, where a and b are schematic diagrams of the results of the SERS spectra of 40 sites selected by axial scanning of the capillary and statistical analysis of the two characteristic peaks using AuTOH / Cap as the substrate. Figure 2 a in the middle is the characteristic peak 447cm recorded at 40 sites -1 The intensity change of the peak intensity, Figure 2 b is the characteristic peak 498cm recorded at 40 sites -1 Peak intensity variation. From the figure, it can be seen that the relative standard deviation of the intensity of different characteristic peaks at 40 consecutive sites on the capillary SERS substrate is less than 10%, indicating that the enhancement effect of the capillary SERS substrate at each location is similar, with good uniformity and reproducibility.

[0072] Based on Au TOH / Cap, 447 cm -1 、498 cm -1 The relative standard deviation of the peak intensity at 447 cm was less than 10%, showing good uniformity and reproducibility. Another Au TOH / Cap substrate was used to collect MB solutions of the same concentration for 20 consecutive days to obtain a series of spectra to evaluate its stability. -1 The characteristic peak at 20° had almost no change in 20 days. Figure 2 As shown in (c) and (d), c and d are schematic diagrams of the statistical results of the SERS spectrum and characteristic peak intensity values ​​of Au TOH / Cap within 20 days. Figure 2 Figure c is a collection of 20 spectra obtained by testing the same concentration of methylene blue using different Au TOH / Caps from the same batch for 20 consecutive days. Figure 2Figure d shows the statistical analysis of the intensity changes of the characteristic peak 447 cm-1 in Figure c, and found that its relative standard deviation is less than 15%, indicating that the enhancement effect of the capillary SERS substrate is stable over a period of time and has good stability.

[0073] The above shows that the Au TOH / Cap substrate has good uniformity, reproducibility and stability.

[0074] The third step is chip production: make multiple semicircular grooves with a depth of 0.5 mm on the PDMS (polydimethylsiloxane) chip (75 mm*25 mm*3 mm). The length of the groove is 15 mm. The groove is parallel to the long side of the chip and is 5 mm away from the port. The etched groove can place the quartz capillary in the middle and fit it perfectly. Open a cylindrical groove with a diameter of 3 mm and a depth of 1.1 mm at one end of the 15 mm long groove, ensuring that the edge of the 15 mm long groove is exactly on the tangent of the cylindrical groove. And so on, a total of 10 grooves of the same size are made on the chip.

[0075] Step 4: Preparation of the injection device: Take a microinjection needle with a needle tube length of 10 cm and a maximum volume of 50 uL, bend the top of the needle about 1 cm toward the back of the needle 90°, so that the needle of the microinjection needle is perpendicular to the cylindrical groove on the chip, and repeatedly ultrasonically clean the above-mentioned microinjection needle with deionized water to remove all bubbles, and set it aside.

[0076] Step 5: Combine the injection pump and chip

[0077] Place the capillary prepared in the second step into the groove with a length of 15 mm in the chip, and place the prepared injection device on the LongerPump micro-injection pump (TS-2A Syringe Pump Controller), which has precise stroke control and an ultra-wide range of linear speed (7.9 μm / min-79.4 μm / min). After the above injection device is assembled, suspend the needle of the micro-injection needle on the side of the cylindrical groove on the chip away from the 15 mm groove to reduce the effect of liquid flow on molecular diffusion. Set the injection speed on the LongerPump micro-injection pump so that the liquid in the injection device can be injected into the sample pool (i.e., the cylindrical groove) at a uniform and slow speed.

[0078] Step 6: Temperature Control Device

[0079] A semiconductor temperature control platform (purchased from Wentian Precision Instrument Technology (Suzhou) Co., Ltd.) was used to cool or heat the entire detection platform, and the predetermined temperature was set at 25 °C, and the temperature difference was controlled within + / -0.1 °C. Figure 5 Schematic diagram of the detection platform.

[0080] Step 7: Laser confocal Raman time series detection of analyte signal changes

[0081] Confocal Raman detector: HORIBA XploRA PLUS, parameter setting: Range: 300~1200 cm -1 ;Acq time(s): 1; Accumulation: 1; Objective: 10X; Grating: 1200(750 nm);Filter: 1%; Laser: 785nm; Slit: 100um; Hole: 300um.

[0082] The object to be tested was placed on the stage, and a 10x microscope was used to find the center point of the upper edge of the inner wall of the capillary in the groove for Raman spectrum acquisition. The LongerPump micro-injection pump was used to draw the solution of the object to be tested into the injection device, and the needle of the injection device was suspended on the side of the cylindrical groove on the chip away from the 15 mm groove. The LongerPump micro-injection pump was turned on, and the solution of the object to be tested was slowly and uniformly injected into the sample pool (i.e., the cylindrical groove). At the same time, the confocal Raman detector was used to start the time series acquisition at the center point of the upper edge of the inner wall of the capillary in the groove, and the integration time and spectrum acquisition speed were set to obtain a series of spectrum information at the observation point within a certain time. After removing the background, the series of spectra were analyzed, and the liquid phase diffusion coefficient of the analyte was calculated based on the molecular diffusion state, combined with information such as temperature, liquid viscosity, solution density, molecular weight and molecular volume. According to the obtained Raman spectrum, it was found by calculation that this method can accurately determine the liquid phase diffusion coefficient of the object to be tested at two different concentration levels. This proves that this method can accurately measure the liquid phase diffusion coefficient under certain conditions.

[0083] Step 8: Derivation of the formula for calculating the diffusion coefficient of the physical-liquid phase

[0084] ① Experimental setup

[0085] Use a capillary with uniform pore size to place the diffusion medium (aqueous solution). The total length of the capillary is l (cm) and the pore size is d (m). The volume V1 of the analyte solution (concentration C0) is instantaneously injected into one end of the capillary (x = 0). The analyte begins to diffuse unidirectionally in the aqueous solution medium with a volume V2, from one end of the capillary (x=0) to the other end (x=l). Let the volume unit be L and the concentration unit be M. Set the signal detection point of the analyte at a certain position x in the middle of the capillary, that is, When the diffusion time , the concentration of the analyte in the capillary will tend to be consistent everywhere. Let this equilibrium concentration be C e , calculated by the following formula:

[0086] (1)

[0087] ②Basic diffusion equation

[0088] The diffusion of the analyte in the solution medium is a phenomenon of material migration caused by the thermal motion of the microscopic particles of the system. According to the details and conditions of the experimental setup, the unidirectional diffusion of the analyte in the capillary aqueous solution (medium) can be regarded as a one-dimensional non-steady statediffusion process starting from an instantaneous point source. Therefore, Fick's second law is used to describe the diffusion process of molecules. The one-dimensional form of Fick's second law is:

[0089] (2)

[0090] in:

[0091] C(x,t) is the (local) concentration of the analyte at capillary position x with diffusion time t.

[0092] D is the diffusion coefficient of the analyte.

[0093] Note: It is assumed here that the capillary length l is much larger than x, that is, within the detection time, the analyte has not diffused to the capillary end point l.

[0094] ③ Initial conditions for molecular diffusion

[0095] Ⅰ Instantaneous point source release: When t = 0, at x = 0, the concentration of the analyte is C0, that is, C(0,0) = C0.

[0096] This means that all analyte molecules are initially concentrated at the origin of the capillary. In other words, it is assumed that the origin of the capillary is completely occupied by the analyte solution, thus forming an ideal instantaneous point source.

[0097] Ⅱ Initial spatial distribution: When t = 0, in the region of x > 0, the concentration of the analyte is zero at the initial moment, that is This means that the analyte molecules are initially concentrated only at the origin of the capillary (x = 0) and have not yet distributed to other areas.

[0098] ④Boundary conditions for molecular diffusion

[0099] IC(x→∞,t) = 0. At time t, the light has not yet diffused to the infinitely far place.

[0100] Ⅱ The actual length of the capillary is much greater than x, then as time goes by, the system reaches a dynamic equilibrium state, at which time the concentration of the analyte becomes uniform throughout the system and there is no longer any concentration gradient. This can be expressed as:

[0101] when , where Ce is the final equilibrium concentration.

[0102] ⑤Diffusion equation

[0103] According to the experimental setting conditions, the unidirectional diffusion of the analyte in the aqueous solution in the capillary is regarded as a one-dimensional non-steady state diffusion process starting from an instantaneous point source, that is, it is expressed as a one-dimensional diffusion problem in a semi-infinite medium. Assuming the initial conditions are C(x,0) = 0 (except x=0), C(0,t) = C0, and the boundary conditions are C(x→∞, t) = 0, solve the diffusion equation (2) and obtain the equation describing the diffusion of the analyte molecules:

[0104] (3)

[0105] Where erf(·) is the error function, which is defined as:

[0106]

[0107] The influence of interfering factors on the spontaneous diffusion process of the analyte:

[0108] The object to be tested may be affected by two factors during the spontaneous diffusion process. First, the thermal effect of the Raman laser may cause the local temperature of the diffusion pool to be high, which in turn leads to abnormal diffusion. The present invention uses an ultra-high-definition infrared thermal imager to monitor the temperature of the laser point for up to 2 minutes while the Raman is performing a time-series scan. The results show that: except for the temperature, the temperature of the laser point changes by less than ±0.5 °C under the premise that all parameters and actual experimental conditions remain consistent. Therefore, during the entire acquisition period, the thermal effect of the laser point has almost no effect on the observation position of the diffusion pool, eliminating the influence of the thermal effect of the laser point on the diffusion of the liquid in the diffusion pool. Figure 3 As shown in d. The second consideration is the effect of the inner wall of the capillary diffusion cell on the viscous resistance of the fluid. The capillary is 15.0 mm long, 1.0 mm in outer diameter, 0.8 mm in inner diameter, and is completely filled with liquid. The total volume V1 = 7.54 mm 3 , the diameter of a water molecule is a = 4.0*10 -7 mm, the volume of a single molecule is V2= 3.35*10 -20 mm3 Based on this, the total number of water molecules in the capillary is calculated to be N0 = 2.25*10 20 Assuming that the inner wall of the capillary tube has a viscous effect on the expected contact layer of liquid molecules, the surface area of ​​the inner wall of the capillary tube S1 = 37.70 mm 2 , the area occupied by a single molecule is S2 = 1.26*10 -3 mm 2 , then the number of molecules adhering to the inner surface of the capillary is N1= 3.00*10 5 . It can be seen that the number of molecules with viscosity accounts for 10 of the total number of molecules. 15 This influence is extremely small and can be ignored. In summary, the diffusion state of the analyte in the capillary is only affected by the concentration gradient.

[0109] Detection of liquid phase diffusion coefficient of the object to be tested:

[0110] Use the LongerPump micro-injection pump to draw urea aqueous solution into the injection device, and suspend the needle of the injection device in the cylindrical groove on the chip, away from the side of the 15 mm long groove. Turn on the LongerPump micro-injection pump, slowly and evenly inject the urea aqueous solution into the sample pool, and use the confocal Raman detector to start time-series acquisition at the center point of the upper edge of the inner wall of the capillary in the groove, and set the Raman parameters (Range: 300~1200 cm -1 ; Acq time(s): 1;Accumulation: 1; Objective: 10X; Grating: 1200(750 nm); Filter: 1%; Laser:785nm; Slit: 100um; Hole: 300um). Thus, a series of spectral information at the observation points within 100s was obtained.

[0111] According to the above method, the diffusion coefficient of urea (Ns) solution in water was measured. First, the linear relationship between the concentration (0.5M-5M) of urea solution at 298.15K and the relative intensity of SERS was measured at certain intervals. The results are as follows: Figure 3 Then the microchannel SERS substrate is filled with aqueous solution, and a certain amount of urea solution (initial concentration is 5 M) is drawn into the microinjection needle, and the temperature is set to 298.15K. The spectrum is collected while the injection is started, and the following is obtained: Figure 3 The Raman relative intensity is then converted into concentration, and the relationship between the concentration of the analyte and time is substituted into the above formula to obtain the following: Figure 3The graph of the diffusion coefficient changing with time shown in f is shown in the figure. Since the diffusion coefficient is a characteristic value of the analyte and is only related to the analyte itself and the temperature, the change in the diffusion coefficient is caused by the "turbulence" during the injection. However, as time goes on, the influence of this "turbulence" is gradually weakened. About 50 seconds after the start of diffusion, the influence of the "turbulence" is already very weak and can be almost ignored. Based on the above situation, taking into account the normal volatility of SERS detection data, which will lead to a small amount of data affecting the accuracy of the fitting results, and the fact that the liquid in the open platform will continue to evaporate and the detection time should not be too long, etc., the starting point of the fitting is set at any point between 50 s and 80 s, and the end point of the fitting is 100 s. The SD value of each interval is used as the consideration standard, and the interval with the lowest SD value is selected as the optimal data interval for fitting the diffusion coefficient of the substance under this condition, as shown in FIG. Figure 3 Finally, at 298.15k, the time interval of urea fitting with C0=5M is 76s-100s, as shown in Figure 3 As shown in f, the diffusion coefficient is 8.04*10 -6 cm 2 / s, which is very close to the value reported in the literature [doi:10.1021 / ja01128a060].

[0112] Figure 3The following is a schematic diagram of the detection results of the liquid phase diffusion coefficient of the urea solution to be tested, where (a) is a schematic diagram of the SERS relative intensity of the Ns solution at 450μm from the capillary injection port at a temperature of 298.15k. It is a diagram of the SERS relative intensity over time during the entire detection time. It can be seen from the figure that the signal intensity of the test object is gradually increasing over time; (b) is a schematic diagram of the linear relationship between the concentration (0.5-5M) of the Ns solution and the SERS relative intensity at a temperature of 298.15k, reflecting the signal intensity of the test object corresponding to different test object concentrations. The two have a linear relationship within a certain range; y=0.219+0.173x, R2=0.98998. In (c): a is a schematic diagram of the urea concentration change curve over time; b is a schematic diagram of the ideal curve of the urea concentration change over time. The black curve in the figure converts the SERS relative intensity in figure a into concentration information, and obtains the relationship between the concentration of the analyte and time during the detection period. The red curve in figure c is the standard curve obtained by substituting the calculation formula derived in the eighth step. There is a relatively obvious deviation between the two, indicating that there is "turbulence" interference at the beginning, which causes the experimental data to not completely match the ideal data; (d) is a schematic diagram of the laser point thermal effect monitoring. It can be seen from the figure that during the entire detection period, the thermal effect of the laser hardly causes temperature changes, so the influence of the laser thermal effect is excluded. (e) is the mean diffusion coefficient and its SD value of all fitting intervals from 50-80s when the fitting starting point is used. According to the calculation logic, the fitting starting point is used as the X-axis, the fitted diffusion coefficient is used as the Y-axis, and the SD value is used as the Z-axis to draw the obtained three-dimensional graph. It can be seen from the figure that different fitting starting points will fit different diffusion coefficients, and the SD values ​​are also different. (f) is a schematic diagram of the diffusion coefficient diagram of urea fitting at a temperature of 298.15k, the noise reduction curve (red), the interval with the lowest SD value (76s-100s) and the diffusion coefficient value (yellow area) obtained by fitting. Figure f selects the point with the lowest SD value in figure e as the standard value for measuring the diffusion coefficient of the object to be measured.

[0113] The present invention also measured the temperature at 293.15K, the initial concentration was 1.0*10 -5 M is the diffusion coefficient of methylene blue solution in water.

[0114] Use the LongerPump microinjection pump to draw the methylene blue solution into the injection device, and suspend the needle of the injection device in the cylindrical groove on the chip, away from the side of the 15 mm long groove. Turn on the LongerPump microinjection pump, slowly and evenly inject the urea aqueous solution into the sample pool, and use the confocal Raman detector to start time series acquisition at the center point of the upper edge of the inner wall of the capillary in the groove, and set the Raman parameters (Range: 300~1200 cm -1; Acq time(s): 1;Accumulation: 1; Objective: 10X; Grating: 1200(750 nm); Filter: 1%; Laser:785nm; Slit: 100um; Hole: 300um). Thus, a series of spectral information at the observation points within 100s was obtained.

[0115] First, the diffusion rate of methylene blue aqueous solution in water was measured (e.g. Figure 4 Then the linear relationship between the relative SERS intensity and concentration was detected (as shown in (a)). Figure 4 Then convert it into a schematic diagram of concentration variation over time (as shown in (b) and (c)). Figure 4 Finally, according to the same data processing logic, the interval with the lowest SD value within the limited range is selected: 80s-100s (as shown in (d)). Figure 4 As shown in (e) and (f) above, the diffusion coefficient of methylene blue under this condition is 1.65*10 -5 cm 2 / s. The diffusion coefficient obtained is 10 -5 -10 -6 cm 2 / s range, which is a typical value for dyes in aqueous solution.

[0116] Figure 4 Schematic diagram of the detection results of the liquid phase diffusion coefficient of the methylene blue solution to be tested, where (a) is a schematic diagram of the change in the relative intensity of the MB solution at 450 um from the capillary inlet over time at a temperature of 298.15 K. (b) is a schematic diagram of the linear relationship between the MB solution concentration (2.5-10 uM) and the SERS relative intensity at a temperature of 298.15 K. (c) is a schematic diagram of the linear relationship between the MB solution concentration (0.5-2.5 uM) and the SERS relative intensity at a temperature of 298.15 K. (d) is a schematic diagram of the experimental curve of the change in MB concentration over time. (e) is a schematic diagram of the average diffusion coefficient and its SD value for all fitting intervals from 50-80 s to 100 s. (f) is a schematic diagram of the diffusion coefficient diagram of MB fitting and its noise reduction curve (red) at a temperature of 298.15 K, as well as the interval of the SD minimum value (80-100 s) and the diffusion coefficient value obtained by fitting (yellow area).

[0117] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A method for rapid detection of liquid phase diffusion coefficient of analytes based on microchannel surface enhanced Raman spectroscopy, characterized in that: The following steps are involved: In the first step, the capillary is soaked in acetone, ethanol and pure water solutions in turn and ultrasonically cleaned several times, the capillary is dried, the dried capillary is immersed in piranha solution for 0.5 to 2 h, ultrasonically cleaned with pure water until there are no bubbles on the inner wall of the capillary, and then dried. The dried capillary is immersed in a 0.5 to 3% volume fraction 3-aminopropyltrimethoxysilane ethanol solution for 0.5 to 24 h, repeatedly cleaned with ethanol and dried again to obtain a capillary with positively charged amino groups on the inner wall; The Au TOH solution is sucked into the capillary tube, left to stand, repeatedly washed with pure water, and dried to obtain a treated capillary tube; Step 2: Chip production: A first groove is made on the polydimethylsiloxane chip, the first groove is parallel to the long side of the chip and is at least 4 mm away from the port, and a second groove is set at one end of the first groove to ensure that the edge of the first groove is exactly on the tangent line of the second groove; Step 3: Preparation of the injection device: bend the tip of the microinjection needle 90 degrees at 0.8-1.3 cm from the top to the back of the needle, so that the tip of the microinjection needle is perpendicular to the second groove on the chip, and the microinjection needle is repeatedly ultrasonically cleaned with deionized water to remove all bubbles; Step 4: Combine the injection pump and chip Place the capillary prepared in the first step into the first groove of the chip prepared in the second step, place the prepared injection device on the LongerPump microinjection pump, and suspend the needle of the injection device in the second groove on the chip away from the first groove; Step 5: Laser confocal Raman time series detection of analyte signal changes Use the LongerPump micro-injection pump to draw the solution of the object to be tested into the injection device, turn on the LongerPump micro-injection pump, slowly and evenly inject the solution of the object to be tested into the second groove, use the confocal Raman detector to start time-series acquisition at the center point of the upper edge of the inner wall of the capillary in the first groove, set the integration time and the spectrum acquisition speed, and then obtain a series of spectrum information at the observation point within a certain period of time, and calculate the liquid phase diffusion coefficient of the analyte; Step 6: Derivation of the formula for calculating the diffusion coefficient of the physical-liquid phase ① Experimental setup The total length of the capillary is 1 cm, and the pore diameter is dm. The volume V1 of the analyte solution with a concentration of C0 is instantaneously injected into one end of the capillary, x = 0, and the analyte begins to diffuse unidirectionally in the aqueous solution medium with a volume V2, from one end of the capillary x = 0 to the other end x = l; let the volume unit be L, the concentration unit be M, and set the signal detection point of the analyte at a certain position x in the middle of the capillary, that is, When the diffusion time , the concentration of the analyte in the capillary will tend to be consistent everywhere. Let this equilibrium concentration be C e , calculated by the following formula: （1） ②Basic diffusion equation Use Fick's second law to describe the diffusion process of molecules. The one-dimensional form of Fick's second law is: （2） in: C(x,t) is the (local) concentration of the analyte at capillary position x with diffusion time t; D is the diffusion coefficient of the analyte; Note: It is assumed here that the capillary length l is much greater than x, that is, within the detection time, the analyte has not diffused to the capillary end point l; ③ Initial conditions for molecular diffusion Ⅰ Instantaneous point source release: When t = 0, at x = 0, the concentration of the analyte is C0, that is, C(0,0) = C0; This means that all analyte molecules are initially concentrated at the origin of the capillary; in other words, it is assumed that the origin of the capillary is completely occupied by the analyte solution, thus forming an ideal instantaneous point source; Ⅱ Initial spatial distribution: When t = 0, in the region of x > 0, the concentration of the analyte is zero at the initial moment, that is ; This means that the analyte molecules are initially concentrated only at the origin of the capillary (x = 0) and have not yet distributed to other areas; ④Boundary conditions for molecular diffusion ⅠC(x→∞,t) = 0, at time t, the infinite distance has not yet diffused; Ⅱ The actual length of the capillary is much greater than x. Then, as time goes by, the system reaches a dynamic equilibrium state, at which the concentration of the analyte becomes uniform throughout the system and there is no longer any concentration gradient; this can be expressed as: when , where Ce is the final equilibrium concentration; ⑤Diffusion equation According to the experimental setting conditions, the unidirectional diffusion of the analyte in the aqueous solution in the capillary is regarded as a one-dimensional non-steady-state diffusion process starting from an instantaneous point source, that is, it is expressed as a one-dimensional diffusion problem in a semi-infinite medium; assuming that the initial conditions are C(x,0) = 0 (except at x=0), C(0,t) = C0, and the boundary conditions are C(x→∞, t) = 0, the diffusion equation (2) is solved, and the equation describing the diffusion of the analyte molecules is obtained as follows: （3） Where erf(·) is the error function, which is defined as: 。 2. The method for rapid detection of liquid phase diffusion coefficient of analytes by microchannel-based surface enhanced Raman spectroscopy according to claim 1, characterized in that: The first groove is a semicircular groove with a depth of 0.5 mm and a length of 15 mm.

3. The method for rapid detection of liquid phase diffusion coefficient of analytes by microchannel-based surface enhanced Raman spectroscopy according to claim 1, characterized in that: The second groove is a cylindrical groove with a diameter of 3 mm and a depth of 1.1 mm.

4. The method for rapid detection of liquid phase diffusion coefficient of analytes by microchannel-based surface enhanced Raman spectroscopy according to claim 1, characterized in that: The needle tube length of the microinjection needle is 10 cm and the maximum volume is 50 uL.

5. The method for rapid detection of liquid phase diffusion coefficient of analytes by microchannel-based surface enhanced Raman spectroscopy according to claim 1, characterized in that: The setting parameters of the confocal Raman detector are: HORIBA XploRA PLUS, and the parameters are set as follows: Range: 300~1200 cm -1 ; Acq time(s): 1; Accumulation: 1; Objective: 10X; Grating: 1200(750 nm); Filter: 1%; Laser: 785nm; Slit: 100um; Hole: 300um.

6. The method for rapid detection of liquid phase diffusion coefficient of analytes by microchannel-based surface enhanced Raman spectroscopy according to claim 1, characterized in that: In the method, a semiconductor temperature control platform is used to cool or heat the entire detection platform, the predetermined temperature is set at 25°C, and the temperature difference is controlled within + / -0.1°C.

7. The method for rapid detection of liquid phase diffusion coefficient of analytes by microchannel-based surface enhanced Raman spectroscopy according to claim 1, characterized in that: The preparation method of the Au TOH solution comprises the following steps: Dissolve hexadecyltrimethylammonium bromide (CTAB) in water and stir to dissolve at a temperature of 25-30°C; add HAuCl4 solution to the above solution, then quickly add NaBH4 solution under stirring, stir and stand for 3-5 hours to obtain a CTAB-coated gold nanocluster solution; The hexadecyltrimethylammonium chloride solution was mixed with the CTAB-coated gold nanocluster solution, and then ascorbic acid solution was added. After the reaction was complete, HAuCl4 solution was added. After reacting for 10 to 30 minutes, the mixture was centrifuged and resuspended in the hexadecyltrimethylammonium chloride solution to obtain an Au NSs solution. The Au NSs solution was added to the hexadecyltrimethylammonium chloride solution, and then the ascorbic acid solution was added to the mixed solution. After mixing, the HAuCl4 solution was added to the above solution, and the reaction was carried out at a temperature of 25-30°C for 10-30 minutes. The mixture was centrifuged, and then the precipitate was dispersed in a PVP ethanol solution with a mass fraction of 0.5-2%. The mixture was ultrasonicated and centrifuged. The supernatant was removed and the precipitate was dispersed in ethanol to obtain an Au TOH solution.

8. The method for rapid detection of liquid phase diffusion coefficient of analytes by microchannel-based surface enhanced Raman spectroscopy according to claim 1, characterized in that: The dimensions of the capillary are: outer diameter 1.0 mm, inner diameter 0.8 mm, and length 15 mm.

9. The method for rapid detection of liquid phase diffusion coefficient of analytes by microchannel-based surface enhanced Raman spectroscopy according to claim 1, characterized in that: The piranha solution is prepared from concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 1 to 10:

1.

10. The method for rapid detection of liquid phase diffusion coefficient of analytes by microchannel-based surface enhanced Raman spectroscopy according to claim 1, characterized in that: The size of the polydimethylsiloxane chip is: 75 mm*25 mm*3 mm.

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