A method for biologic fluorescence characterization of flow rate in a transparent formation

Abstract

The present application relates to geotechnical engineering disaster whole process monitoring technical field, and disclose a kind of transparent stratum seepage flow rate bioluminescence characterization method, comprising the following steps: S1. extract luciferin and luciferase;S2. preparation has different shear failure intensity of biocompatibility package microcapsule sequence, and the luciferase is wrapped in the biocompatibility package microcapsule sequence inside.This transparent stratum seepage flow rate bioluminescence characterization method, based on the biochemical self-luminous characteristic, uses luciferin-luciferase system to self-catalyze oxidation luminescence after microcapsule rupture, completely replaces the external excitation light source required by traditional fluorescence tracing.This design fundamentally eliminates the shadow zone and stray light interference caused by particle refraction and scattering in complex porous medium, significantly improves the signal-to-noise ratio and image clarity of weakly cemented rock mass flow field monitoring, and high-fidelity luminescence image can be obtained in the full depth range of model.

Description

Technical Field

[0001] This invention relates to the field of whole-process monitoring technology for geological disasters in geotechnical engineering, specifically a biofluorescence characterization method for seepage velocity in transparent strata. Background Technology

[0002] Weakly cemented aquifers are widely distributed in arid mining areas of western my country. These aquifers are characterized by loose structure, low cementation strength, and high pore connectivity. Under the combined effects of mining unloading and groundwater seepage, they are prone to structural damage, erosion and increased permeability, abrupt changes in runoff, and formation collapse. Studying the pore water seepage velocity characteristics in weakly cemented aquifers is crucial for revealing their instability and collapse mechanisms. In recent years, the development of transparent geophysical model testing technology has provided new insights into the visualization of complex seepage processes in weakly cemented aquifers. By constructing pore fluids with refractive indices matching those of the framework particles and combining this with methods such as fluorescence tracing, ultraviolet excitation, and high-resolution imaging, the dynamic evolution and connectivity topology changes of seepage channels can be monitored in real time.

[0003] However, existing methods for testing seepage velocity have significant limitations. Differential pressure methods and tracer penetration methods are mostly used for macroscopic average velocity measurements, making it difficult to reflect the non-uniform pore velocity distribution within porous media. Particle image velocimetry typically relies on external light sources and transparent media conditions, making it unsuitable for complex porous media systems such as weakly cemented aquifers. Traditional fluorescent tracers primarily reflect solute concentration migration processes and require external excitation sources; in transparent formations, they are prone to refracted shadows and stray light interference, making it impossible to directly establish a quantitative correlation between flow velocity and hydraulic shear. Therefore, there is currently a lack of a highly sensitive in-situ monitoring method that can respond to local shear stress changes, requires no external excitation, and enables quantitative characterization of flow velocity. Summary of the Invention

[0004] To address the problems mentioned in the background section, this invention provides the following technical solution: a method for characterizing the biofluorescence of seepage velocity in transparent formations, comprising the following steps:

[0005] S1. Extraction of luciferin and luciferase;

[0006] S2. Prepare biocompatible encapsulated microcapsule sequences with different shear failure strengths, and encapsulate the luciferase inside the biocompatible encapsulated microcapsule sequences;

[0007] S3. Dissolve the luciferin in the infiltration fluid to form a continuous medium, and disperse the biocompatible microcapsule sequence containing the luciferase therein to form a tracer suspension, and then inject it into a transparent formation physical model;

[0008] S4. When the tracer suspension flows through the transparent formation pores, the biocompatible encapsulated microcapsule sequence is triggered to rupture by hydraulic shear force. The released luciferase comes into contact with the luciferin in the continuous medium and emits light. The luminescence image is collected, and the light intensity signal field matrix is ​​obtained.

[0009] S5. Establish a quantitative relationship model between the light intensity signal field matrix and the seepage velocity field matrix, and transform the collected light intensity signal field matrix into the seepage velocity field matrix to characterize the seepage velocity.

[0010] Preferably, in step S1, luciferin and luciferase are extracted from the single-celled planktonic organism Noctiluca scintillans using a fractional extraction method and an organic solvent method. The luciferase is purified and stored in a buffer solution, and the luciferin is used as the stock solution.

[0011] Preferably, in step S2, biocompatible encapsulated microcapsule sequences with a particle size of 50-500 μm and a specific shear failure strength are prepared using microfluidic technology, and the luciferase is injected into biocompatible encapsulated microcapsule sequences of different strengths.

[0012] Preferably, the biocompatible encapsulated microcapsule sequence is prepared from sodium alginate, gelatin, chitosan, divalent calcium ions and transglutaminase in an environment with a pH of 6.0-8.0 and a temperature of 4℃-37℃.

[0013] Preferably, the biocompatible encapsulated microcapsule sequence is made of the following components in parts by weight: 1-5 parts sodium alginate, 2-10 parts gelatin, and 0.5-2 parts chitosan, and is solidified by a divalent calcium ion solution with a concentration of 0.1-0.5 mol / L, and further cross-linked by transglutaminase with an activity unit of 10-100 U / g.

[0014] Preferably, in step S3, the injection process requires that the tracer suspension be kept in a light-protected environment and the injection flow rate be lower than the lowest shear failure threshold in the biocompatible encapsulated microcapsule sequence, so as to ensure that the luciferase does not come into contact with luciferin before entering the transparent formation.

[0015] Preferably, in step S4, an EMCCD camera is used to capture bioluminescent images within the flow field in real time, and the light intensity signal field matrix is ​​represented as:

[0016]

[0017] in, The light intensity signal field matrix, Indicates the first Liede The light intensity value of the line, From 1 to Any integer between From 1 to Any integer between , The value is known.

[0018] Preferably, in step S5, the quantitative relationship model is established based on the Weber distribution and the Beer-Lambert optical attenuation principle, specifically expressed as follows:

[0019]

[0020] in, The seepage velocity field matrix is... Light intensity signal field matrix, The maximum intensity signal field matrix, The characteristic intensity modulus of the microcapsule sequence, For gradient shape factor, The dynamic viscosity of the pore fluid. The pore diameter is Porosity This is the magnification factor of the pore structure. , , , , All values ​​are known.

[0021] Preferably, in step S4, during the acquisition of bioluminescence images, the luminescence intensity under different pH environments is calibrated in real time through intelligent spectroradiometric brightness analysis to eliminate the influence of environmental acidity and alkalinity changes on chemiluminescence dynamics.

[0022] Preferably, the method is used for non-contact in-situ quantitative characterization of the seepage velocity field in a physical simulation test model of a weakly cemented aquifer.

[0023] Compared with existing technologies, this invention provides a method for characterizing the biofluorescence of seepage velocity in transparent formations, which has the following advantages:

[0024] 1. This bioluminescence characterization method for seepage velocity in transparent formations, based on the biochemical self-luminescence properties, employs a luciferin-luciferase system that spontaneously catalyzes oxidation and emission after microcapsule rupture, completely replacing the external excitation light source required for traditional fluorescence tracers. This design fundamentally eliminates shadow areas and stray light interference caused by particle refraction and scattering in complex porous media, significantly improving the signal-to-noise ratio and image clarity for flow field monitoring in weakly cemented rock masses, and enabling the acquisition of high-fidelity luminescence images across the entire depth range of the model.

[0025] 2. This method for characterizing the biofluorescence of seepage velocity in transparent formations utilizes microfluidic technology to prepare biocompatible encapsulated microcapsule sequences with different shear failure intensities, encapsulating luciferase within them. The microcapsules are then ruptured using pore hydraulic shear force, converting invisible local hydraulic shear stress into quantifiable biofluorescence signals. The faster the seepage velocity, the more ruptured microcapsules there are, resulting in stronger luminescence intensity. This establishes a direct logical correlation between hydrodynamic characteristics and luminescence intensity, enabling sensitive capture and quantitative characterization of seepage velocities over a wide range (0.01 mm / s to 20 mm / s).

[0026] 3. This bioluminescence characterization method for seepage velocity in transparent formations, based on the Weber distribution and the Beer-Lambert optical attenuation principle, establishes a quantitative relationship model between the light intensity signal field matrix and the seepage velocity field matrix, considering hydraulic shear stress and environmental pH corrections. By acquiring luminescence images in real time using an EMCCD camera and retrieving the velocity field pixel-by-pixel, a non-contact, high spatial resolution (sub-millimeter level) in-situ quantitative characterization of seepage velocity in transparent formations is achieved. This provides a precise quantitative monitoring method for studying the instability evolution mechanism of weakly cemented aquifers under the combined effects of mining unloading and groundwater seepage. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the steps in the biofluorescence characterization method for seepage velocity in transparent formations according to the present invention. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1

[0030] Please see Figure 1 This embodiment provides a biofluorescence characterization method for seepage velocity in transparent formations, used for non-contact in-situ quantitative characterization of the seepage velocity field in a physical simulation test model of weakly cemented aquifers. Specifically, it includes the following steps:

[0031] S1. Extraction of luciferin and luciferase

[0032] Luciferin and luciferase were extracted from the single-celled planktonic algae *Noctilucascintillans* using a fractional extraction method and an organic solvent extraction method. The specific procedures were as follows: *Noctilucascintillans* cells cultured to the logarithmic growth phase were collected by centrifugation and washed twice with pre-chilled phosphate-buffered saline (PBS, pH 7.4). The cells were resuspended in lysis buffer containing 1% Triton X-100 and sonicated on ice. The lysis buffer was centrifuged at 12,000 rpm for 20 minutes at 4°C, and the supernatant was the crude enzyme solution. Luciferase was purified by ammonium sulfate fractionation (40%-60% saturation) and DEAE-Sepharose anion exchange chromatography. The purified luciferase was stored in PBS buffer containing 50% glycerol at -20°C for later use. Luciferin was extracted using ethyl acetate extraction: the above lysis buffer was mixed with an equal volume of ethyl acetate and shaken. After standing and separation, the organic phase was collected and concentrated by rotary evaporation to obtain the luciferin stock solution, which was stored at -80°C in the dark.

[0033] S2. Preparation of biocompatible microcapsule sequences encapsulating luciferase

[0034] Biocompatible microcapsule sequences with different shear failure strengths were prepared using microfluidic technology, and the luciferase extracted in step S1 was injected into the microcapsules. The materials and proportions for microcapsule preparation are as follows: 3 parts sodium alginate, 6 parts gelatin, and 1 part chitosan were dissolved in deionized water, and the pH was adjusted to 7.0 to form an aqueous phase. Microcapsules with a particle size of 50-500 μm were prepared using a microfluidic chip (channel width 200 μm) with the aqueous phase as the inner phase and vegetable oil containing 0.3 mol / L CaCl2 as the outer phase. After collecting the microcapsules, they were solidified in a 0.3 mol / L CaCl2 solution for 30 minutes, and then transferred to a solution containing 50 U / g transglutaminase. Cross-linking was enhanced at 25 °C for 12 hours to obtain microcapsule sequences with specific shear failure strengths. By adjusting the ratio of gelatin to sodium alginate and the cross-linking time, five microcapsules with shear failure thresholds of 0.5 Pa, 1.0 Pa, 2.0 Pa, 5.0 Pa, and 10.0 Pa, respectively, were prepared. The luciferase solution purified in step S1 (concentration 0.5 mg / mL) was injected into these microcapsules using microinjection technology, with the injection volume approximately 30% of the volume of each microcapsule. The injected microcapsules were stored at 4°C protected from light for later use.

[0035] S3. Prepare the tracer suspension and inject it into the physical model.

[0036] The luciferin stock solution extracted in step S1 was diluted 100 times with deionized water, and then dissolved in the infiltration fluid (deionized water) at a volume ratio of 1:1000 to form a continuous medium. The microcapsule sequences encapsulating luciferase prepared in step S2 were mixed in equal mass ratios and dispersed in the above continuous medium at a total concentration of 1 g / L to form a tracer suspension. The entire preparation process was carried out in a dark room and stored in brown containers.

[0037] A constant-flow-driven device (injection pump) was used to inject the tracer suspension into a physical simulation model of a weakly cemented aquifer. The physical model was filled with transparent quartz sand (particle size 0.5-1.0 mm), with a porosity of approximately 0.38, and the model dimensions were 300 mm × 200 mm × 50 mm. The injection flow rate was controlled at 0.1 mL / min, which is lower than the minimum shear failure threshold of the microcapsule sequence prepared in step S2 (the critical flow rate corresponding to 0.5 Pa is approximately 0.2 mL / min), thus ensuring that the luciferase would not leak prematurely due to tubular shear before entering the aquifer.

[0038] S4. Acquire bioluminescence images and obtain the light intensity signal field matrix.

[0039] When the tracer suspension entered the physical model, microcapsules with different intensity thresholds ruptured sequentially under the action of pore hydraulic shear force. In areas with higher local seepage velocities, more microcapsules ruptured, releasing luciferase which contacted luciferin in the continuous medium, undergoing a catalytic oxidation luminescence reaction in the presence of oxygen, producing strong bioluminescence. An EMCCD camera (Andor iXon Ultra 888) was used to capture real-time images of the bioluminescence within the flow field. The camera exposure time was set to 1 second, the gain to 300, and the acquisition frequency to 1 frame / second. During acquisition, the pH value at different locations within the model was monitored in real-time using a smart spectroradiometer (CS-2000), and the luminescence intensity was calibrated based on the pH value using the following calibration formula: ,in The neutral reference value is 7.0. After denoising and background subtraction, the acquired image yields the light intensity signal field matrix. Its size is In this embodiment , .

[0040] S5. Establish a quantitative model of light intensity-flow velocity and characterize the flow velocity field.

[0041] Based on the Weber distribution and the Beer-Lambert optical attenuation principle, a relationship model between the light intensity signal field matrix and the seepage velocity field matrix is ​​established. Let the maximum light intensity signal field matrix be... This value was obtained through a prior calibration experiment at a known maximum flow rate (10 mm / s in this embodiment). The known parameters in the model are as follows: microcapsule characteristic intensity modulus. Gradient shape factor Pore ​​fluid dynamic viscosity pore diameter Porosity Pre-obtained via CT scan (in this embodiment) (a constant), pore structure amplification factor Substitute the above parameters into the relational model:

[0042]

[0043] The seepage velocity field matrix is ​​calculated pixel by pixel. Finally, the velocity field is output as a pseudo-color image, realizing a visualized and quantitative characterization of seepage velocity in transparent formations. Compared with the standard microelectrode velocimetry method, the average relative error of the velocity measured by the method in this embodiment is less than 8%, and the spatial resolution reaches the sub-millimeter level.

[0044] Effect verification

[0045] To verify the technical effectiveness of this invention, a control group was set up: a conventional sodium fluorescein tracer (excitation wavelength 488 nm) was used in conjunction with ultraviolet light irradiation, and flow velocity measurements were performed in the same physical model. The results showed that the conventional method, due to refraction and scattering by transparent quartz sand particles, resulted in blurred images at model depths exceeding 2 cm, and could not establish a direct quantitative relationship between fluorescence intensity and flow velocity, only obtaining the average velocity of solute migration. In contrast, the method of this invention utilizes bioluminescence, eliminating the need for an external excitation light source and avoiding refraction and scattering interference, achieving clear images across the entire model depth (5 cm). Furthermore, due to the shear-triggered rupture mechanism of the microcapsules, the luminescence intensity exhibits a monotonic relationship with the local flow velocity, enabling quantitative characterization of flow velocity. Moreover, the method of this invention has a minimum measurable flow velocity of 0.01 mm / s and a maximum measurable flow velocity of 20 mm / s, covering the typical range of seepage in weakly cemented aquifers.

[0046] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for characterizing the biofluorescence of seepage velocity in transparent formations, characterized in that, Includes the following steps: S1. Extraction of luciferin and luciferase; S2. Prepare biocompatible encapsulated microcapsule sequences with different shear failure strengths, and encapsulate the luciferase inside the biocompatible encapsulated microcapsule sequences; S3. Dissolve the luciferin in the infiltration fluid to form a continuous medium, and disperse the biocompatible microcapsule sequence containing the luciferase therein to form a tracer suspension, and then inject it into a transparent formation physical model; S4. When the tracer suspension flows through the transparent formation pores, the biocompatible encapsulated microcapsule sequence is triggered to rupture by hydraulic shear force. The released luciferase comes into contact with the luciferin in the continuous medium and emits light. The luminescence image is collected, and the light intensity signal field matrix is ​​obtained. S5. Establish a quantitative relationship model between the light intensity signal field matrix and the seepage velocity field matrix, and transform the collected light intensity signal field matrix into the seepage velocity field matrix to characterize the seepage velocity.

2. The method for characterizing biofluorescence of seepage velocity in transparent formations according to claim 1, characterized in that, In step S1, luciferin and luciferase are extracted from the single-celled planktonic algae Noctiluca scintillans using a fractional extraction method and an organic solvent method. The luciferase is purified and stored in a buffer solution, while the luciferin is used as the stock solution.

3. The method for characterizing biofluorescence of seepage velocity in transparent formations according to claim 1, characterized in that, In step S2, biocompatible encapsulated microcapsule sequences with a particle size of 50-500 μm and specific shear failure strength are prepared using microfluidic technology, and the luciferase is injected into biocompatible encapsulated microcapsule sequences with different strengths.

4. The method for characterizing biofluorescence of seepage velocity in transparent formations according to claim 1, characterized in that, The biocompatible encapsulated microcapsule sequence was prepared from sodium alginate, gelatin, chitosan, divalent calcium ions, and transglutaminase at a pH of 6.0-8.0 and a temperature of 4℃-37℃.

5. The method for characterizing biofluorescence of seepage velocity in transparent formations according to claim 1, characterized in that, The biocompatible encapsulated microcapsule sequence is made of the following components in parts by weight: 1-5 parts sodium alginate, 2-10 parts gelatin, and 0.5-2 parts chitosan, which are solidified by a divalent calcium ion solution with a concentration of 0.1-0.5 mol / L and cross-linked with transglutaminase with an activity unit of 10-100 U / g.

6. The method for characterizing biofluorescence of seepage velocity in transparent formations according to claim 1, characterized in that, In step S3, the injection process requires the tracer suspension to be kept in a light-protected environment, and the injection flow rate to be lower than the lowest shear failure threshold in the biocompatible encapsulated microcapsule sequence, so as to ensure that luciferase does not come into contact with luciferin before entering the transparent formation.

7. The method for characterizing biofluorescence of seepage velocity in transparent formations according to claim 1, characterized in that, In step S4, an EMCCD camera is used to capture bioluminescent images of the flow field in real time, and the light intensity signal field matrix is ​​represented as follows: in, The light intensity signal field matrix, Indicates the first Liede The light intensity value of the line, From 1 to Any integer between From 1 to Any integer between , The value is known.

8. The method for characterizing biofluorescence of seepage velocity in transparent formations according to claim 1, characterized in that, In step S5, the quantitative relationship model is established based on the Weiber distribution and the Beer-Lambert optical attenuation principle, specifically expressed as follows: in, The seepage velocity field matrix is... Light intensity signal field matrix, The maximum light intensity signal field matrix, The characteristic intensity modulus of the microcapsule sequence, For gradient shape factor, The dynamic viscosity of the pore fluid. The pore diameter is Porosity This is the magnification factor of the pore structure. , , , , All values ​​are known.

9. The method for characterizing biofluorescence of seepage velocity in transparent formations according to claim 1, characterized in that, In step S4, during the acquisition of bioluminescence images, the luminescence intensity under different pH environments is calibrated in real time through intelligent spectroradiometric brightness analysis to eliminate the influence of environmental acidity and alkalinity changes on chemiluminescence dynamics.

10. The method for characterizing biofluorescence of seepage velocity in transparent formations according to claim 1, characterized in that, The method is used for non-contact in-situ quantitative characterization of seepage velocity field in physical simulation test models of weakly cemented aquifers.

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