Microchannel-based high-resolution confocal surface-enhanced Raman method for rapidly detecting intermolecular interaction and application of microchannel-based high-resolution confocal surface-enhanced Raman method

Abstract

The invention discloses a microchannel-based high-resolution confocal surface-enhanced Raman rapid intermolecular interaction detection method, which does not depend on molecular markers or molecular immobilization, is based on the principle of molecular dynamic interaction diffusion, is ingeniously combined with the characteristics of microchannel microscale and high-throughput chips, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically, it relates to a method and application for rapid detection of intermolecular interactions using high-resolution confocal surface-enhanced Raman spectroscopy based on microchannels. Background Technology

[0002] The study of intermolecular interactions has always been a fundamental research area in biochemistry and molecular biology. This research is crucial for a deeper understanding of biomolecules and their selective action mechanisms, the elucidation of gene-protein interaction networks, the discovery of biomarkers, and drug development and discovery—all vital aspects of life science research. In recent years, with the deepening of research, several biophysical techniques for studying molecular interactions have flourished, such as biomembrane interferometry (BLI), surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), backscattering interferometry (BSI), and microthermophoresis (MST). The analytical principles of these techniques are based on changes caused by molecular interactions, such as changes in light, electricity, heat, nuclear, and magnetism when molecules approach each other. However, these analytical techniques all have some drawbacks in practical applications. For example, SPR mainly detects binding affinity by measuring changes in the refractive index of a chip surface; however, SPR is a heterogeneous method requiring complex surface chemistry, necessitating the immobilization of the smaller of the two interacting molecules onto the sensor chip. This molecular immobilization method can affect molecular binding to some extent. Furthermore, false positives due to nonspecific binding and the high cost of precious metal sensor chips are drawbacks of SPR. BLI detects interactions by monitoring changes in biofilm thickness in real time as molecular binding events occur. BLI overcomes some limitations of SPR, but still requires surface immobilization of molecules. Typically, the smaller of the two interacting molecules is immobilized on the biofilm to detect larger changes in membrane thickness. Like SPR, immobilization causes minor structural changes that can significantly impact affinity, thus affecting the accuracy of the results. FCS (Fluorescence Correlation Spectroscopy) has low accuracy for large molecules or molecules with extreme diffusion rates and cannot detect interactions between molecules of similar size. ITC is a widely accepted method for measuring intermolecular interaction rates and affinities. However, this method suffers from low throughput, relatively large sample sizes, and detection limits. MST is a relatively new free-solution method that can be performed in almost any buffer solution and has no limitations on the molecular size and relative molecular mass of the analytes. However, MST requires fluorescent labeling in most cases, increasing the cost and complexity of the assay. BSI is a label-free, free-solution assay that can rapidly measure a wide range of binding affinities with only small sample sizes. However, this method is difficult to commercialize and is expensive due to its demanding experimental conditions, hindering the development of standardized and regulated assay procedures.

[0003] Raman spectroscopy is a light scattering technique. It involves the emission of scattered light carrying characteristic information about a substance after its interaction with the light. A Raman spectrum typically consists of a number of Raman peaks, each representing the wavelength and intensity of the scattered light. Each peak corresponds to a specific molecular bond vibration, including not only single chemical bonds such as C=C, C=C, and CH, but also vibrations of groups composed of multiple bonds, such as the breathing vibration and lattice vibration of the benzene ring. Therefore, Raman spectroscopy can accurately identify and analyze substances. Furthermore, it is non-contact, non-destructive, real-time, in-situ, and requires very small sample sizes. Laser confocal Raman spectroscopy offers higher spatial and spectral resolution than traditional Raman spectroscopy, making it possible to observe the dynamic changes of molecules at micro-range distances.

[0004] Existing interaction analysis techniques mostly rely on molecular markers, require molecular immobilization, or involve large sample volumes, making it impossible to conduct detection under natural biological conditions. These techniques alter the natural state of molecules to some extent, thus introducing unavoidable errors in the results. Therefore, combining Raman spectroscopy with dynamic monitoring of molecular interactions is a promising and urgently needed technology. Summary of the Invention

[0005] The purpose of this invention is to provide a method for rapid detection of intermolecular interactions using high-resolution confocal surface-enhanced Raman spectroscopy based on microchannels.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a method for rapid detection of intermolecular interactions using high-resolution confocal surface-enhanced Raman spectroscopy based on microchannels, comprising the following steps:

[0008] The first step involves immersing a capillary tube (outer diameter 1.0 mm, inner diameter 0.8 mm, length 15 mm) sequentially in acetone, ethanol, and pure aqueous solution, followed by ultrasonic cleaning several times. The capillary tube is then dried (at a temperature of 100-120°C, preferably 110°C). After drying, the capillary tube is immersed in a piranha solution (concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 1-10:1, preferably 3:1) for 0.5-2 h (preferably 1 h). It is then ultrasonically cleaned with pure water until no bubbles remain on the inner wall of the capillary tube, and dried (at a temperature of 100-120°C, preferably 110°C). The dried capillary tube is then immersed in a 0.5-3% (preferably 1%) 3-aminopropyltrimethoxysilane ethanol solution for 0.5-24 h (preferably 12 h). It is repeatedly cleaned with ethanol and dried again to obtain a capillary tube with positively charged amino groups on its inner wall.

[0009] The Au TOH solution is drawn into the above capillary and left to stand (for 10 to 40 minutes, preferably 30 minutes). The capillary is then repeatedly washed with pure water and dried to obtain the treated capillary (i.e., SERS substrate, abbreviated as Au TOH / Cap).

[0010] The second step, chip manufacturing:

[0011] A first groove is formed 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. A second groove is provided at one end of the first groove, ensuring that the edge of the first groove is exactly on the tangent of the second groove.

[0012] The third step is the preparation of the injection device: bend the tip of the micro-injection needle 0.8~1.3 cm (preferably 1 cm) towards the back of the needle at a 90° angle, so that the tip of the micro-injection needle is perpendicular to the second groove on the chip. The micro-injection needle is then repeatedly ultrasonically cleaned with deionized water to remove air bubbles.

[0013] The fourth step involves using the injection pump and the chip in combination.

[0014] Place the capillary tube 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 of the chip on the side away from the first groove.

[0015] Step 5: Laser confocal Raman time-series detection of signal changes in the analyte.

[0016] The analyte solution was drawn into the injection device using a LongerPump micro-injection pump. The LongerPump was then turned on, and the analyte solution was slowly and uniformly injected into the second groove. A confocal Raman spectrometer was used to begin timing acquisition from the center point of the upper edge of the capillary inner wall in the first groove. The integration time and acquisition rate were set to obtain a series of spectra at the observation points within a certain time period, and the intermolecular equilibrium dissociation constant was calculated. ;

[0017] Step 6: Quantitative calculation of intermolecular interactions

[0018] (1) Experimental determination of diffusion coefficient

[0019] ① Experimental Design

[0020] Select small organic molecules, proteins, and solvent systems (usually water), and determine the diffusion coefficient of the molecules in solution at different temperatures T (e.g., 280 K, 300 K, 320 K, etc.). ;

[0021] The average intermolecular interaction between small organic molecules and proteins is activated by the diffusion of ligand molecules in solution. calculate;

[0022] ② Measurement method

[0023] Surface-enhanced Raman spectroscopy (SERS) is used to calculate the diffusion coefficient by measuring the directional diffusion of molecules in the system.

[0024] ③ Data Recording

[0025] Record the diffusion coefficient Di at different temperatures Ti to obtain a set of data points (Ti, Di);

[0026] (2) Fitting the relationship between diffusion coefficient and temperature

[0027] ① Linearization of the Arrhenius equation

[0028] diffusion coefficient The relationship with temperature T is described by the Arrhenius equation, namely:

[0029] ;

[0030] Taking the natural logarithm, we obtain the linearized form:

[0031] ;

[0032] In the formula, It is the diffusion activation energy. Pre-exponential factors are determined by the nature of the receptor system. It is the ideal gas constant. It is absolute temperature;

[0033] ② Data Fitting

[0034] calculate and ;

[0035] By fitting the data using linear regression, we obtain the slope m and the intercept b, i.e.:

[0036] ;

[0037] in:

[0038] slope ,

[0039] intercept ;

[0040] ③ Calculate the activation energy of diffusion :

[0041] The diffusion activation energy is obtained from the slope m. :

[0042] ;

[0043] (3) Calculate the average interaction energy

[0044] ① Relationship between diffusion activation energy and average interaction energy

[0045] Assuming diffusion activation energy It is mainly composed of the average interaction energy of small organic molecules and proteins in solvents. <eint>Decide:

[0046] ;

[0047] Where α is a proportionality constant; according to the traditional definition <eint>It refers to the average interaction energy between a small organic molecule and a protein in a solvent;

[0048] ②The physical meaning of α

[0049] When organic small molecules and proteins are at equal concentrations during the experiment, the protein concentration is uniform throughout the microchannel. However, at the signal detection site, organic small molecules diffuse from the sample cell, resulting in a lower local concentration of organic small molecules compared to the protein concentration. Therefore, the diffusion activation energy obtained from the diffusion experiment... This reflects the average interaction energy between a small organic molecule and multiple proteins at the signal detection point. Therefore, the proportionality constant α is less than 1.0.

[0050] ③ Calculate the average interaction energy:

[0051] ;

[0052] (4) Calculate the equilibrium dissociation constant :

[0053] ① Equilibrium dissociation constant With average interaction energy <eint>The relationship is:

[0054] ;

[0055] in:

[0056] It is a constant related to the entropy change of the binding reaction, and its value needs to be determined experimentally. And obtain; It is the entropy change of the combination reaction, and K0 is the entropy change constant.

[0057] It is the ideal gas constant, R = 8.314 J·mol⁻¹ -1 ·K -1 ;

[0058] It is absolute temperature;

[0059] ② Determine the entropy constant K0

[0060] The entropy change of the combination reaction was measured experimentally. Then calculate:

[0061] ;

[0062] If K0 is unknown, we can assume K0=1, that is, ignore the effect of entropy change, and the calculation result is a relative value.

[0063] ③ Substitute into the formula to calculate

[0064] Known <eint>Substituting T, R, and K0 into the formula, the equilibrium dissociation constant is obtained. ;

[0065] ;

[0066] Controlled reaction free energy and relational formula It can be found that, .

[0067] The preparation method of the Au-TOH solution includes the following steps:

[0068] Hexadecyltrimethylammonium bromide (CTAB) was dissolved in water and stirred at a temperature of 25-30°C (preferably 27°C). HAuCl4 solution was added to the above solution, and then NaBH4 solution was rapidly added while stirring. The mixture was stirred and left to stand for 3-5 hours to obtain a solution of gold nanoclusters coated with CTAB.

[0069] A hexadecyltrimethylammonium chloride solution was mixed with a CTAB-coated gold nanocluster solution, and then ascorbic acid solution was added. After the reaction was complete, HAuCl4 solution was added. The reaction was carried out for 10-30 min (preferably 15 min), and then centrifuged (centrifuged at 10000 rpm for 25 min). The mixture was then resuspended in a hexadecyltrimethylammonium chloride solution to obtain an Au NSs solution.

[0070] Au NSs solution was added to hexadecyltrimethylammonium chloride solution, and then ascorbic acid solution was added to the mixed solution. After mixing, HAuCl4 solution was added to the above solution. The reaction was carried out at a temperature of 25~30℃ (preferably 27℃) for 10~30 min (preferably 15 min). The mixture was centrifuged (7000 rpm for 10 min), and then the precipitate was dispersed in a PVP ethanol solution with a mass fraction of 0.5~2% (preferably 1%). The mixture was sonicated (10 min), centrifuged (7000 rpm for 10 min), and the supernatant was removed to disperse the precipitate in ethanol to obtain Au TOH (nano gold trioctahedral) solution.

[0071] The first groove is a semi-circular groove with a depth of 0.5 mm and a length of 15 mm.

[0072] The second groove is a cylindrical groove with a diameter of 3 mm and a depth of 1.1 mm.

[0073] The micro-injection needle has a tube length of 10 cm and a maximum volume of 50 μL.

[0074] The settings for the confocal Raman detector: HORIBA XploRA PLUS, parameters set as follows: Range: 400~1400 cm -1 ; Acq time(s): 1; Accumulation: 1; Objective: 10X; Grating: 1200(750 nm); Filter: 1%; Laser: 785 nm; Slit: 100 μm; Hole: 300 μm.

[0075] The organic small molecules that can be measured using the method of the present invention should have a certain SERS activity and be able to detect SERS signals.

[0076] The organic small molecules are selected from methylene blue (MB) and crystal violet (CV).

[0077] The protein is selected from bovine serum albumin (BSA).

[0078] The method of this invention for measuring intermolecular interactions has a wide range of applications: at the application level, there is a significant need for quantitative determination or rapid screening of ligand-receptor binding affinity in fields such as new drug discovery and drugability evaluation. The KID (Kinetic Interactive Diffusion) method (the method of this invention based on high-resolution confocal surface-enhanced Raman spectroscopy for rapid detection of intermolecular interactions using microchannels) provides a novel option for efficiently and accurately measuring intermolecular interactions. The KID method is particularly effective for biomolecules with relatively small molecular weights or lacking active groups that are difficult to immobilize on surfaces, and may even be the only solution for intermolecular interaction analysis, thus offering novel advantages in complex biomedical research scenarios.

[0079] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0080] The present invention provides a method for rapid detection of intermolecular interactions using high-resolution confocal surface-enhanced Raman spectroscopy based on microchannels. This method does not rely on molecular labeling or molecular immobilization, but is based on the principle of dynamic molecular interactive diffusion. Furthermore, it is ingeniously combined with the characteristics of microchannels—small quantity and high throughput—and has broad application prospects.

[0081] Essentially, the method of this invention is a homogeneous affinity evaluation method. Homogeneous techniques do not require solid-phase immobilization of the protein or nucleic acid target, thus more accurately reflecting the strength of the interaction between the target ligand molecule and the drug, avoiding steric hindrance and uncertainties that may arise from interface immobilization. The affinity value obtained in this case is called native affinity. Due to the elimination of the aforementioned potential interferences, homogeneous characterization techniques offer better reproducibility and more realistically reflect intermolecular interactions compared to heterogeneous techniques such as SPR and BLI. Furthermore, the KID method possesses the advantage of Raman spectroscopy multiplexing and overcomes the drawbacks of the MST method, such as cumbersome fluorescent labeling, photobleaching, and peak width limitations in fluorescence polychromaticity. Drug molecules and ligand molecules do not require prior mixing / incubation; instead, they interact, diffuse, and are detected simultaneously, significantly improving the analytical throughput. In addition, homogeneous characterization techniques are simpler to operate and have lower testing costs. The KID method adds another biologically orthogonal and complementary approach to the field of molecular interactions, effectively addressing the lack of methods for intrinsic affinity analysis.

[0082] This invention innovatively establishes a new technology and method for analyzing drug-target molecule interactions based on a novel principle of molecular dynamic interaction diffusion. It solves the scientific challenge of innocent, label-free determination of intermolecular interactions while preserving the structure and natural conformation of biomolecules. Attached Figure Description

[0083] Figure 1 This is a schematic diagram of the electron microscope image of nanoparticles and the SERS substrate.

[0084] Figure 2 This is a schematic diagram showing the performance results of the microchannel SERS substrate.

[0085] Figure 3 The graph shows the linear fit between 1 / T and ln D.

[0086] Figure 4 This is a schematic diagram of the testing platform.

[0087] Figure 5 This is a schematic diagram illustrating the principle of dynamic intermolecular interaction and diffusion in this experimental platform.

[0088] Figure 6 The graph shows the linear fitting relationship between 1 / T and lnD in Application Example 2. Detailed Implementation

[0089] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0090] The reagents required for this invention are: methylene blue (MB), bovine serum albumin (BSA), 4-mercaptobenzoic acid (4-MBA), 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.); and distilled water (Guangzhou Watsons Food & Beverage Co., Ltd.).

[0091] Example 1

[0092] Step 1: Preparation and characterization of nanoparticles and microchannels

[0093] Gold nanoparticles (Au NSs) were prepared using a seed-mediated growth method.

[0094] 0.365 g CTAB was dissolved in 5 mL of water and stirred at 27 °C. 5 mL of 0.5 mmol / L HAuCl4 solution was added to the solution, followed by rapid addition of 0.6 mL of 10 mmol / L NaBH4 solution while stirring. The mixture was stirred for 2 min and allowed to stand for 3–5 h to ensure complete reaction, yielding a solution of 3 nm gold nanoclusters coated with CTAB.

[0095] Mix 2 mL of 0.2 mol / L CTAC solution with 50 μL 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, and resuspend in 1 mL of 20 mmol / L CTAC solution to obtain Au NSs solution with a particle size of 10 nm. Store at 4℃ for later use.

[0096] 20 μL of Au NSs solution with a particle size of 10 nm was added to 10 mL of 0.1 mol / L CTAC solution, followed by 650 μL of 0.1 mol / L AA solution. After mixing, 10 mL of 0.5 mmol / L HAuCl4 solution was added to the mixture. The reaction was carried out at 27 °C for 15 min, followed by centrifugation at 7000 rpm for 10 min. The precipitate was then dispersed in 0.5 mL of 1% PVP ethanol solution, sonicated for 10 min, and centrifuged at 7000 rpm for 10 min. The supernatant was removed, and the precipitate was dispersed in 0.5 mL of ethanol to obtain a solution of 75 nm gold trioctahedral nanoparticles (Au TOH). The electron micrograph of these nanoparticles is shown below. Figure 1 As shown in Figure a. Figure 1 This image shows an electron microscope (EM) image of the nanoparticles and a SERS substrate. In the image, 'a' is an EEM image of the trioctahedral gold nanoparticles. The image clearly shows that the prepared nanoparticles are trioctahedral in shape, with a diameter of approximately 75 nanometers.

[0097] The second step involves the pretreatment of the quartz capillary (microchannel) as follows: First, the quartz capillary (outer diameter 1.0 mm, inner diameter 0.8 mm, length 15 mm) is soaked in acetone solution and sonicated. It is then ultrasonically cleaned several times alternately with ethanol and pure water. The capillary is then dried in an oven at 110°C. After drying, the capillary is immersed in a piranha solution (concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 3:1) for 1 hour. It is then ultrasonically cleaned with pure water until no bubbles remain on the inner wall of the capillary, and dried at 110°C with forced air. The dried capillary is then immersed in a 1% (v / v) ATPMS ethanol solution for 12 hours. It is repeatedly cleaned with ethanol to remove excess ATPMS and dried again to obtain a capillary with positively charged amino groups modified on its inner wall. Au-TOH solution is then drawn into the capillary using capillary action, allowing the gold nanoparticles to fill the entire capillary. After standing for 30 minutes, remove excess Au-TOOH solution and wash repeatedly with pure water. Dry the treated capillary (i.e., the SERS substrate, abbreviated as Au-TOOH / Cap) for later use. The resulting SERS substrate is shown below. Figure 1 As shown, Figure 1 The figures show an electron microscope (EM) image of the nanoparticles and a SERS substrate. In the figures, a is an EEM image of Au TOH, b is an EEM image of Au TOH on the inner wall of the capillary (showing that Au TOH has been monolayered on the inner wall of the capillary), c is an EEM image of the capillary modified with Au TOH, and d is an optical image of the capillary modified with Au TOH.

[0098] Stability, reproducibility, uniformity and sensitivity of microchannel SERS substrates

[0099] In SERS substrate detection, substrate homogeneity, reproducibility, and stability are important indicators for evaluating substrate performance. Therefore, to investigate the homogeneity, reproducibility, and stability of Au TOH / Cap, this invention uses methylene blue (MB) as a probe molecule, selecting 40 sites axially on the SERS substrate for SERS signal detection. During signal acquisition, capillary action is utilized, with a 10 μM MB solution directly aspirated into a capillary tube, and then sites are selected transversely along the capillary tube at 5 μm intervals. The results are obtained by analyzing the MB molecules at 447 cm⁻¹. -1 498 cm -1 Statistical analysis of peak intensity was performed, and the relative standard deviation was calculated. The results are as follows: Figure 2 As shown in (a) and (b), Figure 2 This is a schematic diagram of the performance results of the microchannel SERS substrate. Figures a and b show the SERS spectra of 40 sites selected during axial scanning of the capillary using AuTOH / Cap as the substrate, with statistical results of two characteristic peaks. Figure 2 In the middle, 'a' represents the characteristic peak at 447 cm⁻¹ recorded at 40 sites. -1 The intensity change of the peak intensity Figure 2 The peak at 498 cm⁻¹ is a characteristic peak recorded at 40 sites. -1 Peak intensity variation. As shown in the figure, 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 is similar at each location, and has good uniformity and reproducibility.

[0100] Using Au TOH / Cap as a substrate, 447 cm -1 498 cm -1 The relative standard deviations of the peak intensities were all less than 10%, demonstrating good homogeneity and reproducibility. Separately, using an Au-TOH / Cap substrate, MB solutions of the same concentration were collected continuously for 20 days to obtain a series of spectra and evaluate their stability. The measured MB peak intensities at 447 cm⁻¹... -1 The characteristic peak at that location hardly changed over 20 days, as shown in the results. Figure 2 As shown in (c) and (d), c and d are schematic diagrams of the SERS spectra and characteristic peak intensity values ​​of Au TOH / Cap over 20 days. Figure 2 In the middle, c is a collection of 20 spectra obtained by using different Au TOH / Caps to detect the same concentration of methylene blue for 20 consecutive days from the same batch of Au TOH / Caps. Figure 2 In Figure d, the characteristic peak at 447 cm⁻¹ in Figure c is... -1 The intensity variation was statistically analyzed, and the relative standard deviation was found to be less than 15%, indicating that the capillary SERS substrate has a stable reinforcement effect over a period of time and has good stability.

[0101] The above demonstrates that the Au TOH / Cap substrate exhibits good homogeneity, reproducibility, and stability.

[0102] The third step is chip fabrication: Multiple semi-circular grooves, 0.5 mm deep and 15 mm long, are fabricated on a polydimethylsiloxane (PDMS) chip (75 mm × 25 mm × 3 mm). These grooves are parallel to the long side of the chip and 5 mm from the port. The etched grooves allow the quartz capillary to be placed within them and fit completely. At one end of the 15 mm long groove, a cylindrical groove with a diameter of 3 mm and a depth of 1.1 mm is created, ensuring that the edge of the 15 mm long groove lies precisely on the tangent of the cylindrical groove. This process is repeated until a total of 10 grooves of the same size are fabricated on the chip.

[0103] Step 4, preparation of the injection device: Take a micro-injection needle with a length of 10 cm and a maximum volume of 50 uL. Bend the tip of the needle about 1 cm from the back of the needle at a 90° angle so that the tip of the micro-injection needle is perpendicular to the cylindrical groove on the chip. Clean the micro-injection needle repeatedly with deionized water using ultrasonication to remove air bubbles, and then set it aside for use.

[0104] Step 5: Using the injection pump and chip together

[0105] The capillary prepared in the second step was placed into a 15 mm long groove in the chip. The prepared injection device was then placed on a LongerPump micro-injection pump (TS-2A Syringe Pump Controller), which features precise stroke control and an ultra-wide range of linear velocities (7.9 mm / min–79.4 mm / min). After assembling the injection device, the tip of the micro-injection needle was suspended in the cylindrical groove on the chip, away from the 15 mm long groove, to reduce the influence of liquid flow on molecular diffusion. The injection speed was set on the LongerPump micro-injection pump so that the liquid in the injection device could be injected into the sample cell (i.e., the cylindrical groove) at a uniform and slow rate.

[0106] Step 6, Temperature control device

[0107] A semiconductor temperature control platform (customized by Wentian Precision Measurement Instruments Technology (Suzhou) Co., Ltd.) was used to cool or heat the entire testing platform. The temperatures were set to 283K, 293K, 303K, and 313K respectively, with the temperature difference controlled within + / -0.1℃. Figure 4 This is a schematic diagram of the testing platform.

[0108] Step 7: Time-series detection of signal changes in the analyte using laser confocal Raman spectroscopy.

[0109] Confocal Raman spectrometer: HORIBA XploRA PLUS, parameters set as follows: Range: 400~1400 cm -1 ;Acq time(s): 1; Accumulation: 1; Objective: 10X; Grating: 1200(750 nm);Filter: 1%; Laser: 785 nm; Slit: 100 μm; Hole: 300 μm.

[0110] The analyte was placed on the stage, and Raman spectroscopy was performed using a 10x microscope to locate the center point of the upper edge of the capillary wall in the groove. The analyte solution was drawn into the injection device using a LongerPump micropump, and the needle of the injection device was suspended in the cylindrical groove on the chip, away from the 15 mm long groove. The LongerPump micropump was turned on, and the analyte solution was slowly and uniformly injected into the sample cell (i.e., the cylindrical groove). Simultaneously, a confocal Raman detector was used to begin sequential acquisition at the center point of the upper edge of the capillary wall in the groove. The integration time and acquisition speed were set to obtain a series of spectra at the observation point within a certain time. After background removal, the series of spectra were analyzed. Based on the diffusion state of the analyte in the macromolecular solution under different temperature conditions, and combined with information such as liquid viscosity, solution density, and molecular weight, the intermolecular equilibrium dissociation constant was calculated. Based on the obtained Raman spectra, this method can accurately determine intermolecular interaction forces.

[0111] Step 8: Quantitative calculation of intermolecular interactions

[0112] (1) Experimental determination of diffusion coefficient

[0113] ① Experimental Design

[0114] Choose appropriate small organic molecules (ligands), common proteins (receptors), and solvent systems (usually water).

[0115] Organic small molecules that can be measured using the method of this invention should have certain SERS activity and be able to detect SERS signals.

[0116] The diffusion coefficient D of molecules in solution was determined at different temperatures T (such as 280 K, 300 K, 320 K, etc.).

[0117] The average intermolecular interaction between small organic molecules and proteins is activated by the diffusion of ligand molecules in solution, through the energy Q. d calculate.

[0118] ② Measurement method

[0119] Surface-enhanced Raman spectroscopy (SERS) is used to calculate the diffusion coefficient by measuring the directional diffusion of molecules in the system.

[0120] ③ Data Recording

[0121] Record the diffusion coefficient Di at different temperatures Ti to obtain a set of data points (Ti, Di).

[0122] (2) Fitting the relationship between diffusion coefficient and temperature

[0123] ① Linearization of the Arrhenius equation

[0124] diffusion coefficient The relationship with temperature T is described by the Arrhenius equation, namely:

[0125]

[0126] Taking the natural logarithm, we obtain the linearized form:

[0127]

[0128] In the formula, It is the diffusion activation energy. Pre-exponential factors are determined by the nature of the receptor system. It is the ideal gas constant. It is absolute temperature.

[0129] ② Data Fitting

[0130] calculate and ;

[0131] By fitting the data using linear regression, we obtain the slope m and the intercept b, i.e.:

[0132]

[0133] in:

[0134] slope ,

[0135] intercept .

[0136] ③ Calculate the activation energy of diffusion

[0137] The diffusion activation energy is obtained from the slope m. :

[0138]

[0139] (3) Calculate the average interaction energy

[0140] ① Relationship between diffusion activation energy and average interaction energy

[0141] Since the diffusion of ligand molecules requires overcoming the attraction from acceptors in solution, we assume a diffusion activation energy. It is mainly composed of the average interaction energy of small organic molecules and proteins in solvents. <eint>Decide:

[0142]

[0143] Where α is a proportionality constant. According to the traditional definition, <eint>It refers to the average interaction energy between a small organic molecule and a protein in a solvent.

[0144] ②The physical meaning of α

[0145] When organic small molecules and proteins are at equal concentrations during the experiment, the protein concentration is uniform throughout the microchannel. However, at the signal detection site, organic small molecules diffuse from the sample cell, resulting in a lower local concentration of organic small molecules compared to the protein concentration. Therefore, the diffusion activation energy obtained from the diffusion experiment... This reflects the average interaction energy between a small organic molecule and multiple proteins at the signal detection point. Therefore, the proportionality constant α is less than 1.0.

[0146] ③ Calculate the average interaction energy:

[0147]

[0148] (4) Calculate the equilibrium dissociation constant

[0149] ① Equilibrium dissociation constant With average interaction energy <eint>The relationship is:

[0150]

[0151] in:

[0152] K0 is a constant related to the entropy change of the binding reaction. This value needs to be obtained by experimentally measuring the entropy change ΔS of the binding reaction; ΔS is the entropy change of the binding reaction, and K0 is the entropy change constant.

[0153] It is the ideal gas constant, R = 8.314 J·mol⁻¹ -1 ·K -1 ;

[0154] It is absolute temperature.

[0155] ② Determine the entropy constant K0

[0156] The entropy change ΔS of the binding reaction was measured experimentally, and then calculated:

[0157]

[0158] If K0 is unknown, we can assume K0=1, that is, ignore the effect of entropy change, and the calculation result is a relative value.

[0159] ③ Substitute into the formula to calculate

[0160] Known <eint>Substituting T, R, and K0 into the formula, the equilibrium dissociation constant is obtained. .

[0161]

[0162] Controlled reaction free energy and relational formula It can be found that, Equilibrium dissociation constant The magnitude of the constant reflects the strength of intermolecular forces. A large constant indicates weak intermolecular forces, while a small constant indicates strong intermolecular forces.

[0163] Application Example 1

[0164] Detection of intermolecular interactions between methylene blue (MB) and bovine serum albumin (BSA) in water:

[0165] Following the method in Example 1, the concentration was measured to be 1×10⁻⁶. -5 Methylene blue (MB) solution (water as solvent) and a concentration of 1×10 -5 The equilibrium dissociation constant between bovine serum albumin (BSA) solutions (with water as the solvent).

[0166] The semiconductor chip was subjected to different temperature conditions (283K, 293K, 303K, 313K), and the diffusion of MB solution in BSA solution was measured under different temperature conditions. The diffusion coefficient was then calculated. The diffusion coefficients of MB solution in BSA solution at different temperatures were obtained, and the results are shown in Table 1.

[0167] Table 1

[0168]

[0169] Substitute different temperatures and the diffusion coefficient values ​​at those temperatures into the formula. In the middle, the diffusion activation energy is obtained. .

[0170] According to the following formula:

[0171]

[0172] In the MB and BSA solution system, it is assumed that the average concentration ratio at the signal detection point is 1 small molecule to 4.5 protein molecules (average value) (α is a proportionality constant less than 1; during the experiment, the concentrations of ligand and protein are set to be equal, and the protein concentration is consistent throughout the microchannel). However, at the signal detection point, the ligand small molecule diffuses from the sample cell, and the local concentration of the small molecule is lower than the protein concentration. Therefore, the diffusion activation energy obtained from the diffusion experiment... This reflects the average interaction energy between a single ligand molecule and several proteins at the signal detection point. Therefore, the proportionality constant α is less than 1.0. Molecular simulations show that the average concentration ratio of MB to BSA at the signal detection point in this solution system is... Substituting into the above formula, we obtain:

[0173]

[0174] Calculate using the following formula:

[0175]

[0176] Finally, the solutions of MB and BSA at 303 K were obtained. The value is 8.11 × 10 -6 The value was consistent with the reported value in the literature (Zhao Gang, Yang Dan, He Qian, et al., "Study on the Interaction between Methylene Blue and its Metabolites with Bovine Serum Albumin") (5.0 × 10⁻⁶). -6 (Very close) Figure 3 The graph shows the linear fitting relationship between 1 / T and ln D in Application Example 1. Figure 5 This is a schematic diagram illustrating the principle of dynamic intermolecular interaction and diffusion in this experimental platform.

[0177] Application Example 2

[0178] Detection of the intermolecular interaction between crystal violet (CV) and bovine serum albumin (BSA):

[0179] Following the method in Example 1, the concentration was measured to be 1×10⁻⁶. -5 M crystal violet (CV) solution (solvent: water) and a concentration of 1×10 -5 The equilibrium dissociation constant between bovine serum albumin (BSA) solutions (with water as the solvent).

[0180] The semiconductor chip was subjected to different temperature conditions (288K, 298K, 308K, 318K, 328K), and the diffusion of the CV solution in the BSA solution was measured under different temperature conditions. The diffusion coefficient was then calculated. The diffusion coefficients of CV solution in BSA solution at different temperatures were obtained, and the results are shown in Table 2.

[0181] Table 2

[0182]

[0183] Substitute different temperatures and the diffusion coefficient values ​​at those temperatures into the formula. In the middle, the diffusion activation energy is obtained. .

[0184] According to the following formula:

[0185]

[0186] In the CV and BSA solution system, assuming the average concentration ratio at the signal detection point is 1 small molecule to 4.5 protein molecules (average), substituting into the above formula yields:

[0187]

[0188] Calculate according to the following formula

[0189]

[0190] Finally, the solutions for CV and BSA at 298K were obtained. The value is 5.99 × 10 -6 The value reported in the literature (Kong Dexian, Zhou Xuechou, Wang Yulin, et al. Study on the interaction between crystal violet and bovine serum albumin [J]. Strait Pharmaceutical Journal, 2013, 25(07):200-202.) is 6.71×10 -6 (Very close) Figure 6 The graph shows the linear fitting relationship between 1 / T and lnD in Application Example 2.

[0191] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.< / eint> < / eint> < / eint> < / eint> < / eint> < / eint> < / eint> < / eint>

Claims

1. A method for rapid detection of intermolecular interactions using high-resolution confocal surface-enhanced Raman spectroscopy based on microchannels, characterized in that, Includes the following steps: The first step involves immersing the capillary tube sequentially in acetone, ethanol, and pure water, followed by ultrasonic cleaning several times. The capillary tube is then dried and immersed in a piranha solution for 0.5–2 hours. After ultrasonic cleaning with pure water until no bubbles remain on the inner wall of the capillary tube, it is dried again. The dried capillary tube is then immersed in a 0.5–3% (v / v) 3-aminopropyltrimethoxysilane ethanol solution for 0.5–24 hours. It is then repeatedly cleaned with ethanol and dried again to obtain a capillary tube with positively charged amino groups on its inner wall. The Au-TOH solution was drawn into the capillary tube, allowed to stand, and repeatedly washed with pure water and dried to obtain the treated capillary tube. The second step, chip manufacturing: 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. 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 of the second groove. The third step is the preparation of the injection device: bend the tip of the micro-injection needle 0.8~1.3 cm towards the back of the needle at a 90° angle so that the tip of the micro-injection needle is perpendicular to the second groove on the chip. The micro-injection needle is then repeatedly ultrasonically cleaned with deionized water to remove air bubbles. The fourth step involves using the injection pump and the chip in combination. The capillary tube prepared in the first step is placed in the first groove of the chip prepared in the second step. The prepared injection device is placed on the LongerPump micro-injection pump. The needle of the injection device is suspended in the second groove of the chip on the side away from the first groove. Step 5: Laser confocal Raman time-series detection of signal changes in the analyte. The analyte solution was drawn into the injection device using a LongerPump micro-injection pump. The LongerPump was then turned on, and the analyte solution was slowly and uniformly injected into the second groove. A confocal Raman spectrometer was used to begin timing acquisition from the center point of the upper edge of the capillary inner wall in the first groove. The integration time and acquisition rate were set to obtain a series of spectra at the observation points within a certain time period, and the intermolecular equilibrium dissociation constant was calculated. ; Step 6: Quantitative calculation of intermolecular interactions (1) Experimental determination of diffusion coefficient ① Experimental Design By selecting small organic molecules, proteins, and solvent systems, the diffusion coefficients of the molecules in solution were determined at different temperatures (T). ; The average intermolecular interaction between small organic molecules and proteins is activated by the diffusion of ligand molecules in solution. calculate; ② Measurement method Surface-enhanced Raman spectroscopy is used to calculate the diffusion coefficient by measuring the directional diffusion of molecules in the solution within the system. ③ Data Recording Record the diffusion coefficient Di at different temperatures Ti to obtain a set of data points (Ti, Di); (2) Fitting the relationship between diffusion coefficient and temperature ① Linearization of the Arrhenius equation diffusion coefficient The relationship with temperature T is described by the Arrhenius equation, namely: ； Taking the natural logarithm, we obtain the linearized form: ； In the formula, It is the diffusion activation energy. Pre-exponential factors are determined by the nature of the receptor system. It is the ideal gas constant. It is absolute temperature; ② Data Fitting calculate and ; By fitting the data using linear regression, we obtain the slope m and the intercept b, i.e.: ； in: slope , intercept ; ③ Calculate the activation energy of diffusion : The diffusion activation energy is obtained from the slope m. : ； (3) Calculate the average interaction energy ① Relationship between diffusion activation energy and average interaction energy Assuming diffusion activation energy It is mainly composed of the average interaction energy of small organic molecules and proteins in solvents. <eint> Decide:< / eint> ； Where α is a proportionality constant; according to the traditional definition <eint> It refers to the average interaction energy between a small organic molecule and a protein in a solvent;< / eint> ②The physical meaning of α The proportionality constant α is less than 1.0; ③ Calculate the average interaction energy: ； (4) Calculate the equilibrium dissociation constant : ① Equilibrium dissociation constant With average interaction energy <eint> The relationship is:< / eint> ； in: It is a constant related to the entropy change of the binding reaction, and its value needs to be determined experimentally. And obtain; It is the entropy change of the combined reaction, and K0 is the entropy change constant; It is the ideal gas constant, R = 8.314 J·mol⁻¹ -1 ·K -1 ; It is absolute temperature; ② Determine the entropy constant K0 The entropy change of the combination reaction was measured experimentally. Then calculate: ； If K0 is unknown, we can assume K0=1, that is, ignore the effect of entropy change, and the calculation result is a relative value. ③ Substitute into the formula to calculate : Known <eint>Substituting T, R, and K0 into the formula, the equilibrium dissociation constant is obtained. ;< / eint> ； Controlled reaction free energy and relational formula It can be found that, .

2. The method for rapid detection of intermolecular interactions based on high-resolution confocal surface-enhanced Raman spectroscopy using microchannels according to claim 1, characterized in that, The preparation method of the Au-TOH solution includes the following steps: Hexadecyltrimethylammonium bromide (CTAB) was dissolved in water and stirred at 25-30°C. HAuCl4 solution was added to the above solution, followed by rapid addition of NaBH4 solution under stirring. The mixture was stirred and allowed to stand for 3-5 hours to obtain a solution of gold nanoclusters coated with CTAB. Hexadecyltrimethylammonium chloride solution was mixed with CTAB-coated gold nanoclusters solution, then ascorbic acid solution was added. After the reaction was complete, HAuCl4 solution was added. After reacting for 10-30 min, the mixture was centrifuged and resuspended in hexadecyltrimethylammonium chloride solution to obtain Au NSs solution. Au NSs solution was added to hexadecyltrimethylammonium chloride solution, and then ascorbic acid solution was added to the mixture. After mixing, HAuCl4 solution was added to the above solution. The reaction was carried out at 25~30℃ for 10~30 min. After centrifugation, the precipitate was dispersed in a 0.5~2% PVP ethanol solution, sonicated, centrifuged, and the supernatant was removed to disperse the precipitate in ethanol to obtain Au TOH solution.

3. The method for rapid detection of intermolecular interactions based on high-resolution confocal surface-enhanced Raman spectroscopy using microchannels according to claim 1, characterized in that, The first groove is a semi-circular groove with a depth of 0.5 mm and a length of 15 mm.

4. The method for rapid detection of intermolecular interactions based on high-resolution confocal surface-enhanced Raman spectroscopy using microchannels 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.

5. The method for rapid detection of intermolecular interactions based on high-resolution confocal surface-enhanced Raman spectroscopy using microchannels according to claim 1, characterized in that, The micro-injection needle has a tube length of 10 cm and a maximum volume of 50 μL.

6. The method for rapid detection of intermolecular interactions based on high-resolution confocal surface-enhanced Raman spectroscopy using microchannels according to claim 1, characterized in that, The settings for the confocal Raman detector: HORIBA XploRA PLUS, parameters set as follows: Range: 400~1400 cm -1 ; Acq time(s): 1; Accumulation: 1; Objective: 10X; Grating: 1200(750 nm); Filter: 1%; Laser: 785 nm; Slit: 100 μm; Hole: 300 μm.

7. The method for rapid detection of intermolecular interactions based on high-resolution confocal surface-enhanced Raman spectroscopy using microchannels according to claim 1, characterized in that, The organic small molecules that can be measured using the method of the present invention should have a certain SERS activity and be able to detect SERS signals.

8. The method for rapid detection of intermolecular interactions based on high-resolution confocal surface-enhanced Raman spectroscopy using microchannels according to claim 7, characterized in that, The organic small molecules are selected from methylene blue and crystal violet.

9. The method for rapid detection of intermolecular interactions based on high-resolution confocal surface-enhanced Raman spectroscopy using microchannels according to claim 1, characterized in that, The protein was selected from bovine serum albumin.

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