Fluorescent DNA probe based on hairpin structure and application thereof

Abstract

The invention discloses a fluorescent DNA probe based on a hairpin structure, molecular stability, reaction dynamics and detection sensitivity of the fluorescent DNA probe are improved by screening and optimizing the length of a stem region, GC content, a loop sequence and thermodynamic parameters of the probe, and the fluorescent DNA probe with the sequence of 5 '-FAM-GCAGCTATTTTTAGCTGC-DABCYL-3' is finally obtained. Meanwhile, the invention further researches and develops a detection method and a fluorescence detection kit based on the probe, and measures such as a dual-wavelength ratio detection strategy, an improved temperature control system adapted to a Siemofen LUX microplate reader, a dynamic continuous detection mode, a four-parameter logistic regression algorithm correction fitting standard curve and the like are adopted. Finally, high-sensitivity, wide-dynamic-range, simple, convenient and rapid detection of DNAse enzyme can be realized, and the kit has the advantages of low cost, high stability, high precision and the like, and has pioneering clinical application value and wide market prospect.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, relates to the field of DNase fluorescence detection, and specifically relates to a fluorescent DNA probe based on a hairpin structure and an application thereof. Background Art

[0002] DNAse I (deoxyribonuclease I) is a nonspecific endonuclease that digests single-stranded or double-stranded DNA to produce mono- and oligodeoxynucleotides containing a 5'-phosphate group and a 3'-OH group.

[0003] The detection methods of DNAse I enzyme mainly include the following:

[0004] (1) Fluorescence Resonance Energy Transfer (FRET) method: In this method, a synthetic DNA oligonucleotide probe is used, which has a fluorescent group (such as VIC) on one end and a quencher group (such as BHQ1) on the other end. When DNAse I cuts this substrate, the fluorescent group and the quencher group separate, resulting in an increase in the fluorescence signal. By measuring the change in fluorescence intensity, the activity of DNAse I can be detected very sensitively. (2) DNase I Footprinting Assay: This is an assay used to determine the binding sites of DNA binding proteins on DNA molecules. By pre-incubating DNA with protein and then treating it with DNAse I, regions that are protected by protein and not cut by DNAse I can be identified. These regions are displayed by autoradiography or fluorescence detection after gel electrophoresis. (3) Electrophoretic Mobility Shift Assay (EMSA): Although this is not a direct method for detecting DNAse I, it can be used in conjunction with the DNase I footprinting assay to determine whether transcription factors bind directly to DNA. By comparing the changes in electrophoretic mobility of proteins before and after binding to DNA, the presence of DNA-binding proteins can be inferred.

[0005] Fluorescence resonance energy transfer is widely used due to its high sensitivity and simple operation. However, the current problems of fluorescence detection methods include: high detection cost, detection sensitivity and specificity need to be improved, high detection limit, consumption of precious samples, and narrow detection range.

[0006] In view of this situation, there is an urgent need to provide a fluorescence detection method and a corresponding kit with high sensitivity, wide dynamic range, strong stability and economical and practical cost. Summary of the invention

[0007] To solve the above problems, the purpose of the present invention is to provide a fluorescent DNA probe based on a hairpin structure and its application. The fluorescent DNA probe and related products and methods provided in this application have the advantages of being simple, economical, wide in detection range, strong in specificity and high in stability.

[0008] The technical contents of the present invention are as follows:

[0009] In a first aspect, the present invention provides a fluorescent DNA probe based on a hairpin structure, the sequence of the probe being shown in SEQ ID NO.1: GCAGCTATTTTTAGCTGC.

[0010] Preferably, the 5' end of the fluorescent DNA probe is a fluorescent group, and the 3' end is a quenching group.

[0011] Preferably, the fluorescent group includes but is not limited to any one of FAM, HEX, TET and ROX.

[0012] Preferably, the quenching group includes but is not limited to any one of DABCYL, BHQ1, BHQ2, and TAMRA.

[0013] Preferably, the fluorescent group is FAM, and the quenching group is DABCYL.

[0014] Specifically, the fluorescent substrate probe of the present invention is an optimized hairpin structure DNA probe, and its sequence is:

[0015] 5'-FAM-GCAGCTATTTTTAGCTGC-DABCYL-3';

[0016] The characteristics of the fluorescent DNA probe of the present invention are as follows: a. stem region length: 7 base pairs; b. loop region size: 4 nucleotides; c. stem region GC content: 57%; d. predicted melting temperature: 65°C; e. predicted ΔG value: -15.8 kcal / mol.

[0017] Fluorescence Resonance Energy Transfer (FRET) is a physical phenomenon based on energy transfer between two fluorescent molecules, which is used to study intermolecular interactions and intramolecular conformational changes. In FRET technology, commonly used fluorescent groups (donors) and quenching groups (acceptors) include:

[0018] Fluorophores (donors): 6-FAM (6-carboxyfluorescein): commonly used to label DNA or proteins, with an emission wavelength of about 520-530nm. TET (tetramethylrhodamine): a red fluorophore, commonly used in FRET experiments, with an emission wavelength of about 550nm. JOE (4-methylumbelliferone): a fluorophore with an emission wavelength of about 530nm. HEX (6-carboxy-2',4',5',7'-tetrachlorofluorescein): a fluorophore with an emission wavelength of about 565nm. Cy3: a near-infrared fluorophore with an emission wavelength of about 570nm.

[0019] Quencher group (acceptor): DABCYL (4-(4-dimethylaminophenyl)-3-cyano-5-isothiocyanatebenzoic acid): a commonly used non-fluorescent quencher group because of its small quenching spatial range and the fact that it does not emit fluorescence itself. TAMRA (6-carboxytetramethylrhodamine): a fluorophore that can also be used as a quencher group with an emission wavelength of approximately 580nm. BHQ1 (Black Hole Quencher 1): a very effective non-fluorescent quencher group suitable for applications requiring long-distance quenching. BHQ2 (Black Hole Quencher 2): similar to BHQ1, it is also a highly efficient non-fluorescent quencher group.

[0020] The combination of these groups can be selected according to the specific needs of the experiment to achieve the best FRET efficiency and signal ratio. The efficiency of FRET depends on the overlap between the emission spectrum of the donor and the excitation spectrum of the acceptor, as well as the distance and relative orientation between the donor and the acceptor. By selecting appropriate fluorescent groups and quenching groups, accurate detection of dynamic changes in biomolecules can be achieved.

[0021] During the long-term scientific research process, the applicant has carried out numerous experimental screening and verification and finally obtained a probe with the above-mentioned excellent performance, which has important practical application value.

[0022] The substrate design principles of DNAse I enzyme mainly consider the following aspects:

[0023] DNA structure: The substrate should contain single-stranded or double-stranded DNA, because DNAse I can digest both forms of DNA. For specific applications, such as removing DNA templates in in vitro transcription reactions, the substrate should mimic the DNA structure generated by in vitro transcription. DNA length: The DNA length of the substrate should be suitable for the cleavage characteristics of DNAse I. For applications that produce fragments of specific sizes, a DNA substrate of appropriate length needs to be designed. Specificity: The substrate design should ensure the specificity of DNAse I and avoid nonspecific cleavage, especially in experiments that require precise removal of specific DNA sequences. Salt tolerance: Considering that the activity of DNAse I may be affected under different salt concentrations, the substrate design should consider the salt concentration that may exist in the experiment, and choose a salt-tolerant DNAse I or design a substrate that can remain stable under high salt conditions. Fluorescent label and quencher group: For FRET experiments, the substrate DNA should contain a fluorescent label and a quencher group to achieve the change of the fluorescent signal after DNAse I cleavage. The distance and sequence between the fluorescent label and the quencher group should be designed appropriately to ensure that the fluorescent signal is effectively quenched when not cleaved, and the fluorescent signal is enhanced after cleavage. Stability: The substrate DNA should be stable enough to resist physical and chemical changes under experimental conditions, while being effectively cleaved by DNAse I. Non-nuclease activity: The substrate design should avoid introducing nonspecific nuclease activity to ensure the accuracy of the experimental results. Ease of detection: The design of the substrate DNA should facilitate the detection of the cleavage product, whether by electrophoresis, fluorescence reading or other analytical methods. Biosafety: The design of the substrate DNA should take biosafety into consideration and avoid the use of sequences that may cause immune responses or have potential pathogenicity. Economy and reproducibility: The synthesis and purification of the substrate DNA should be economical and efficient to facilitate large-scale experiments and reproducible studies. In summary, the substrate design of the DNAse I enzyme requires comprehensive consideration of multiple factors to ensure the success of the experiment and the reliability of the results.

[0024] Specifically, the inventors developed a fluorescent DNA probe based on a hairpin structure and systematically optimized its molecular stability, reaction dynamics and detection sensitivity. The core improvements in the probe design are reflected in the following aspects:

[0025] 1. Optimization of stem length and GC content: The length of the probe stem was set to 7 base pairs. After screening experiments, it was verified that this length can provide sufficient conformational stability to ensure that the probe maintains a hairpin conformation ratio of more than 95% at 25°C, and avoid the reduction of enzyme cleavage efficiency caused by an excessively long stem. The GC content of the stem region was optimized to 57%, which was proven to maximize the balance between conformational stability and dynamic response capability at room temperature. Experiments showed that when the GC content was less than 50%, the hairpin conformation ratio of the probe at 25°C dropped to 78.2%±2.4%, significantly increasing the background fluorescence; and when the GC content exceeded 65%, the enzyme cleavage efficiency decreased by more than 15% (p<0.01).

[0026] 2. Loop sequence selection: The loop region is composed of 4 consecutive thymines (T). This design ensures that the loop region has good spatial flexibility and effectively avoids nonspecific pairing or the formation of secondary structures. Compared with randomly selected loop sequences (such as AGTC, CAGC, etc.), the kinetic response speed of the TTTT loop region increased by about 12% and the background signal decreased by 8%.

[0027] 3. Optimization of thermodynamic parameters: The inventors used NUPACK software to screen and verify the melting temperature (Tm) and dissociation energy (ΔG) of multiple candidate probe sequences, and finally selected a sequence with a melting temperature of 65°C and a dissociation energy of -15.8 kcal / mol. This thermodynamic property ensures that the probe has high conformational stability at conventional detection temperatures (25-37°C) and can maintain activity during transportation and storage. Further studies showed that the background signal of this sequence only increased by 6.8% ± 2.5% under simulated transportation conditions (48 hours of shaking, room temperature).

[0028] 4. Storage and background signal processing: Experimental verification shows that when the probe is stored at -20°C for 3 months, the activity retention rate is 89.5% ± 3.2%, and the background signal fluctuation is controlled within ± 5%. However, after storage at room temperature for more than 48 hours, the background signal increases significantly (+ 18.6% ± 3.5%). In response to this problem, the inventors suggest: a. The probe should be ready for use before use; b. Use a dual-wavelength ratio detection strategy to correct the background signal drift in real time through a 590nm reference wavelength.

[0029] The second invention is to provide a use of the fluorescent DNA probe as described in the first aspect in preparing a fluorescent detection kit.

[0030] In a third aspect, the present invention provides a kit, the kit comprising the fluorescent DNA probe described in the first aspect;

[0031] Preferably, the kit further comprises DNase I standard, reaction buffer, and DEPC water;

[0032] Preferably, the reaction buffer comprises Tris-HCl, MgCl2, CaCl2;

[0033] Specifically, this kit contains the following components:

[0034] DNase I standard: Purified recombinant bovine pancreatic DNase I (G8263-1500U, P3196129, Adamas

[0035] Life

[0036] Fluorescent substrate probe: Optimized design of hairpin DNA probe

[0037] Sequence: 5′-FAM-GCAGCTATTTTTAGCTGC-DABCYL-3′ (Qingke Biotechnology);

[0038] Features: a. Stem region length: 7 base pairs; b. Loop region size: 4 nucleotides; c. Stem region GC content: 57%; d. Predicted melting temperature: 65℃; e. Predicted ΔG value: -15.8kcal / mol.

[0039] Reaction buffer (10×):

[0040] 100mM Tris-HCl (pH 7.5, 82997H, P3118149, Adamas Life);

[0041] 25mM MgCl2 (83143L, P2374530, Adamas Life);

[0042] 5mM CaCl2 (G10299K, P2895661, Adamas Life).

[0043] DEPC water (G8010-100mL, P3002894, Adamas Life).

[0044] In a fourth aspect, the present invention provides a detection method, which uses the fluorescent DNA probe described in the first aspect or the kit described in the third aspect.

[0045] Preferably, the detection method adopts a dual-wavelength ratio detection strategy;

[0046] Preferably, the dual wavelength ratio detection uses 495 nm as the excitation wavelength and 590 nm as the reference wavelength;

[0047] Definition of dual-wavelength ratio detection: Dual-wavelength ratio detection is an analytical technique that reflects the concentration or activity of a target substance by measuring the ratio of the signal intensity of the same substance at two different wavelengths. This method can reduce the impact of environmental changes on the test results and improve the accuracy of the test.

[0048] Principle of dual-wavelength ratio detection: The principle of dual-wavelength ratio detection is based on measuring the signal intensity of the same substance at two different wavelengths, and then calculating the ratio of the signal intensity at these two wavelengths. This ratio is proportional to the concentration of the target substance and can be used for quantitative analysis. In fluorescent probes, this principle can use the fluorescence intensity ratio of a fluorescent dye at two different wavelengths to reflect the concentration or activity of the target substance. In spectrophotometry, by selecting two wavelengths, the absorbance of the measured component at these two wavelengths is large enough, and the absorbance of the interfering component and the background at these two wavelengths is the same, thereby achieving accurate determination of the target substance.

[0049] Application of dual-wavelength ratio detection in fluorescent probe detection: After the fluorescent probe binds to the reactant, the excitation or emission spectrum may shift. At this time, the fluorescence intensity ratio measured at two different wavelengths can be used for measurement, which is called ratio measurement. This method can reduce data distortion caused by factors such as probe concentration, optical path length of the excitation light, excitation intensity, and detection efficiency, and improve the accuracy and reliability of detection. For example, Fura-2 is a fluorescent probe used to measure the concentration of intracellular free calcium ions. The ratio of its fluorescence intensity at two excitation wavelengths of 340nm and 380nm is directly related to the concentration of intracellular free calcium ions. By measuring the fluorescence intensity ratio at these two wavelengths, the change in calcium ion concentration can be obtained, thereby achieving accurate determination of intracellular calcium ion concentration. This technology has applications in many fields such as biological stains, living cell applications, and gene expression level determination.

[0050] Preferably, the method comprises a preheating equilibrium step;

[0051] Preferably, the preheating time is 3-10 min; for example, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 or 10 min; the preheating equilibrium is stable at 37±0.2°C;

[0052] Preferably, the preheating time is 5 minutes.

[0053] Preferably, the method adopts a kinetic continuous monitoring mode;

[0054] Preferably, the total detection time in the kinetic continuous monitoring mode is 20-40 min, for example, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min or 40 min; the sampling interval is set to 50-70 seconds; for example, 50 seconds, 52 seconds, 54 seconds, 56 seconds, 58 seconds, 60 seconds, 62 seconds, 64 seconds, 66 seconds, 68 seconds or 70 seconds.

[0055] Preferably, the total detection time is 30 minutes, and the sampling interval is set to 60 seconds.

[0056] Preferably, the method uses a four-parameter logistic regression algorithm to fit and analyze the standard curve;

[0057] Preferably, the four-parameter logistic regression algorithm adopts the following mathematical model:

[0058] y=d+(ad) / (1+(x / c)^b)

[0059] Among them, a represents the asymptote under the curve, d represents the asymptote above the curve, c is the EC50 value, and b is the slope factor of the curve.

[0060] The basic form of the four-parameter logistic regression model can be divided into two types: one is the form in which the concentration X has been logarithmically transformed with a base of 10, and the other is the form in which X has not been transformed. The specific formula is as follows:

[0061] Logarithmic transformation form (base 10):

[0062] Y=Bottom+(Top-Bottom) / (1+10^((LogEC50-X)*HillSlope))

[0063] Unlogarithmically transformed form:

[0064] Y=Bottom+(Top-Bottom) / (1+10^((LogEC50-X)*HillSlope))

[0065] Application of four-parameter logistic regression algorithm in fluorescent probe detection: In fluorescent probe detection, the four-parameter logistic regression algorithm can be used to analyze the law of fluorescence signal change with concentration, especially in dose-response experiments. For example, the model can be used to estimate the EC50 value of the fluorescent probe with or without an enhancer, that is, the probe concentration that causes half the intensity of the maximum fluorescent signal. This is very useful for determining the sensitivity and efficacy of fluorescent probes. Through the four-parameter logistic regression model, researchers can more accurately understand the interaction between fluorescent probes and target molecules, thereby optimizing the design and application of probes.

[0066] Specifically, the present invention provides a detection method, comprising the following steps:

[0067] (1) Sample preparation: adding a standard or a sample to be tested to a reaction buffer, and then adding a fluorescent substrate probe working solution containing the fluorescent substrate probe described in the first aspect;

[0068] (2) ELISA: The test was performed using a preheated ELISA reader with a preheating time of 5 min and a stable preheating equilibrium of 37 ± 0.2 °C.

[0069] Among them, the fluorescence detection adopts a dual-wavelength detection strategy, with 495nm as the excitation wavelength, and simultaneously collects the main emission signal of 528nm and the reference signal of 590nm; the kinetic detection adopts a continuous monitoring mode, with a total detection time of 30 minutes and a sampling interval set to 1 minute;

[0070] (3) Data processing: A four-parameter logistic regression algorithm was used to fit and analyze the standard curve.

[0071] The fluorescence detection method of DNase is mainly based on the Fluorescence Resonance Energy Transfer (FRET) technology. The following are the detailed steps and principles of this method:

[0072] (1) Substrate design: DNase substrate is a synthetic DNA oligonucleotide probe with a fluorescent group (donor, such as VIC) at one end and a quenching group (acceptor, such as BHQ1) at the other end. The absorption spectra of these two groups overlap to a certain extent. When the distance between them is appropriate, the fluorescence energy is transferred from the donor to the acceptor, resulting in the attenuation of the fluorescence intensity of the donor fluorescent molecule itself. (2) Fluorescence quenching: When DNase is not acting, the VIC fluorescent group and the BHQ1 quenching group are closely connected, and the fluorescence is quenched, so the fluorescence of VIC cannot be detected. (3) DNase action: When DNase acts on the substrate and cuts the DNA, VIC and BHQ1 separate, and the fluorescence of VIC is no longer quenched by BHQ1, so the fluorescence of VIC can be detected. (4) Fluorescence detection: DNase enzyme activity can be detected very sensitively by fluorescence detection. (5) Standard curve: The kit provides a DNase I standard, and the DNase I enzyme activity of the sample can be calculated by setting a standard curve. (6) Operation steps: Mix the DNase substrate, sample and reaction solution, and use a fluorescence microplate reader to perform fluorescence measurement. (7) Calculation: Compare the relative enzyme activity of DNase in the sample based on the detected fluorescence intensity. If DNase I detection is performed, the DNase I enzyme activity in the sample can be calculated using the drawn standard curve and the sample fluorescence intensity value.

[0073] This fluorescence detection method has the advantages of high sensitivity, small sample amount, wide application range, flexible use and fast detection speed. It is not only suitable for detecting DNase I, but also can detect other types of DNase, including nucleases that can cut single-stranded and double-stranded DNA.

[0074] Improvement of detection strategy: In order to improve the sensitivity, accuracy and anti-interference ability of detection, the inventors systematically optimized the reaction conditions and detection strategy:

[0075] 1. Improvement of the temperature control system: The method / kit detection adopted in this application is fully compatible with the Thermo Fisher LUX microplate reader, and its temperature control system accuracy reaches ±0.2°C, ensuring the reliability of the enzymatic reaction. In the experimental operation, a 5-minute preheating equilibrium step was set to allow the sample to reach a temperature control stable state (37±0.2°C). This process showed significant advantages in the experiment: short-term fluctuations in the temperature control system were controlled within 0.1°C, ensuring the reproducibility of the kinetic data. Experiments without preheating equilibrium showed that the intra-batch coefficient of variation increased to 6.8% (p<0.05).

[0076] 2. Dual-wavelength ratio detection strategy: The detection strategy uses 495nm as the excitation wavelength and 590nm as the reference wavelength, and calculates the ratio to correct the influence of light source fluctuation, instrument thermal noise and sample autofluorescence. Compared with the single-wavelength detection method, this strategy reduces the background interference rate from 18.2% to 7.4%, and reduces the detection limit to 0.0625mU / μl.

[0077] 3. Optimization of dynamic detection parameters: In the kinetic detection, the inventors set the sampling once per minute, and the total detection time was 30 minutes. By fitting the initial rate of the reaction curve, accurate enzyme activity values ​​can be obtained. This high-frequency sampling strategy provides sufficient data support for subsequent quantitative analysis. At the same time, the inventors optimized the time window selection in the algorithm (5-15 minutes), effectively avoiding the influence of signal attenuation caused by long-term detection on the results.

[0078] 4. Linear range and high concentration correction: The detection range covers 0.0625-10mU / μl, which can meet the analysis requirements of most actual samples. In the high concentration area (>5.0mU / μl), due to substrate depletion and product accumulation, the fluorescence signal tends to saturation (such as 10mU / μl signal increase is only 3.2%). In response to this, the inventor introduced a four-parameter logistic regression algorithm to correct the signal saturation effect and ensure the accuracy of concentration calculation. Experimental results show that the fitting deviation of this method at the high concentration point of 10mU / μl is less than 5%.

[0079] (3) Improvement of data processing method: In terms of data processing method, the present invention selects a four-parameter logistic regression algorithm for fitting and analyzing the standard curve by deeply analyzing the characteristics of DNase activity detection. This selection is based on the following considerations: In enzyme activity detection, the relationship between the fluorescence signal and the enzyme concentration often presents a typical S-shaped curve characteristic, especially in the higher concentration area, due to the consumption of substrate and the accumulation of product, the signal response will gradually deviate from the linear relationship. The traditional linear regression method has obvious limitations in dealing with this nonlinear relationship, especially when the detection range is expanded, the reliability of the data will be significantly reduced.

[0080] The four-parameter logistic regression algorithm uses the following mathematical model: y = d + (ad) / (1 + (x / c) ^ b) where a represents the asymptote below the curve, d represents the asymptote above the curve, c is the EC50 value, and b is the slope factor of the curve.

[0081] This model can more accurately describe the kinetic characteristics of the enzymatic reaction. By optimizing the algorithm parameters, the inventors systematically verified and expanded the detection range. The experimental results showed that the method exhibited excellent linear response in the range of 0.0625-10mU / μl, and the correlation coefficient (R 2 ) remained stable above 0.998, significantly better than the 0.25-5.0 mU / μl detection range of the traditional method. This expansion of the detection range provides greater flexibility for the analysis of samples of different concentrations.

[0082] Among them, the optimization method of the algorithm is as follows: the basic algorithm framework is determined through preliminary experiments, and the least squares method based on regression analysis is selected as the preliminary algorithm framework to establish a linear relationship between concentration and reaction rate. Then, according to the actual performance of the experimental data, the key parameters in the model are optimized, including the regression coefficient, the slope factor of the curve (b), the EC50 value (c), and the setting of the asymptote (a and d). The adjustment of these parameters is intended to ensure a higher fit of the standard curve, so that the detection range can show a stable and accurate linear response within 0.0625 to 10mU / μl.

[0083] The parameter optimization method: Through iterative adjustment, the optimal parameter combination is obtained, so that the high concentration sample can effectively perform signal correction, and the detection sensitivity of the low concentration sample is improved. The initial parameters include regression step, tolerance, number of iterations, etc. First, the regression step is adjusted so that the algorithm can fit the relationship between concentration and reaction rate faster and more accurately. By gradually adjusting the regression step and tolerance threshold, the detection range is successfully expanded and the correlation coefficient (R 2), which significantly improves the detection sensitivity, reduces signal deviation, and effectively improves the reliability of experimental results. Optimized parameters: regression step: 0.01; maximum number of iterations: 1000; tolerance threshold: 1e-6; smoothing algorithm type: weighted smoothing.

[0084] In order to comprehensively evaluate the precision of the method, the inventors selected eight concentration levels of quality control products, 0.0625, 0.125, 0.25, 0.625, 1.25, 2.5, 5.0 and 10.0 mU / μl, for system verification. The results showed that the intra-batch coefficient of variation (CV) showed a significant downward trend with increasing concentration: in the low concentration area (0.0625-0.25 mU / μl), the intra-batch CV fluctuated between 5.0-6.3%; in the medium concentration area (0.625-2.5 mU / μl), the intra-batch CV dropped to 3.8-4.6%; in the high concentration area (5.0-10.0 mU / μl), the intra-batch CV further decreased to 3.5-3.6%. The inter-batch coefficient of variation also showed a similar pattern, which was 6.2-7.5%, 5.2-5.8% and 4.6-4.8% in the low, medium and high concentration areas, respectively. These data indicate that this method can extend the detection range while maintaining good precision characteristics.

[0085] To verify the accuracy of the method, the inventors used a spike recovery test for evaluation. Spiked experiments were conducted at three concentration levels of 0.125, 1.25 and 5.0 mU / μl, and the recovery rates were 91.5-106.8%, 94.3-103.7% and 95.8-102.2%, respectively, with an average recovery rate of 97.8%. At the same time, 30 clinical samples were tested in parallel, and the results showed that the correlation coefficient with the reference method (agarose gel electrophoresis combined with gel imaging quantitative analysis system) reached 0.992 (p<0.001), of which 82.2% of the sample results had a deviation of less than 10%, 17.8% of the sample results had a deviation of between 10-20%, and no sample deviation exceeded 20%. These results fully confirm the accuracy and reliability of this method in practical applications.

[0086] In addition, the algorithm showed good robustness when processing large batches of samples. In the validation experiment using 96-well plates for continuous sample testing (n=96), the system deviation of the entire process was controlled between ±3.7-4.1%, which is comparable to the precision level of single sample testing. The data collection interval was 1 minute, the total detection time was 30 minutes, and the data processing time was less than 1 minute, which fully met the daily high-throughput testing needs of the laboratory. During the batch processing, the results of the three levels of quality control products, high, medium, and low, always remained within ±5% of the target value, confirming the stability of the method in continuous testing. This improved data processing method not only ensures the reliability of batch test results, but also greatly improves the detection efficiency, providing a more practical solution for DNase activity detection of clinical samples.

[0087] Beneficial effects:

[0088] Probe design: The innovation in probe design is mainly reflected in the rational optimization of the stem and loop sequences, as well as the precise layout of the fluorescent group and the quenching group. The stem region of the probe consists of 7 base pairs, and the GC content is optimized to 57%, which provides sufficient conformational stability while avoiding the effect of excessive stability on enzyme cleavage efficiency; the loop region consists of 4 consecutive thymine nucleotides. This design effectively avoids non-specific binding that may be formed between the loop region and the stem region or other probes. Experimental data show that the background fluorescence signal of the probe has good controllability under 25°C detection conditions, and the coefficient of variation (CV) is maintained within 5%, ensuring the reliability of the results.

[0089] Optimization of detection process: This kit combines the temperature control and kinetic detection functions of the Thermo Fisher LUX microplate reader to ensure the efficiency and reliability of experimental operations. Based on the temperature control accuracy (37±0.2℃) and detection system of the platform, data is collected once a minute, and fluorescence kinetic monitoring is completed within 30 minutes to obtain reliable enzyme activity data. This process not only meets the needs of conventional laboratories, but also adapts to 96-well plate high-throughput operations, significantly improving the sample processing efficiency of the laboratory.

[0090] Compatibility: The detection system used in this application is fully compatible with the Thermo Fisher LUX microplate reader and is compatible with other mainstream fluorescence detection platforms. Users do not need to invest in additional dedicated equipment or perform system modifications. In addition, the kit exhibits high stability under transportation and storage conditions. Experimental verification shows that under storage conditions of -20°C, the activity retention rate of probes and enzyme reagents is maintained at 89.5% ± 3.2% within 3 months, and the background signal increase is less than ± 5%. This provides a guarantee for the industrial application and logistics distribution of the product.

[0091] Economical and practical: The fluorescent DNA probe and its detection method and kit provided in this application are not only significantly optimized in functionality, but also significantly reduce the cost of raw material procurement by adopting economical fluorescent groups and quenching groups (FAM-DABCYL). This combination is widely used in the industry and has relatively low cost, and shows good stability and batch consistency in industrial production. Compared with other expensive fluorescent labeling reagents, this design provides a significant advantage in reducing product costs. Compared with traditional probe designs, the cost of a single test of this combination is reduced by about 15%-20%, which has obvious advantages in large-scale production and promotion. In addition, the production process does not involve complex purification steps, which further improves the efficiency of large-scale production. This kit achieves a balance between performance and cost through a probe design that is both economical and practical. While meeting the industry's detection sensitivity and accuracy requirements, the production cost of the kit is further reduced, providing a realistic and feasible solution for large-scale promotion and industrial application. This design and manufacturing concept is particularly suitable for laboratories with limited resources, as well as industrial or clinical testing scenarios that require processing of a large number of samples, and has significant market competitiveness and application potential.

[0092] In summary, the present invention provides a fluorescent DNA probe based on a hairpin structure, which improves its molecular stability, reaction dynamics and detection sensitivity by screening and optimizing the stem region length, GC content, loop region sequence and thermodynamic parameters of the probe, and finally obtains a fluorescent DNA probe with the sequence: 5'-FAM-GCAGCTATTTTTAGCTGC-DABCYL-3'. At the same time, the present invention also develops a detection method and a fluorescent detection kit based on the probe, adopting a dual-wavelength ratio detection strategy, an improved temperature control system adapted to the Thermo Fisher LUX microplate reader, a kinetic continuous detection mode, and a four-parameter logistic regression algorithm to correct and fit the standard curve. Each step cooperates with each other on the basis of the fluorescent DNA probe and works together to finally achieve high sensitivity, wide dynamic range, simple and rapid detection of DNAse enzymes, with the advantages of low cost, high precision, strong stability, etc. The method shows excellent linear response in the range of 0.0625-10mU / μl, and the correlation coefficient (R 2 ) stably maintained above 0.998, significantly better than the detection range of 0.25-5.0mU / μl of the traditional method, with an average recovery rate of 97.8%, which has pioneering clinical application value and broad market prospects. DETAILED DESCRIPTION

[0093] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention. The raw materials and reagents used in the present invention are all commercially available.

[0094] Unless otherwise specified, the reagents and instruments used in this experiment are from the following sources:

[0095]

[0096] Example 1 Test method

[0097] The detection method of this application has been strictly optimized and verified, and a standardized operating procedure has been established.

[0098] This kit contains the following components:

[0099] 1. DNase I standard: Purified recombinant bovine pancreatic DNase I (G8263-1500U, P3196129, Adamas

[0100] Life

[0101] 2. Fluorescent substrate probe: Optimized hairpin DNA probe

[0102] Sequence: 5′-FAM-GCAGCTATTTTTAGCTGC-DABCYL-3′ (Qingke Biotechnology);

[0103] Features: a. Stem region length: 7 base pairs; b. Loop region size: 4 nucleotides; c. Stem region GC content: 57%; d. Predicted melting temperature: 65℃; e. Predicted ΔG value: -15.8kcal / mol.

[0104] 3. Reaction buffer (10×):

[0105] 100mM Tris-HCl (pH 7.5, 82997H, P3118149, Adamas Life)

[0106] 25mM MgCl2 (83143L, P2374530, Adamas Life)

[0107] 5mM CaCl2 (G10299K, P2895661, Adamas Life)

[0108] 4. DEPC water (G8010-100mL, P3002894, Adamas Life).

[0109] Before starting the test, ensure that all reagents return to room temperature (20-25°C). First, prepare the standard. Use 1×Reaction Buffer to dilute the DNase I stock solution (10 mg / ml) step by step to prepare a series of standards with a concentration gradient of 10, 5, 2.5, 1.25, 0.625, 0.25, 0.125, and 0.0625 mU / μl. To ensure data reliability, set up three parallel wells for each concentration point, and set up a blank control.

[0110] The experiment was conducted in a 96-well black flat-bottom microplate. The reaction system was established in the following order: Add DEPC water to the fluorescent substrate probe to a final concentration of 10 μmol / L. Add 80 μL of 1× reaction buffer (pH 7.5) pre-warmed to room temperature, then add 10 μL of standard or sample to be tested and mix gently, and finally add 10 μL of fluorescent substrate probe working solution (the fluorescent substrate probe has been dissolved in DEPC water to a substrate probe concentration of 1 μmol / L). The entire sample addition process should be gentle and smooth to avoid bubbles. If necessary, centrifuge at 1000 rpm for 5 seconds in a centrifuge to remove bubbles.

[0111] The test was performed using an ELISA reader (Thermo Fisher LUX ELISA reader) with temperature control and kinetic detection functions. Temperature control is a key factor in ensuring the accuracy of the test. The system needs to be preheated for 5 minutes before the formal test to stabilize the reaction temperature at 37±0.2℃. This preheating step is essential for obtaining stable enzyme activity and consistent kinetic data. Before the experiment, the sample plate was shaken at medium speed (2mm amplitude) for 3 seconds to ensure that the reaction system was fully mixed.

[0112] Fluorescence detection uses a dual-wavelength detection strategy, with 495nm as the excitation wavelength, while collecting the main emission signal of 528nm and the reference signal of 590nm. This dual-wavelength ratio method can effectively eliminate the interference caused by environmental factors (such as temperature fluctuations, unstable light sources, etc.). The gain value adopts an automatic setting mode. The system will automatically select the optimal gain value in the range of 80-90 according to the background signal and the maximum signal intensity in the sample plate to ensure that the detection signal falls within the linear response range of the instrument.

[0113] The kinetic detection adopts a continuous monitoring mode, with a total detection time of 30 minutes and a sampling interval of 1 minute. This high-frequency sampling strategy can accurately capture the initial rate and kinetic characteristics of the reaction, providing reliable data support for subsequent quantitative analysis. Each data collection point simultaneously records the fluorescence values ​​of the main wavelength and the reference wavelength, and ultimately eliminates systematic errors through ratio calculation, thereby improving the accuracy and repeatability of the data.

[0114] Performance Verification Data

[0115] The performance of the kit was systematically evaluated. The DNase I stock solution (10 mg / ml) was serially diluted using 1× Reaction Buffer to prepare a total of 8 concentration gradients of 0.0625-10 mU / μl. Three parallel wells were set for each concentration, and the experiment was repeated 5 times (a total of 15 data points) (the operation method is the same as above).

[0116] Table 1. Comprehensive performance evaluation results of DNase fluorescence detection kit

[0117] Concentration (mU / μl) Mean RFU RFU standard deviation Intra-assay CV (%) Inter-assay CV (%) Recovery rate range (%) 0.0625 21.8 2.1 9.6 12.3 85.4-112.6 0.125 43.2 3.4 7.8 9.5 88.7-108.4 0.25 76.5 4.8 6.3 8.4 91.2-106.8 0.625 122.3 6.7 5.5 7.2 93.5-104.2 1.25 165.8 7.9 4.8 6.5 94.2-103.8 2.5 192.4 8.8 4.6 6.1 95.1-102.9 5.0 208.6 9.5 4.5 5.9 94.8-103.2 10.0 215.3 10.2 4.7 6.2 93.6-104.5

[0118] Note: Data are based on 15 independent experiments (n=15), RFU is relative fluorescence unit

[0119] This kit exhibits excellent linear response and a wide range of application in terms of detection performance. In the concentration range of 0.0625-10mU / μl, the correlation coefficient (R 2 ) reaches 0.998, indicating that this method has good quantitative ability.

[0120] For high-concentration samples (such as 10mU / μl), although the signal growth tends to saturate (the signal increase is only 3.2%), accurate quantitative results can still be obtained after correction by the four-parameter logistic regression algorithm. This correction strategy effectively solves the signal deviation problem in high-concentration areas of traditional methods, further expanding the detection range to meet the analysis needs of samples of different concentrations.

[0121] The four-parameter logistic regression algorithm uses the following mathematical model: y = d + (ad) / (1 + (x / c) ^ b) where a represents the asymptote below the curve, d represents the asymptote above the curve, c is the EC50 value, and b is the slope factor of the curve.

[0122] For low-concentration samples (such as 0.0625 mU / μl), although the method can still detect fluorescence signals, the intra-batch coefficient of variation (CV) reaches 9.6%, showing a high uncertainty. This phenomenon may be related to the signal being close to the lower limit of instrument detection sensitivity. In order to improve the reliability of the results in the low-concentration area, it is recommended to repeat the test of the sample three times and take the average as the final result. This strategy has been verified in the experiment and can effectively reduce the detection error and control the quantitative deviation of low-concentration samples within an acceptable range.

[0123] Example 2 Stability Study

[0124] The stability of the kit was systematically evaluated under different conditions:

[0125] The main components of the kit (DNase standard and fluorescent DNA probe) were packaged into small tubes and stored at -20°C, 4°C and room temperature. Three parallel samples were taken for testing at each time point, and the experiment was repeated twice (n=6). The activity of the DNase standard was determined by the classic Kunitz method, and the stability of the fluorescent DNA probe was evaluated by measuring its fluorescence signal intensity and background signal. A freshly prepared standard was used as a control for each test, and the test results were converted into retention rates.

[0126] Table 2. Results of stability study of kit components (n=6)

[0127]

[0128]

[0129] Note: "-" indicates not applicable; data are mean ± standard deviation

[0130] The results of the stability study showed that the kit components exhibited the best stability characteristics under -20°C storage conditions, but there was still significant time-dependent degradation. The activity of the DNase I standard remained relatively stable within 3 months of storage, and the activity retention rate was maintained above 88.7±4.1%. However, the activity dropped significantly to 82.4±4.8% after 6 months of storage. It is worth noting that the fluorescent DNA probe exhibited a faster decay characteristic under this temperature condition, and the signal retention rate dropped to 85.6±4.4% after 3 months, while the background fluorescence value increased by 12.3±3.1%. This result shows that the stability of the probe may be a key factor restricting the storage period of the kit.

[0131] Under 4°C storage conditions, the stability of the kit components decreased significantly. Although the indicators were basically stable within 1 week, significant performance degradation was observed after 2 weeks of storage: the activity of the DNase I standard dropped to 85.6±4.2%, and the probe fluorescence signal retention rate dropped to 81.2±4.6%. After 1 month of storage, the probe background fluorescence value increased by 23.7±4.1%, which was beyond the methodologically acceptable range. This significant temperature sensitivity suggests that stable control of storage temperature has an important impact on product performance.

[0132] The experimental data under room temperature storage conditions revealed the time limit of the kit during use. Within 24 hours, the activity retention rate of the DNase I standard was 89.4±3.8%, and the probe fluorescence signal retention rate was 85.7±4.2%, which basically met the detection requirements. However, after 48 hours, various indicators deteriorated sharply. At 72 hours, the DNase I activity dropped to 73.5±5.4%, and the probe background fluorescence value increased by 25.8±4.3%, which could no longer guarantee the accuracy of quantitative detection. These data show that the performance decay of the kit under room temperature conditions showed significant time-dependent characteristics.

[0133] Example 3 Four-parameter logistic regression algorithm calibration.

[0134] In order to further optimize the detection accuracy of high-concentration samples, this kit introduces a four-parameter logistic regression algorithm to effectively correct the saturation effect of the fluorescence signal. In conventional kinetic detection, the increase in the fluorescence signal of high-concentration samples (>5.0mU / μl) tends to saturate due to substrate depletion and product accumulation, resulting in increased detection errors. To this end, we designed a correction experiment and verified the effectiveness of the four-parameter logistic regression algorithm by comparing the data before and after correction.

[0135] In the experiment, DNase I standards with concentration gradients of 5.0, 7.5, 10.0, 12.5, and 15.0 mU / μl were selected, and the concentrations were calculated using the linear fitting method and the four-parameter logistic regression method, respectively.

[0136] Table 3. Comparison of calculation results and deviations of the two algorithms

[0137]

[0138] In addition, we further verified the effect of different sample dilution strategies on the correction of high-concentration signals. Parallel detection was performed under dilution multiples of 1:1, 1:2, and 1:4.

[0139] Table 4. Calculated concentration values ​​and deviations after four-parameter correction

[0140]

[0141] The experimental results show that under different dilution conditions, the four-parameter logistic regression algorithm can stably correct the concentration value, and the deviation is controlled within +0.3%. This method not only effectively solves the problem of high-concentration detection error caused by signal saturation, but also adapts to the high-concentration sample dilution operation requirements commonly used in industrial production.

[0142] Comparative Example 1

[0143] In order to fully verify the innovation and practical application value of this product, we selected three mainstream DNase detection products on the market (numbered P1, P2, and P3) for comparative research. These products all use traditional linear probe technology and are widely used in the field of nucleic acid sample detection. The comparative experiment was carried out under standard conditions (37°C, pH 7.5), and all samples used the same batch of quality control products.

[0144] Table 5. Comparison of core performance indicators of different products (n=6)

[0145] Performance Indicators P1 P2 P3 Industry standard requirements Background interference rate 28.6±2.4％ 31.2±2.8％ 29.5±2.5％ ≤25％ 2h fluorescence decay 18.5±2.1％ 21.3±2.4％ 19.8±2.2％ ≤20％ Minimum detection amount 0.38mU / μl 0.45mU / μl 0.41mU / μl ≤0.25mU / μl

[0146] Through a systematic evaluation of mainstream DNase activity detection products on the market, we found that current products generally have some technical bottlenecks. Although most products basically meet the requirements of industry standards, they still face some challenges in practical applications. In terms of background interference rate, existing products generally fluctuate between 28-31%, which is just at the edge of the industry standard (≤25%) or slightly exceeds it. This higher background signal inevitably affects the signal-to-noise ratio of the detection, especially when dealing with low-concentration samples, which may bring the risk of false positives or missed detections.

[0147] In terms of signal stability, the fluorescence decay rate of the tested product within 2 hours is in the range of 18-21%, which is critical compared with the industry standard (≤20%). This signal decay characteristic imposes time constraints on batch sample processing and may affect the comparability of test results. The minimum detection amount indicator shows that the detection sensitivity of the current product is between 0.38-0.45mU / μl. Although it meets the basic standard requirements (≤0.25mU / μl), it is still insufficient in application scenarios such as trace sample analysis.

[0148] These data show that although the existing technology basically meets the needs of conventional detection, there is still room for improvement in high sensitivity, high throughput applications, etc. This is also the technical direction that the present invention is committed to breaking through. By optimizing the probe design and detection strategy, it is expected to further improve product performance on the basis of ensuring that industry standards are met, and provide solutions for a wider range of application scenarios.

[0149] Comparative Example 2 Comparison of Data Processing Methods

[0150] In terms of data processing, the inventors systematically evaluated two commonly used fluorescence quantitative methods. The same standard series (0.0625-10mU / μl, 8 gradients) was used for comparison:

[0151] Table 6. Comparison of quantitative accuracy of different data processing methods

[0152] Concentration (mU / μl) Linear regression Four parameter method of this product 0.125 142.5±9.2％ 97.8±2.4％ 1.25 115.8±6.8％ 98.5±2.2％ 5.0 72.6±7.4％ 96.4±2.8％

[0153] *Data represent recovery rate ± standard deviation (n=8)

[0154] Table 7. Comparison of stability of quantitative methods

[0155] Precision index Linear regression Four parameter method of this product Intra-batch precision 9.8-14.5％ 3.5-5.8％ Batch-to-batch precision 13.5-16.8％ 4.6-6.5％

[0156] *Data are from comprehensive analysis of low, medium and high concentration levels (n=15)

[0157] To verify the reliability of the method, agarose gel electrophoresis combined with gel imaging quantitative analysis system was selected as a control method. Specifically, the Qinxiang Chemical Imaging System was used to collect gel images, grayscale analysis was performed using ImageJ software, and semi-quantitative analysis was performed based on the degree of DNA degradation. Under standard conditions (0.8% agarose gel, 120V, 45min), 45 samples from the same batch were tested in parallel, and the comparison results are as follows:

[0158] Table 8. Analysis of deviations from electrophoresis results (n=45)

[0159] Deviation range Linear regression Four parameter method of this product <10％ 40.0％(18 / 45) 82.2％(37 / 45) 10-20％ 31.1％(14 / 45) 17.8％(8 / 45) >20％ 28.9％(13 / 45) 0％(0 / 45)

[0160] Note: 1. Deviation calculation is based on the relative quantitative results of gel electrophoresis grayscale analysis

[0161] 2. Grayscale value standardization and data analysis using ImageJ software

[0162] 3. Repeat the measurement for each sample 3 times and take the average value for comparison

[0163] The correlation analysis of the two methods showed that the four-parameter logistic regression method of this product had good consistency with the electrophoresis grayscale analysis method (R 2 =0.992, p<0.001). Especially in the low concentration region (<1mU / μl), this method showed better quantitative accuracy and repeatability. This result verifies the reliability and superiority of this method in the quantitative detection of DNase activity.

[0164] In summary, the present invention provides a fluorescent DNA probe based on a hairpin structure, which improves its molecular stability, reaction dynamics and detection sensitivity by screening and optimizing the stem region length, GC content, loop region sequence and thermodynamic parameters of the probe, and finally obtains a fluorescent DNA probe with a sequence of 5'-FAM-GCAGCTATTTTTAGCTGC-DABCYL-3'. At the same time, the present invention also develops a detection method and a fluorescent detection kit based on the probe, adopts a dual-wavelength ratio detection strategy, an improved temperature control system adapted to the Thermo Fisher LUX microplate reader, a dynamic continuous detection mode, and a four-parameter logistic regression algorithm to correct the fitting standard curve and other means. Each step cooperates with each other on the basis of the fluorescent DNA probe and works together to finally achieve high sensitivity, wide dynamic range, simple and rapid detection of DNAse enzymes, with the advantages of low cost, high precision, strong stability, etc., and has pioneering clinical application value and broad market prospects.

[0165] After the preferred embodiments are described in detail, those familiar with the technology can clearly understand that various changes and modifications can be made without departing from the scope and spirit of the above patent application, and any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are within the scope of the technical solution of the present invention. The present invention is not limited to the example implementation methods given in the specification.

Claims

1. A fluorescent DNA probe based on a hairpin structure, characterized in that: The sequence of the probe is shown in SEQ ID NO.

1.

2. The fluorescent DNA probe according to claim 1, characterized in that The 5' end of the fluorescent DNA probe is a fluorescent group, and the 3' end is a quenching group; Preferably, the fluorescent group includes: any one of FAM, HEX, TET, and ROX; Preferably, the quenching group includes: any one of DABCYL, BHQ1, BHQ2, TAMRA; Preferably, the fluorescent group is FAM, and the quenching group is DABCYL.

3. Use of the fluorescent DNA probe as claimed in claim 1 or 2 in the preparation of a fluorescence detection kit.

4. A kit, characterized in that: The kit comprises the fluorescent DNA probe according to claim 1 or 2; Preferably, the kit further comprises DNase I standard, reaction buffer, and DEPC water; Preferably, the reaction buffer comprises Tris-HCl, MgCl2, and CaCl2.

5. A detection method, characterized in that: The method adopts the fluorescent DNA probe described in claim 1 or 2 or the kit described in claim 4.

6. The detection method according to claim 5, characterized in that: The detection method adopts a dual-wavelength ratio detection strategy; Preferably, the dual-wavelength ratio detection uses 495 nm as the excitation wavelength and 590 nm as the reference wavelength.

7. The method according to claim 5 or 6, characterized in that: The method includes a preheating equilibrium step; Preferably, the preheating time is 3-10 minutes; the preheating equilibrium is stable at 37±0.2°C; Preferably, the preheating time is 5 minutes.

8. The method according to any one of claims 5 to 7, characterized in that: The method adopts a kinetic continuous monitoring mode; Preferably, the total detection time of the kinetic continuous monitoring mode is 20-40 minutes, and the sampling interval is set to 50-70 seconds; Preferably, the total detection time is 30 minutes, and the sampling interval is set to 60 seconds.

9. The method according to any one of claims 5 to 8, characterized in that: The method uses a four-parameter logistic regression algorithm to fit and analyze the standard curve; Preferably, the four-parameter logistic regression algorithm adopts the following mathematical model: y=d+(ad) / (1+(x / c)^b) Among them, a represents the asymptote under the curve, d represents the asymptote above the curve, c is the EC50 value, and b is the slope factor of the curve.

10. A detection method, characterized in that: The steps include: (1) Sample preparation: adding a standard or a sample to be tested to a reaction buffer, and then adding a fluorescent substrate probe working solution containing the fluorescent substrate probe according to claim 1 or 2; (2) ELISA: The test was performed using a preheated ELISA reader with a preheating time of 5 min and a stable preheating equilibrium of 37 ± 0.2 °C. Among them, the fluorescence detection adopts a dual-wavelength detection strategy, with 495nm as the excitation wavelength, and simultaneously collects the main emission signal of 528nm and the reference signal of 590nm; the kinetic detection adopts a continuous monitoring mode, with a total detection time of 30 minutes and a sampling interval set to 1 minute; (3) Data processing: A four-parameter logistic regression algorithm was used to fit and analyze the standard curve.

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