A method and system for quantifying the luminescence intensity of a fluorescent probe molecule

Abstract

The application discloses a fluorescent probe molecule luminous intensity quantification method and system, and belongs to the technical field of fluorescent probes. The fluorescent probe molecule luminous intensity quantification method comprises the following steps: building an initial structure of a fluorescent probe molecule and performing molecular dynamics simulation, uniformly extracting a plurality of molecular conformations from a motion trajectory; performing excitation state property calculation on each molecular conformation, extracting an excitation wavelength and an excitation intensity corresponding to a maximum excitation state oscillator strength; performing excitation state geometry optimization on each molecular conformation, extracting an emission wavelength, an emission intensity and an emission energy; calculating a luminous intensity coefficient based on the wavelength parameter, the intensity parameter and the energy parameter; evaluating the luminous performance of the probe molecule according to the luminous intensity coefficient, and screening a probe molecule with the largest luminous intensity coefficient as a target probe. The application can convert a dynamic effective object which is difficult to directly measure into a numerical form which can be measured and calculated, and realizes the standardization, dynamicization and repeatable quantitative characterization of the luminous intensity of the probe molecule.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a method and system for quantifying the luminescence intensity of fluorescent probe molecules. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Fluorescent probe molecules are a class of functional molecules that can specifically recognize target substances and produce changes in fluorescence signals. As a core tool in modern life science and medical research, they play an important role in life sciences, medical diagnosis, drug development, environmental monitoring, food safety and forensic identification.

[0004] Luminescence intensity refers to the strength of the fluorescence signal emitted by a probe molecule after being irradiated with excitation light, and it is one of the core indicators for evaluating probe performance. Given that accurately quantifying the luminescence intensity of fluorescent probe molecules is a crucial foundation for the transition of fluorescence technology from qualitative observation to quantitative science, establishing a standardized quantitative evaluation system for the luminescence performance of fluorescent probes has significant scientific importance and value.

[0005] However, existing methods for quantifying the luminescence intensity of fluorescent probes still suffer from the following bottlenecks: (1) The power of the measuring instrument and the sensitivity of the detector are different, and there is a lack of unified standards for parameters such as excitation wavelength and sample concentration, which makes the measurement results of the luminescence intensity of different probe molecules incomparable. (2) The experimental measurement process is complex, the operation is cumbersome, the human error is large, the cost is high, and the repeatability is poor, which seriously restricts the research and development efficiency of fluorescent probes. (3) Existing methods mostly focus on measuring "steady-state luminescence intensity", ignoring the influence of time evolution and dynamic fluctuations in molecular structure caused by environmental perturbations on luminescence intensity, making it difficult to accurately assess the performance of probes in dynamic processes such as actual in vivo imaging. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method and system for quantifying the luminescence intensity of fluorescent probe molecules. This method and system can transform dynamic and effective objects that are difficult to measure directly into measurable and calculable numerical forms, and realize the standardization, dynamization and repeatable quantitative characterization of the luminescence intensity of probe molecules. This provides a scientific tool for comparing the luminescence performance of fluorescent probes and high-throughput screening.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the technical solution of the present invention provides a method for quantifying the luminescence intensity of a fluorescent probe molecule, comprising: The initial structure of the fluorescent probe molecule was constructed and molecular dynamics simulations were performed to uniformly extract multiple molecular conformations from the motion trajectory. The excited-state properties of each molecular conformation are calculated, and the excitation wavelength and excitation intensity corresponding to the excited state with the maximum oscillator strength are extracted. Excited-state geometry optimization is performed on each molecular conformation to extract emission wavelength, emission intensity, and emission energy; The luminous intensity coefficient is calculated based on wavelength, intensity, and energy parameters; where wavelength parameters are the excitation and emission wavelengths, intensity parameters are the excitation and emission intensities, and energy parameters are the emission energy. The luminescence performance of probe molecules is evaluated based on the luminescence intensity coefficient, and the probe molecule with the largest luminescence intensity coefficient is selected as the target probe.

[0008] In at least one embodiment, an initial structure of the fluorescent probe molecule is constructed and molecular dynamics simulations are performed to uniformly extract multiple molecular conformations from the motion trajectory, specifically including: Construct a three-dimensional structural model of the fluorescent probe molecule and export it as a molecular structure file format; The exported molecular structure file is processed using a molecular force field parameter conversion tool to generate the topology file corresponding to the fluorescent probe molecule; Create a periodic water box in the shape of a cube, and fill the water box with water molecules, ensuring that the distance from the solute molecule to the boundary of the box is not less than a first set distance, and output the system architecture file containing the solvent and solute system; Based on the architecture and topology files, the steepest descent method is used to perform energy minimization calculations on the system in order to eliminate unreasonable atomic contacts and optimize the initial configuration. Starting with the configuration obtained after minimizing energy, molecular dynamics simulations are performed based on the set simulation time step and number of steps to generate molecular dynamics simulation trajectories. Multiple molecular conformations are extracted uniformly at equal time intervals from the molecular dynamics simulation trajectory and saved as a molecular structure exchange file format.

[0009] In at least one embodiment, excited-state properties are calculated for each molecular conformation, and the excitation wavelength and excitation intensity corresponding to the excited state with the maximum oscillator intensity are extracted, specifically including: The extracted structural files of multiple molecular conformations are batch converted into quantum chemical calculation input files; The calculation instructions in each input file are uniformly set to include time-dependent density functional theory calculations, keywords for specifying functionals and basis sets, and submitted to the quantum chemistry calculation program to perform molecular excited state property calculations; By parsing each output file, the corresponding field of the excited state is retrieved, the oscillator strength values ​​in the row containing the field are compared, and the state with the largest oscillator strength is selected as the target excited state. Record the transition wavelength corresponding to the target excited state as the excitation wavelength, and record its oscillator strength value as the excitation intensity.

[0010] In at least one embodiment, excited-state geometry optimization is performed on each molecular conformation to extract emission wavelength, emission intensity, and emission energy, specifically including: The calculation instructions in the quantum chemical calculation input files of multiple molecular conformations are uniformly set to include keywords for geometric structure optimization, time-dependent density functional theory calculation, and specifying functionals and basis sets, and then submitted to the quantum chemical calculation program to perform excited-state geometric structure optimization. By analyzing the output file obtained from the excited-state structure optimization calculation, the excitation energy and oscillator strength fields that appear last are located. The transition wavelength, oscillator strength and energy recorded after this field are extracted and used as the emission wavelength, emission intensity and emission energy, respectively.

[0011] In at least one embodiment, the luminous intensity coefficient is calculated based on wavelength parameters, intensity parameters, and energy parameters, specifically including: Statistically analyze the excitation wavelength, excitation intensity, emission wavelength, emission intensity, and emission energy for each molecular conformation; An excitation light scattering intensity vector is constructed based on excitation wavelength data; an emission light scattering intensity vector is constructed based on emission wavelength data; an excitation intensity vector is constructed based on excitation intensity data; and an emission intensity vector is constructed based on emission intensity data. The Stokes shifts of each molecular conformation are calculated based on the excitation and emission wavelengths, and the Stokes shift vectors are constructed. The radiation coefficients of each molecular conformation are calculated based on the emission intensity and emission energy, and a radiation coefficient vector is constructed. The first element-wise product is obtained by performing an element-wise product operation on the reciprocal of the excitation light scattering intensity vector, the excitation intensity vector, and the radiation coefficient vector. The second element-wise product is obtained by performing an element-wise product operation on the reciprocal of the emitted light scattering intensity vector and the Stokes displacement vector. The luminescence intensity coefficient is calculated based on the first element-wise product, the second element-wise product, and the number of extracted molecular conformations.

[0012] In at least one embodiment, the first element-wise product is specifically represented as:

[0013] In the formula, Represents the first element-wise product; Represents the excitation intensity vector; Represents the radiation coefficient vector; This represents the intensity vector of the excitation light scattering; This represents element-wise product operation; Indicates the excitation intensity of molecular conformation; The radiation coefficient representing the molecular conformation; Indicates the intensity of excitation light scattering based on molecular conformation; Indicates the molecular conformation number.

[0014] In at least one embodiment, the second element-wise product is specifically represented as:

[0015] In the formula, This represents the second element-wise product; Represents the Stokes displacement vector; This represents the vector of emitted light scattering intensity; Stokes shifts, representing molecular conformations; This indicates the intensity of emitted light scattering.

[0016] In at least one embodiment, the luminous intensity coefficient is specifically expressed as:

[0017] In the formula, This represents the luminescence intensity coefficient of the probe molecule.

[0018] In at least one embodiment, the luminescence performance of the probe molecule is evaluated based on the luminescence intensity coefficient, and the probe molecule with the largest luminescence intensity coefficient is selected as the target probe, specifically including: A lateral comparison of the luminescence intensity coefficients of the probe molecules shows that a larger luminescence intensity coefficient indicates a stronger luminescence intensity and better luminescence performance. The probe molecules are sorted according to their luminescence intensity coefficient values, and the probe molecules with the largest luminescence intensity coefficient values ​​are selected as the target probes.

[0019] Secondly, the technical solution of the present invention also provides a system for quantifying the luminescence intensity of fluorescent probe molecules, comprising: The conformation generation module is configured to: build the initial structure of the fluorescent probe molecule and perform molecular dynamics simulation, and uniformly extract multiple molecular conformations from the motion trajectory; The parameter calculation module is configured to: calculate the excited-state properties of each molecular conformation, extract the excitation wavelength and excitation intensity corresponding to the excited state with the maximum oscillator strength; and optimize the excited-state geometry of each molecular conformation to extract the emission wavelength, emission intensity, and emission energy. The luminescence intensity coefficient quantization module is configured to calculate the luminescence intensity coefficient based on wavelength parameters, intensity parameters, and energy parameters; wherein the wavelength parameters are the excitation wavelength and emission wavelength, the intensity parameters are the excitation intensity and emission intensity, and the energy parameter is the emission energy; The performance evaluation and output module is configured to: evaluate the luminescence performance of probe molecules based on the luminescence intensity coefficient, and select the probe molecule with the largest luminescence intensity coefficient as the target probe.

[0020] The beneficial effects of the above-described technical solution of the present invention are as follows: This invention discloses a method for quantifying the luminescence intensity of fluorescent probe molecules. By analyzing the evolutionary conformation of probe molecules at different times in a sampled water environment and integrating the influence of parameters such as wavelength, intensity, and energy, a quantitative factor is constructed to characterize the average effect of the dynamic luminescence brightness of fluorescent probe molecules. This invention eliminates interference from sample concentration and instrument dependence, unifies the luminescence brightness of fluorescent probe molecules into traceable and reproducible absolute brightness values, and achieves quantitative analysis of the brightness at the single-molecule level. This significantly reduces research and development costs and time, and helps improve the reliability of in vivo imaging probe performance evaluation. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 This is a schematic diagram of a method for quantifying the luminescence intensity of a fluorescent probe molecule disclosed in Embodiment 1 of the present invention; Figure 2 This is a schematic diagram of the chemical structure of ClO-1 disclosed in Example 1 of the present invention; Figure 3 This is a schematic diagram of the molecular conformation of ClO-1 in aqueous solution disclosed in Example 1 of the present invention; Figure 4 This is a schematic diagram of the chemical structure of ClO-2 disclosed in Embodiment 1 of the present invention; Figure 5 This is a schematic diagram of the chemical structure of ClO-3 disclosed in Example 1 of the present invention; Figure 6 This is a schematic diagram of the chemical structure of Fe-0 disclosed in Embodiment 1 of the present invention; Figure 7 This is a schematic diagram of a fluorescent probe molecule luminescence intensity quantification system disclosed in Embodiment 2 of the present invention. Detailed Implementation

[0023] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] Example 1 As introduced in the background section, fluorescent probes, as core tools in modern life science and medical research, have permeated various fields of basic research and clinical diagnosis and treatment. In life science research, fluorescent probes enable real-time, dynamic, and visualized monitoring of ion concentration, pH value, enzyme activity, and biomolecular interactions within living cells, allowing researchers to analyze the spatiotemporal patterns of life processes at the single-cell or even single-molecule level. In medical diagnostics, fluorescent probes are key components of in vitro diagnostic reagents and are widely used in immunofluorescence detection, nucleic acid detection, and pathological tissue imaging. Their high sensitivity and specificity enable early tumor marker screening and rapid pathogen identification, providing "intraoperative visualization" for surgical navigation and significantly improving the accuracy of tumor resection. In drug development, fluorescent probes support high-throughput screening, target validation, and drug distribution and metabolism studies, accelerating the transformation of new molecular entities into clinical drug candidates. In public safety fields such as environmental monitoring, food safety, and forensic identification, fluorescent probes also play a crucial role in the rapid detection of trace hazardous substances.

[0025] To overcome the bottlenecks in existing methods for quantifying the luminescence intensity of fluorescent probes, a typical embodiment of the present invention is as follows: Figures 1 to 6 As shown in the figure, this embodiment discloses a method for quantifying the luminescence intensity of a fluorescent probe molecule, which specifically includes the following steps: S1. Construct the initial structure of the fluorescent probe molecule and perform molecular dynamics simulation to uniformly extract multiple molecular conformations from the motion trajectory; S2. Calculate the excited state properties of each molecular conformation and extract the excitation wavelength and excitation intensity corresponding to the excited state with the maximum oscillator strength; S3. Perform excited-state geometry optimization on each molecular conformation to extract emission wavelength, emission intensity, and emission energy; S4. Calculate the luminous intensity coefficient based on wavelength parameters, intensity parameters, and energy parameters; where wavelength parameters are the excitation wavelength and emission wavelength, intensity parameters are the excitation intensity and emission intensity, and energy parameter is the emission energy. S5. Evaluate the luminescence performance of probe molecules based on the luminescence intensity coefficient, and select the probe molecule with the largest luminescence intensity coefficient as the target probe.

[0026] The following detailed description of the above-mentioned method for quantifying the luminescence intensity of fluorescent probe molecules, in conjunction with specific implementation methods, provides a detailed explanation.

[0027] S1. Construct the initial structure of the fluorescent probe molecule and perform molecular dynamics simulations to uniformly extract multiple molecular conformations from the motion trajectory.

[0028] In this step, a three-dimensional structural model of the fluorescent probe molecule is first constructed and exported as a molecular structure file. Then, a molecular force field parameter conversion tool is used to process the exported molecular structure file, generating a topology file corresponding to the fluorescent probe molecule. Next, a cubic-shaped periodic water box is created and filled with water molecules, ensuring that the distance from the solute molecule to the box boundary is not less than a first predetermined distance. Simultaneously, a system architecture file containing the solvent and solute system is output. Then, based on the system architecture file and topology file, the steepest descent method is used to perform energy minimization calculations on the system to eliminate unreasonable atomic contacts and optimize the initial configuration. Next, using the configuration obtained after energy minimization as the starting structure, molecular dynamics simulations are performed based on the set simulation time step and number of steps, generating molecular dynamics simulation trajectories. Finally, the molecular dynamics simulation trajectory is extracted uniformly at equal time intervals from the molecular dynamics simulation trajectory. The molecular conformations are analyzed and saved in a molecular structure exchange file format for subsequent quantum chemical calculations. .

[0029] As a further implementation, the first set distance is preferably 1.0 nm, the simulation time step is preferably 2 fs, and the number of steps is not less than 5000.

[0030] In one specific embodiment, the initial structure of the fluorescent probe molecule is constructed and molecular dynamics simulations are performed to uniformly extract multiple molecular conformations from the motion trajectory, specifically including: (1) Build a three-dimensional structural model of the fluorescent probe molecule in GaussView 6.0 software and export it as a .mol2 format file; (2) Process the .mol2 format file using the acpype.py program to generate the topology file corresponding to the fluorescent probe molecule; (3) Use the editconf command in GROMACS software to create a cube-shaped water box; (4) Use the solvate command in GROMACS software to fill the water box with water molecules, the distance from the solute to the edge of the box is not less than 1.0 nm, and output the architecture file in .gro format; (5) Based on the above architecture and topology files, the steepest descent method is used in GROMACS software to minimize the system energy, eliminate unreasonable atomic contacts, and optimize the initial configuration; (6) Using the configuration after energy minimization as the starting structure, set the simulation time step to 2fs, run a dynamic simulation of no less than 5000 steps, and generate the molecular dynamics simulation trajectory; (7) Extract at least 5 molecular conformations from the molecular dynamics simulation trajectory at equal time intervals and save them as .pdb format files for subsequent quantum chemical calculations.

[0031] S2. Calculate the excited state properties of each molecular conformation and extract the excitation wavelength and excitation intensity corresponding to the excited state with the maximum oscillator strength.

[0032] In this step, the extracted data will be processed first. The structural files of individual molecular conformations are batch-converted into input files for quantum chemical calculations. Then, the calculation instructions in each input file are uniformly set to include keywords for time-dependent density functional theory calculations and specifying functionals and basis sets, and submitted to the quantum chemical calculation program to perform molecular excited-state property calculations. By parsing each output file, the corresponding field for the excited state is retrieved, the oscillator strength values ​​in the row containing that field are compared, the state with the highest oscillator strength is selected as the target excited state, and the transition wavelength corresponding to the target excited state is recorded as the excitation wavelength. And record its oscillator strength value. As the excitation intensity.

[0033] In one specific embodiment, the excited-state properties of each molecular conformation are calculated, and the excitation wavelength and excitation intensity corresponding to the excited state with the maximum oscillator intensity are extracted, specifically including: (1) The .pdb files of individual molecular conformations were batch converted into .gjf format Gaussian software input files using GaussView 6.0 software; (2) Rewrite the calculation instructions in the .gjf file as “#p TD B3LYP / 6-31g(d,p)” and submit it to the Gaussian software to perform molecular excited state property calculations; (3) Parse the output file calculated by the Gaussian software, search for the "Excited state" field, and compare the oscillator strength in the row containing this field. Value, select The state with the largest value is the excited state with the largest oscillator strength, and this is taken as the target excited state; (4) Record the transition wavelength corresponding to the excited state with the highest oscillator intensity. This wavelength is the excitation wavelength. The excited state corresponds to The value is the excitation intensity. .

[0034] S3. Perform excited-state geometry optimization on each molecular conformation to extract emission wavelength, emission intensity and emission energy.

[0035] In this step, The computational instructions in the quantum chemical calculation input file for each molecular conformation are uniformly set to include keywords such as geometry optimization, time-dependent density functional theory calculation, and specified functionals and basis sets, and are submitted to the quantum chemical calculation program to perform excited-state geometry optimization. By parsing the output file obtained from the excited-state structure optimization calculation, the last occurrence of the excitation energy and oscillator strength fields is located, and the transition wavelength, oscillator strength, and energy recorded after this field are extracted and used as the emission wavelengths, respectively. Launch strength and emit energy .

[0036] In one specific embodiment, excited-state geometry optimization is performed on each molecular conformation to extract emission wavelength, emission intensity, and emission energy, specifically including: (1) The calculation command in the .gjf file of each molecular conformation is set to "#p OPT TD B3LYP / 6-31g(d,p)", and then submitted to Gaussian 16 software to perform molecular excited-state geometry optimization. (2) Analyze the output file of the excited-state structure optimization calculation, locate the last occurrence of the "Excitationenergies and oscillator strengths:" field, and extract the transition wavelength, oscillator strength and energy recorded after this field, which is the emission wavelength. Launch strength and emit energy .

[0037] S4. Calculate the luminous intensity coefficient based on wavelength, intensity, and energy parameters.

[0038] In this step, the statistics are first performed. Excitation wavelength of each molecular conformation Excitation intensity Emission wavelength Launch strength and emit energy .

[0039] Then, based on the excitation wavelength data, an excitation light scattering intensity vector is constructed, specifically represented as:

[0040] The emitted light scattering intensity vector is constructed based on the emitted wavelength data, specifically represented as follows:

[0041] An excitation intensity vector is constructed based on the excitation intensity data, specifically represented as follows:

[0042] A transmission intensity vector is constructed based on the transmission intensity data, specifically represented as follows:

[0043] The Stokes shifts for each molecular conformation are calculated based on the excitation and emission wavelengths, and are specifically expressed as follows:

[0044] The Stokes displacement vector is constructed based on the Stokes displacement data, specifically represented as follows:

[0045] The radiation coefficients of each molecular conformation are calculated based on emission intensity and emission energy, and are specifically expressed as follows:

[0046] An emissivity vector is constructed based on the emissivity data, specifically represented as follows:

[0047] The reciprocal of the excitation light scattering intensity vector Excitation intensity vector and radiation coefficient vector Performing element-wise product operation yields the first element-wise product, specifically represented as:

[0048] The reciprocal of the emitted light scattering intensity vector With Stokes displacement vector Performing element-wise product operation yields the second element-wise product, specifically represented as:

[0049] The luminescence intensity coefficient is calculated based on the first element-wise product, the second element-wise product, and the number of extracted molecular conformations, and is specifically expressed as follows:

[0050] In the formula, This represents the luminescence intensity coefficient of the probe molecule.

[0051] S5. Evaluate the luminescence performance of probe molecules based on the luminescence intensity coefficient, and select the probe molecule with the largest luminescence intensity coefficient as the target probe.

[0052] In this step, the luminescence intensity coefficient of the probe molecule is... Conduct a horizontal comparison. A higher value indicates stronger probe luminescence intensity and better luminescence performance. According to... Sort the values ​​and select first. Probe molecules with higher values ​​are selected as candidate probes with better performance. The probe molecule with the highest value is selected as the target probe.

[0053] In one specific embodiment, the fluorescent probe is named ClO-1, and its molecular chemical structure is as follows: Figure 2 As shown.

[0054] S1. Construct the initial structure of the fluorescent probe molecule ClO-1 and perform molecular dynamics simulations to uniformly extract multiple molecular conformations from its trajectory, specifically including: (1) Build a three-dimensional structural model of the fluorescent probe molecule ClO-1 in GaussView 6.0 software and export it as ClO-1.mol2; (2) Process ClO-1.mol2 using the acpype.py program to generate the topology file ClO-1.top corresponding to the fluorescent probe molecule ClO-1; (3) Use the editconf command in GROMACS software to create a cube-shaped water box; (4) Use the solvate command in GROMACS software to fill the water box with water molecules. The distance from the solute to the edge of the box is 1.0 nm. Figure 3 As shown, it also outputs the architecture file ClO-1.gro; (5) Based on the above architecture file ClO-1.gro and topology file ClO-1.top, the steepest descent method is used in GROMACS software to minimize the system energy, eliminate unreasonable atomic contacts, and optimize the initial configuration; (6) Using the configuration after energy minimization as the starting structure, a dynamic simulation with a time step of 2fs and a number of steps of 5000 and a total running time of 1ns was performed to generate the molecular dynamics simulation trajectory; (7) Extract a molecular conformation every 0.1 ns from the molecular dynamics simulation trajectory to obtain a total of 10 molecular conformations, and save them as .pdb format files.

[0055] S2. Calculate the excited-state properties of each molecular conformation, and extract the excitation wavelength and excitation intensity corresponding to the excited state with the maximum oscillator intensity, specifically including: (1) Convert the .pdb files of the 10 molecular conformations of ClO-1 into Gaussian software input files in .gjf format using GaussView 6.0 software; (2) Rewrite the calculation instructions in the .gjf file as “#p TD B3LYP / 6-31g(d,p)” and submit it to the Gaussian software to perform molecular excited state property calculations; (3) Open the results of each excited state property calculation, search for the "Excited state" field, and compare the oscillator strength in the row containing this field. Value, select The state with the largest value is the excited state with the largest oscillator strength, and this is taken as the target excited state; (4) Record the transition wavelength corresponding to the excited state with the highest oscillator intensity. This wavelength is the excitation wavelength. The excited state corresponds to The value is the excitation intensity. .

[0056] S3. Perform excited-state geometry optimization on each molecular conformation to extract emission wavelength, emission intensity, and emission energy, specifically including: (1) Set the calculation command in the .gjf file of 10 molecular conformations to "#p OPT TD B3LYP / 6-31g(d,p)" and submit it to Gaussian 16 software to perform molecular excited state geometry optimization; (2) Open the results of the excited state structure optimization for each molecule, locate the last occurrence of the "Excitation energies and oscillator strengths:" field, and extract the transition wavelength, oscillator strength and energy recorded after this field, which is the emission wavelength. Launch strength and emit energy .

[0057] S4. Calculate the luminous intensity coefficient based on wavelength, intensity, and energy parameters, specifically including: (1) Statistical analysis of the excitation wavelengths of 10 molecular conformations Excitation intensity Emission wavelength Launch strength and emit energy The details are shown in Table 1.

[0058] Table 1. Excitation wavelengths of 10 molecular conformations of the fluorescent probe ClO-1 (Unit: nm) Excitation intensity (Unit: au), Emission wavelength (Unit: nm) Emission Intensity (Unit: au) and emission energy (Unit: eV)

[0059] (2) Construct the excitation light scattering intensity vector based on the excitation wavelength data, specifically as follows:

[0060] The emitted light scattering intensity vector is constructed based on the emitted wavelength data, specifically represented as follows:

[0061] An excitation intensity vector is constructed based on the excitation intensity data, specifically represented as follows:

[0062] A transmission intensity vector is constructed based on the transmission intensity data, specifically represented as follows:

[0063] The Stokes shifts for each molecular conformation are calculated based on the excitation and emission wavelengths, and are specifically expressed as follows: , , , , , , , , , ; The Stokes displacement vector is constructed based on the Stokes displacement data, specifically represented as follows:

[0064] The radiation coefficients of each molecular conformation are calculated based on emission intensity and emission energy, and are specifically expressed as follows: ， ， ， ， ， ， ， ， ， ； An emissivity vector is constructed based on the emissivity data, specifically represented as follows:

[0065] The reciprocal of the excitation light scattering intensity vector Excitation intensity vector and radiation coefficient vector Performing element-wise product operation yields the first element-wise product, specifically represented as:

[0066] The reciprocal of the emitted light scattering intensity vector With Stokes displacement vector Performing element-wise product operation yields the second element-wise product, specifically represented as:

[0067] The luminescence intensity coefficient is calculated based on the first element-wise product, the second element-wise product, and the number of extracted molecular conformations, and is specifically expressed as follows:

[0068] In the formula, This represents the luminescence intensity coefficient of the probe molecule ClO-1.

[0069] S5. Evaluate the luminescence performance of probe molecules based on their luminescence intensity coefficients, and select the probe molecule with the highest luminescence intensity coefficient as the target probe. Specifically, the luminescence intensity coefficient of probe molecule ClO-1 is: .

[0070] In one specific embodiment, the fluorescent probes are named ClO-2 and ClO-3, and their molecular chemical structures are as follows: Figure 4 and Figure 5 As shown.

[0071] S1. Construct the initial structures of fluorescent probe molecules ClO-2 and ClO-3 and perform molecular dynamics simulations. Extract five molecular conformations uniformly from their respective trajectories, specifically including: (1) Build three-dimensional structural models of fluorescent probe molecules ClO-2 and ClO-3 in GaussView 6.0 software and export them as ClO-2.mol2 and ClO-3.mol2; (2) Process ClO-2.mol2 and ClO-3.mol2 using the acpype.py program to generate the topology file ClO-2.top corresponding to the fluorescent probe molecule ClO-2 and the topology file ClO-3.top corresponding to the fluorescent probe molecule ClO-3; (3) Use the editconf command in GROMACS software to create a cube-shaped water box; (4) Use the solvate command in GROMACS software to fill the water box with water molecules. The distance from the solute to the edge of the box is 1.0 nm. Output the architecture files ClO-2.gro and ClO-3.gro respectively. (5) Based on the above architecture file ClO-2.gro and topology file ClO-2.top, the system energy was minimized using the steepest descent method in GROMACS software to eliminate unreasonable atomic contacts and optimize the initial configuration of the fluorescent probe molecule ClO-2 in aqueous solution; based on the above architecture file ClO-3.gro and topology file ClO-3.top, the system energy was minimized using the steepest descent method in GROMACS software to eliminate unreasonable atomic contacts and optimize the initial configuration of the fluorescent probe molecule ClO-3 in aqueous solution; (6) Using the configuration after energy minimization as the starting structure, set the time step to 2fs and the number of steps to 5000, run dynamic simulations for the two fluorescent probe molecules for a total time of 1ns respectively, and generate the molecular dynamic simulation trajectories of the fluorescent probe molecules ClO-2 and ClO-3 respectively. (7) Extract a molecular conformation every 0.2 ns from the molecular dynamics simulation trajectories of fluorescent probe molecules ClO-2 and ClO-3 to obtain 5 conformations of fluorescent probe molecule ClO-2 and 5 conformations of fluorescent probe molecule ClO-3. Save all 10 molecular conformations as .pdb format files.

[0072] S2. Calculate the excited-state properties of various molecular conformations of fluorescent probe molecules ClO-2 and ClO-3, and extract the excitation wavelength and excitation intensity corresponding to the excited state with the maximum oscillator intensity, specifically including: (1) Convert the .pdb files of 5 molecular conformations of fluorescent probe molecules ClO-2 and ClO-3 into .gjf format Gaussian software input files in batches using GaussView6.0 software; (2) Rewrite the calculation instructions in the five .gjf files of the fluorescent probe molecules ClO-2 and ClO-3 as “#p TDB3LYP / 6-31g(d,p)” and submit them to Gaussian 16 software to perform molecular excited state property calculations; (3) Open the results of each excited state property calculation, search for the "Excited state" field, and compare the oscillator strength in the row containing this field. Value, select The state with the largest value is the excited state with the largest oscillator strength, and this is taken as the target excited state; (4) Record the transition wavelength corresponding to the excited state with the highest oscillator intensity. This wavelength is the excitation wavelength. The excited state corresponds to The value is the excitation intensity. .

[0073] S3. Optimize the excited-state geometry of the various molecular conformations of fluorescent probe molecules ClO-2 and ClO-3, and extract the emission wavelength, emission intensity, and emission energy, specifically including: (1) Set the calculation command in the .gjf file of the five molecular conformations of the fluorescent probe molecules ClO-2 and ClO-3 to "#p OPT TD B3LYP / 6-31g(d,p)" and submit it to the Gaussian 16 software to perform molecular excited state geometry optimization; (2) Open the results of the excited state structure optimization for each molecule, locate the last occurrence of the "Excitation energies and oscillator strengths:" field, and extract the transition wavelength, oscillator strength and energy recorded after this field, which is the emission wavelength. Launch strength and emit energy .

[0074] S4. Calculate the luminescence intensity coefficients of probe molecules ClO-2 and ClO-3 based on wavelength, intensity, and energy parameters, respectively, including: (1) Statistical analysis of the excitation wavelengths of the five molecular conformations of the fluorescent probe molecule ClO-2 Excitation intensity Emission wavelength Launch strength and emit energy ; Statistical analysis of the excitation wavelengths of five molecular conformations of the fluorescent probe molecule ClO-3 Excitation intensity Emission wavelength Launch strength and emit energy The details are shown in Table 2.

[0075] Table 2 Excitation wavelengths of the five molecular conformations of the fluorescent probe ClO-2 (Unit: nm) Excitation intensity (Unit: au), Emission wavelength (Unit: nm) Emission Intensity (Unit: au) Emission Energy (Unit: eV) and excitation wavelengths of the five molecular conformations of the fluorescent probe ClO-3. (Unit: nm) Excitation intensity (Unit: au), Emission wavelength (Unit: nm) Emission Intensity (Unit: au) Emission Energy (Unit: eV)

[0076] (2) Based on the excitation wavelength data of the fluorescent probe molecule ClO-2, the excitation light scattering intensity vector of the fluorescent probe molecule ClO-2 is constructed, specifically expressed as:

[0077] The emission light scattering intensity vector of the fluorescent probe molecule ClO-2 is constructed based on the emission wavelength data of ClO-2, and is specifically expressed as follows:

[0078] The excitation intensity vector of the fluorescent probe molecule ClO-2 is constructed based on its excitation intensity data, and is specifically represented as follows:

[0079] The emission intensity vector of the fluorescent probe molecule ClO-2 is constructed based on its emission intensity data, and is specifically represented as follows:

[0080] The Stokes shifts of the five molecular conformations of the fluorescent probe molecule ClO-2 were calculated based on the excitation and emission wavelengths, and are specifically expressed as follows: , , , , ; The Stokes shift vector of the fluorescent probe molecule ClO-2 is constructed based on its Stokes shift data, and is specifically represented as follows:

[0081] The radiation coefficients of five molecular conformations of the fluorescent probe molecule ClO-2 were calculated based on the emission intensity and emission energy, and are specifically expressed as follows: , , , , ; The emissivity vector of the fluorescent probe molecule ClO-2 is constructed based on its emissivity data, and is specifically represented as follows:

[0082] The reciprocal of the excitation light scattering intensity vector of the fluorescent probe molecule ClO-2 Excitation intensity vector and radiation coefficient vector Performing element-wise product operations, we obtain the first element-wise product of the fluorescent probe molecule ClO-2, which is specifically expressed as:

[0083] The reciprocal of the emission light scattering intensity vector of the fluorescent probe molecule ClO-2 With Stokes displacement vector Performing element-wise product operations, we obtain the second element-wise product of the fluorescent probe molecule ClO-2, which is specifically expressed as:

[0084] The luminescence intensity coefficient is calculated based on the first element-wise product, the second element-wise product, and the number of extracted molecular conformations of the fluorescent probe molecule ClO-2, and is specifically expressed as follows:

[0085] In the formula, This represents the luminescence intensity coefficient of the probe molecule ClO-2.

[0086] The excitation light scattering intensity vector of the fluorescent probe molecule ClO-3 is constructed based on the excitation wavelength data of ClO-3, and is specifically expressed as follows:

[0087] The emission wavelength data of the fluorescent probe molecule ClO-3 is used to construct the emission light scattering intensity vector of ClO-3, which is specifically represented as follows:

[0088] The emission light scattering intensity vector of the fluorescent probe molecule ClO-3 is constructed based on the emission wavelength data of ClO-3, and is specifically expressed as follows:

[0089] The excitation intensity vector of the fluorescent probe molecule ClO-3 is constructed based on the excitation intensity data of ClO-3, and is specifically represented as follows:

[0090] The emission intensity vector of the fluorescent probe molecule ClO-3 is constructed based on its emission intensity data, and is specifically represented as follows:

[0091] The Stokes shifts of the five molecular conformations of the fluorescent probe molecule ClO-3 were calculated based on the excitation and emission wavelengths, and are specifically expressed as follows: , , , , ; The Stokes shift vector of the fluorescent probe molecule ClO-3 is constructed based on the Stokes shift data of ClO-3, and is specifically represented as follows:

[0092] The radiative coefficients of five molecular conformations of the fluorescent probe molecule ClO-3 were calculated based on the emission intensity and emission energy, and are specifically expressed as follows: , , , , ; The emissivity vector of the fluorescent probe molecule ClO-3 is constructed based on its emissivity data, and is specifically represented as follows:

[0093] The reciprocal of the excitation light scattering intensity vector of the fluorescent probe molecule ClO-3 Excitation intensity vector and radiation coefficient vector Performing element-wise product operations, we obtain the first element-wise product of the fluorescent probe molecule ClO-3, which is specifically expressed as:

[0094] The reciprocal of the emission light scattering intensity vector of the fluorescent probe molecule ClO-3 With Stokes displacement vector Performing element-wise product operations, we obtain the second element-wise product of the fluorescent probe molecule ClO-3, specifically expressed as:

[0095] The luminescence intensity coefficient is calculated based on the first element-wise product, the second element-wise product, and the number of extracted molecular conformations of the fluorescent probe molecule ClO-3, and is specifically expressed as follows:

[0096] In the formula, This represents the luminescence intensity coefficient of the probe molecule ClO-3.

[0097] S5. Evaluate the luminescence performance of probe molecules based on their luminescence intensity coefficients, and screen the probe molecules with the highest luminescence intensity coefficients as target probes. Specifically, the luminescence intensity coefficients of fluorescent probe molecules ClO-2 and ClO-3 are respectively... and A comparative analysis of the luminescence intensity coefficients of fluorescent probe molecules ClO-2 and ClO-3 revealed that probe ClO-3 exhibited the highest luminescence intensity coefficient, indicating superior luminescence performance. Therefore, fluorescent ClO-3 was preferentially selected as the target probe. The comparison also included the luminescence intensity coefficient of fluorescent probe molecule ClO-1. In comparison, the fluorescent probe molecule ClO-2 has a larger luminescence intensity coefficient. Therefore, the luminescence performance of the three hypochlorous acid probes ClO-1, ClO-2 and ClO-3 is ranked from strongest to weakest as ClO-3>ClO-2>ClO-1.

[0098] In one specific embodiment, the fluorescent probe is named Fe-0, and its molecular chemical structure is as follows: Figure 6 As shown.

[0099] S1. Construct the initial structure of the fluorescent probe molecule Fe-0 and perform molecular dynamics simulations. Extract five molecular conformations uniformly from the trajectory, specifically including: (1) Build a three-dimensional structural model of the fluorescent probe molecule Fe-0 in GaussView 6.0 software and export it as Fe-0.mol2; (2) Process Fe-0.mol2 using the acpype.py program to generate the topology file Fe-0.top corresponding to the fluorescent probe molecule Fe-0; (3) Use the editconf command in GROMACS software to create a cube-shaped water box; (4) Use the solvate command in GROMACS software to fill the water box with water molecules. The distance from the solute to the edge of the box is 1.0 nm. Figure 3 As shown, it also outputs the architecture file Fe-0.gro; (5) Based on the above architecture file Fe-0.gro and topology file Fe-0.top, the steepest descent method is used in GROMACS software to minimize the system energy, eliminate unreasonable atomic contacts, and optimize the initial configuration; (6) Using the configuration after energy minimization as the starting structure, a dynamic simulation with a time step of 2fs and a number of steps of 5000 and a total running time of 1ns was performed to generate the molecular dynamics simulation trajectory; (7) Extract a molecular conformation every 0.2 ns from the molecular dynamics simulation trajectory to obtain a total of 5 molecular conformations, and save them as .pdb format files.

[0100] S2. Calculate the excited-state properties of each molecular conformation, and extract the excitation wavelength and excitation intensity corresponding to the excited state with the maximum oscillator intensity, specifically including: (1) Convert the .pdb files of the five molecular conformations of Fe-0 into Gaussian software input files in .gjf format using GaussView 6.0 software; (2) Rewrite the calculation instructions in the five .gjf files as “#p TD B3LYP / 6-31g(d,p)” and submit them to the Gaussian software to perform molecular excited state property calculations; (3) Open the results of each excited state property calculation, search for the "Excited state" field, and compare the oscillator strength in the row containing this field. Value, select The state with the largest value is the excited state with the largest oscillator strength, and this is taken as the target excited state; (4) Record the transition wavelength corresponding to the excited state with the highest oscillator intensity. This wavelength is the excitation wavelength. The excited state corresponds to The value is the excitation intensity. .

[0101] S3. Perform excited-state geometry optimization on each molecular conformation to extract emission wavelength, emission intensity, and emission energy, specifically including: (1) Set the calculation command in the .gjf file of the 5 molecular conformations to "#p OPT TD B3LYP / 6-31g(d,p)" and submit it to the Gaussian 16 software to perform molecular excited state geometry optimization; (2) Open the results of the excited state structure optimization for each molecule, locate the last occurrence of the "Excitation energies and oscillator strengths:" field, and extract the transition wavelength, oscillator strength and energy recorded after this field, which is the emission wavelength. Launch strength and emit energy .

[0102] (1) Statistical analysis of the excitation wavelengths of the five molecular conformations of the fluorescent probe molecule Fe-0 Excitation intensity Emission wavelength Launch strength and emit energy The details are shown in Table 3.

[0103] Table 3 Excitation wavelengths of the five molecular conformations of the fluorescent probe Fe-0 (Unit: nm) Excitation intensity (Unit: au), Emission wavelength (Unit: nm) Emission Intensity (Unit: au) and emission energy (Unit: eV)

[0104] (2) Based on the excitation wavelength data of the fluorescent probe molecule Fe-0, the excitation light scattering intensity vector of the fluorescent probe molecule Fe-0 is constructed, specifically expressed as:

[0105] The emission wavelength data of the fluorescent probe molecule Fe-0 is used to construct the emission light scattering intensity vector of Fe-0, which is specifically represented as follows:

[0106] The excitation intensity vector of the fluorescent probe molecule Fe-0 is constructed based on its excitation intensity data, and is specifically represented as follows:

[0107] The emission intensity vector of the fluorescent probe molecule Fe-0 is constructed based on its emission intensity data, and is specifically represented as follows:

[0108] The Stokes shifts of the five molecular conformations of the fluorescent probe molecule Fe-0 were calculated based on the excitation and emission wavelengths, and are specifically expressed as follows: , , , , ; The Stokes shift vector of the fluorescent probe molecule Fe-0 is constructed based on its Stokes shift data, and is specifically represented as follows:

[0109] The radiation coefficients of the five molecular conformations of the fluorescent probe molecule Fe-0 were calculated based on the emission intensity and emission energy, and are specifically expressed as follows: , , , , ; The emissivity vector of the fluorescent probe molecule Fe-0 is constructed based on its emissivity data, and is specifically represented as follows:

[0110] The reciprocal of the excitation light scattering intensity vector of the fluorescent probe molecule Fe-0 Excitation intensity vector and radiation coefficient vector Performing element-wise product operations, we obtain the first element-wise product of the fluorescent probe molecule Fe-0, which is specifically expressed as:

[0111] The reciprocal of the emission intensity vector of the fluorescent probe molecule Fe-0 With Stokes displacement vector Performing element-wise product operations, we obtain the second element-wise product of the fluorescent probe molecule Fe-0, which is specifically expressed as:

[0112] The luminescence intensity coefficient is calculated based on the first element-wise product and the second element-wise product of the fluorescent probe molecule Fe-0, as well as the number of extracted molecular conformations, and is specifically expressed as follows:

[0113] In the formula, This represents the luminescence intensity coefficient of the probe molecule Fe-0.

[0114] S5. Evaluate the luminescence performance of probe molecules based on their luminescence intensity coefficients, and select the probe molecule with the highest luminescence intensity coefficient as the target probe. Specifically, the luminescence intensity coefficient of probe molecule Fe-0 is: .

[0115] Example 2 In a typical embodiment of the present invention, such as Figure 7 As shown, this embodiment discloses a fluorescence probe molecule luminescence intensity quantification system, comprising: The conformation generation module is configured to: build the initial structure of the fluorescent probe molecule and perform molecular dynamics simulation, and uniformly extract multiple molecular conformations from the motion trajectory; The parameter calculation module is configured to: calculate the excited-state properties of each molecular conformation, extract the excitation wavelength and excitation intensity corresponding to the excited state with the maximum oscillator strength; and optimize the excited-state geometry of each molecular conformation to extract the emission wavelength, emission intensity, and emission energy. The luminescence intensity coefficient quantization module is configured to calculate the luminescence intensity coefficient based on wavelength parameters, intensity parameters, and energy parameters; wherein the wavelength parameters are the excitation wavelength and emission wavelength, the intensity parameters are the excitation intensity and emission intensity, and the energy parameter is the emission energy; The performance evaluation and output module is configured to: evaluate the luminescence performance of probe molecules based on the luminescence intensity coefficient, and select the probe molecule with the largest luminescence intensity coefficient as the target probe.

[0116] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for quantifying the luminescence intensity of a fluorescent probe molecule, characterized in that, include: The initial structure of the fluorescent probe molecule was constructed and molecular dynamics simulations were performed to uniformly extract multiple molecular conformations from the motion trajectory. The excited-state properties of each molecular conformation are calculated, and the excitation wavelength and excitation intensity corresponding to the excited state with the maximum oscillator strength are extracted. Excited-state geometry optimization is performed on each molecular conformation to extract emission wavelength, emission intensity, and emission energy; The luminous intensity coefficient is calculated based on wavelength, intensity, and energy parameters; where wavelength parameters are the excitation and emission wavelengths, intensity parameters are the excitation and emission intensities, and energy parameters are the emission energy. The luminescence properties of probe molecules are evaluated based on the luminescence intensity coefficient, and the probe molecule with the largest luminescence intensity coefficient is selected as the target probe. The luminous intensity coefficient is calculated based on wavelength, intensity, and energy parameters, specifically including: Statistically analyze the excitation wavelength, excitation intensity, emission wavelength, emission intensity, and emission energy for each molecular conformation; An excitation light scattering intensity vector is constructed based on excitation wavelength data; an emission light scattering intensity vector is constructed based on emission wavelength data; an excitation intensity vector is constructed based on excitation intensity data; and an emission intensity vector is constructed based on emission intensity data. The Stokes shifts of each molecular conformation are calculated based on the excitation and emission wavelengths, and the Stokes shift vectors are constructed. The radiation coefficients of each molecular conformation are calculated based on the emission intensity and emission energy, and a radiation coefficient vector is constructed. The first element-wise product is obtained by performing an element-wise product operation on the reciprocal of the excitation light scattering intensity vector, the excitation intensity vector, and the radiation coefficient vector. The second element-wise product is obtained by performing an element-wise product operation on the reciprocal of the emitted light scattering intensity vector and the Stokes displacement vector. The luminescence intensity coefficient is calculated based on the first element-wise product, the second element-wise product, and the number of extracted molecular conformations.

2. The method for quantifying the luminescence intensity of a fluorescent probe molecule as described in claim 1, characterized in that, The initial structure of the fluorescent probe molecule was constructed and molecular dynamics simulations were performed. Multiple molecular conformations were uniformly extracted from the motion trajectory, specifically including: Construct a three-dimensional structural model of the fluorescent probe molecule and export it as a molecular structure file format; The exported molecular structure file is processed using a molecular force field parameter conversion tool to generate the topology file corresponding to the fluorescent probe molecule; Create a periodic water box in the shape of a cube, and fill the water box with water molecules, ensuring that the distance from the solute molecule to the boundary of the box is not less than a first set distance, and output the system architecture file containing the solvent and solute system; Based on the architecture and topology files, the steepest descent method is used to perform energy minimization calculations on the system in order to eliminate unreasonable atomic contacts and optimize the initial configuration. Starting with the configuration obtained after minimizing energy, molecular dynamics simulations are performed based on the set simulation time step and number of steps to generate molecular dynamics simulation trajectories. Multiple molecular conformations are extracted uniformly at equal time intervals from the molecular dynamics simulation trajectory and saved as a molecular structure exchange file format.

3. The method for quantifying the luminescence intensity of a fluorescent probe molecule as described in claim 2, characterized in that, The excited-state properties of each molecular conformation are calculated, and the excitation wavelength and excitation intensity corresponding to the excited state with the maximum oscillator intensity are extracted, specifically including: The extracted structural files of multiple molecular conformations are batch converted into quantum chemical calculation input files; The calculation instructions in each input file are uniformly set to include time-dependent density functional theory calculations, keywords for specifying functionals and basis sets, and submitted to the quantum chemistry calculation program to perform molecular excited state property calculations; By parsing each output file, the corresponding field of the excited state is retrieved, the oscillator strength values ​​in the row containing the field are compared, and the state with the largest oscillator strength is selected as the target excited state. Record the transition wavelength corresponding to the target excited state as the excitation wavelength, and record its oscillator strength value as the excitation intensity.

4. The method for quantifying the luminescence intensity of a fluorescent probe molecule as described in claim 3, characterized in that, Excited-state geometry optimization is performed on each molecular conformation to extract emission wavelength, emission intensity, and emission energy, specifically including: The calculation instructions in the quantum chemical calculation input files of multiple molecular conformations are uniformly set to include keywords for geometric structure optimization, time-dependent density functional theory calculation, and specifying functionals and basis sets, and then submitted to the quantum chemical calculation program to perform excited-state geometric structure optimization. By analyzing the output file obtained from the excited-state structure optimization calculation, the excitation energy and oscillator strength fields that appear last are located. The transition wavelength, oscillator strength and energy recorded after this field are extracted and used as the emission wavelength, emission intensity and emission energy, respectively.

5. The method for quantifying the luminescence intensity of a fluorescent probe molecule as described in claim 1, characterized in that, The first element-wise product is specifically expressed as: In the formula, Represents the first element-wise product; Represents the excitation intensity vector; Represents the radiation coefficient vector; This represents the intensity vector of the excitation light scattering; This represents element-wise product operation; Indicates the excitation intensity of molecular conformation; The radiation coefficient representing the molecular conformation; Indicates the intensity of excitation light scattering based on molecular conformation; Indicates the molecular conformation number.

6. The method for quantifying the luminescence intensity of a fluorescent probe molecule as described in claim 5, characterized in that, The second element-wise product is specifically expressed as: In the formula, This represents the second element-wise product; Represents the Stokes displacement vector; This represents the vector of emitted light scattering intensity; Stokes shifts, representing molecular conformations; This indicates the intensity of emitted light scattering.

7. The method for quantifying the luminescence intensity of a fluorescent probe molecule as described in claim 6, characterized in that, The luminous intensity coefficient is specifically expressed as: In the formula, This represents the luminescence intensity coefficient of the probe molecule.

8. The method for quantifying the luminescence intensity of a fluorescent probe molecule as described in claim 1, characterized in that, The luminescence properties of probe molecules are evaluated based on their luminescence intensity coefficients, and the probe molecules with the highest luminescence intensity coefficients are selected as target probes. Specifically, this includes: A lateral comparison of the luminescence intensity coefficients of the probe molecules shows that a larger luminescence intensity coefficient indicates a stronger luminescence intensity and better luminescence performance. The probe molecules are sorted according to their luminescence intensity coefficient values, and those with larger luminescence intensity coefficient values ​​are selected as target probes.

9. A system for quantifying the luminescence intensity of a fluorescent probe molecule, characterized in that, include: The conformation generation module is configured to: build the initial structure of the fluorescent probe molecule and perform molecular dynamics simulation, and uniformly extract multiple molecular conformations from the motion trajectory; The parameter calculation module is configured to: calculate the excited-state properties of each molecular conformation, extract the excitation wavelength and excitation intensity corresponding to the excited state with the maximum oscillator strength; and optimize the excited-state geometry of each molecular conformation to extract the emission wavelength, emission intensity, and emission energy. The luminescence intensity coefficient quantization module is configured to calculate the luminescence intensity coefficient based on wavelength parameters, intensity parameters, and energy parameters; wherein the wavelength parameters are the excitation wavelength and emission wavelength, the intensity parameters are the excitation intensity and emission intensity, and the energy parameter is the emission energy; The performance evaluation and output module is configured to: evaluate the luminescence performance of probe molecules based on the luminescence intensity coefficient, and select the probe molecule with the largest luminescence intensity coefficient as the target probe; The luminous intensity coefficient is calculated based on wavelength, intensity, and energy parameters, specifically including: Statistically analyze the excitation wavelength, excitation intensity, emission wavelength, emission intensity, and emission energy for each molecular conformation; An excitation light scattering intensity vector is constructed based on excitation wavelength data; an emission light scattering intensity vector is constructed based on emission wavelength data; an excitation intensity vector is constructed based on excitation intensity data; and an emission intensity vector is constructed based on emission intensity data. The Stokes shifts of each molecular conformation are calculated based on the excitation and emission wavelengths, and the Stokes shift vectors are constructed. The radiation coefficients of each molecular conformation are calculated based on the emission intensity and emission energy, and a radiation coefficient vector is constructed. The first element-wise product is obtained by performing an element-wise product operation on the reciprocal of the excitation light scattering intensity vector, the excitation intensity vector, and the radiation coefficient vector. The second element-wise product is obtained by performing an element-wise product operation on the reciprocal of the emitted light scattering intensity vector and the Stokes displacement vector. The luminescence intensity coefficient is calculated based on the first element-wise product, the second element-wise product, and the number of extracted molecular conformations.

Images