Efficient fluorescence sensor based on TdT / Cas12a cascade amplification and application thereof

Abstract

The invention relates to the technical field of biochemistry, in particular to an efficient fluorescence sensor based on TdT / Cas12a cascade amplification and application of the efficient fluorescence sensor. The invention provides a method for detecting the DNA fragmentation condition of a sample based on the combination of a cascade amplification system of a TdT and CRISPR / Cas12a system and a high-density SNA fluorescent probe. The detection linear range of the detection method is 1 fM to 10 pM, the detection sensitivity reaches 2.5 fM, and the method integrates TdT reaction and CRISPR / Cas12a detection in a single reaction tube, so that the operation process is simplified, the pollution risk and the detection cost are reduced, the result consistency among different laboratories is ensured, a standardized, economical and efficient solution is provided for sperm DNA damage detection, and the method has wide application prospects. The medicine is suitable for wide clinical application.

Description

Technical Field

[0001] The present invention relates to the field of biochemical technology, and in particular to a high-efficiency fluorescent sensor based on TdT / Cas12a cascade amplification and applications thereof. Background Art

[0002] Sperm DNA integrity is an important indicator for assessing male fertility, and damage is closely related to embryo quality and miscarriage rate. The global incidence of infertility has been increasing year by year, of which male factors account for about 40%. The level of sperm DNA fragmentation is closely related to fertilization, embryo development and pregnancy success rate. Excessive sperm DNA damage may lead to reproductive problems such as fertilization failure, decreased embryo quality and spontaneous abortion. Therefore, how to accurately and quickly detect sperm DNA fragmentation has become a key challenge in clinical testing. At present, the mainstream SDF detection method is to evaluate sperm DNA damage through the sperm DNA fragmentation index (DFI), but these methods have problems such as inconsistent standards and complex operations. Summary of the invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide an efficient fluorescent sensor based on TdT / Cas12a cascade amplification and its application.

[0004] The present invention provides a fluorescent sensing system, which includes: terminal deoxynucleotidyl transferase (TdT enzyme), Cas12a, crRNA and SNA fluorescent probe.

[0005] The SNA fluorescent probe includes thiol-modified DNA and a fluorescent group;

[0006] The fluorescent group is selected from FAM, FITC, ROX, RRX, VIC and / or HEX;

[0007] In the present invention, different fluorescent groups are selected, and the excitation wavelength and emission wavelength during detection are also different; in a specific embodiment of the present invention, the fluorescent group is FAM, and correspondingly, the excitation wavelength is set to 488nm, and the emission wavelength is set to 515nm;

[0008] In a specific embodiment of the present invention, the nucleotide sequence of the SNA fluorescent probe is shown in SEQ ID NO:1.

[0009] The SNA fluorescent probe of the present invention is obtained by assembling a thiol-modified DNA fluorescent probe on the surface of gold nanoparticles through freeze compression;

[0010] The freezing compression condition is to freeze at -30°C to -10°C for 0.5h to 1.5h and then compress at 0.5 to 2 MPa; specifically, in the embodiment of the present invention, the freezing compression condition is to freeze at -20°C for 1h and then compress at 1 MPa.

[0011] The nucleotide sequence of the crRNA described in the present invention is shown in SEQ ID NO:2.

[0012] Furthermore, the fluorescence sensing system of the present invention further comprises dATP and Buffer 3;

[0013] The Buffer 3 comprises: potassium acetate, Tris-acetic acid, magnesium acetate, NaCl, Tris-HCl, MgCl2 and recombinant albumin;

[0014] Further, the Buffer 3 comprises: 20mM to 40mM potassium acetate, 10mM to 15mM Tris-acetic acid, 3mM to 9mM magnesium acetate, 122mM to 20mM NaCl, 1mM to 5mM Tris-HCl, 1mM to 5mM MgCl2 and 25μg / ml to 40μg / ml recombinant albumin;

[0015] Furthermore, the Buffer 3 comprises: 33.33 mM potassium acetate, 13.33 mM Tris-acetic acid, 6.66 mM magnesium acetate, 16.66 mM NaCl, 3.33 mM Tris-HCl, 3.33 mM MgCl2 and 33.33 μg / ml recombinant albumin;

[0016] In a specific embodiment of the present invention, the Buffer 3 is obtained by mixing the TdT buffer and the NEBuffer 2.1 buffer in a volume ratio of 2:1; specifically, the TdT buffer includes 50mM potassium acetate, 20mM Tris-acetic acid and 10mM magnesium acetate, pH 7.9 at 25°C; the NEBuffer 2.1 buffer includes 50mM NaCl, 10mM Tris-HCl, 10mMMgCl2 and 100μg / ml recombinant albumin, pH 7.9 at 25°C.

[0017] In the present invention, the concentration of the deoxynucleotidyl transferase is 0.010U / μL to 0.030U / μL, and the optimal concentration is 0.0167U / μL.

[0018] The Cas12a is used at a concentration of 400 nM to 600 nM, and the optimal concentration is 500 nM.

[0019] The ratio of the concentration of Cas12a to the concentration of crRNA is 10:1 to 1:2; specifically, 1:1 is optimal;

[0020] The SNA fluorescent probe is used at a concentration of 50 nM to 150 nM, and is optimally 100 nM;

[0021] The dATP is used at a concentration of 0.01 mM to 0.5 mM, and specifically at 0.1 mM for optimal use.

[0022] The present invention obtains Cas12 rather than other Cas9 proteins for sperm DNA integrity detection by screening of Cas proteins; the test results show that the trans-cleavage activity of Cas12 realizes signal amplification by cleaving multiple ssDNA reporter molecules, greatly improving the detection sensitivity, and is more suitable for ultra-sensitive analysis of sperm DNA fragmentation. In addition, the T enrichment PAM dependence of Cas12 broadens the targeting range, which is convenient for identifying complex biological samples. On this basis, the present invention uses TdT to extend the 3'-OH site of sperm DNA fragments, generates a poly-A tail, and Cas12 / crRNA complexes recognize and activate, thereby realizing signal amplification. Based on TdT / Cas12a cascade amplification and high-density SNA probes (prepared by freezing compression, better performance than non-frozen compression, while selecting the surface area of ​​gold nanoparticles with a particle size of 45nm to be large enough, more DNA or RNA probes can be loaded) combination in improving detection sensitivity, signal amplification and ease of operation have advantages, and the present invention further improves the performance of the detection reaction by optimizing the reaction conditions and the concentration of the participating reaction substances, and obtains a detection method for accurate quantification of DNA fragmentation.

[0023] The present invention provides application of the fluorescence sensing system of the present invention in detecting DNA fragmentation.

[0024] The present invention provides a method for detecting DNA fragmentation for non-diagnostic purposes, which comprises detecting a sample using the fluorescent sensing system of the present invention to obtain the DNA fragmentation status;

[0025] Furthermore, the DNA fragmentation condition includes the average number of breakpoints on the DNA chain;

[0026] Furthermore, in an embodiment of the present invention, the method comprises the following steps:

[0027] Step 1, reacting the sample, deoxynucleotidyl transferase, dATP, Cas12a, crRNA and SNA fluorescent probe in buffer 3 and detecting the fluorescent signal; determining the total concentration Y of the DNA breakpoint of the sample according to the fluorescent signal;

[0028] Step 2: Calculate the average number of breakpoints MDB on each complete sperm DNA chain in the sample according to the following formula:

[0029] MDB=Y×2.4375×10 5 .

[0030] The reaction conditions are 25°C to 42°C, 30min to 60min;

[0031] Specifically, the reaction conditions are: 37° C., 45 min. The method for determining the total concentration Y of the DNA breakpoints of the sample according to the fluorescence signal is a standard curve method.

[0032] The standard curve method is: by establishing a standard curve of samples with known DNA breakpoint numbers and fluorescence signal intensity, a linear equation is obtained, and the fluorescence intensity of the test sample is substituted into the linear equation to obtain the breakpoint number of the test sample;

[0033] The standard curve equation is: FI = 35.6·log (C target )+195.2

[0034] FI is fluorescence intensity;

[0035] C target is the breaking point concentration of the standard sample, the unit of the concentration is nM;

[0036] Furthermore, the sample includes blood, tissue, cells or cell extracts.

[0037] The cells include reproductive cells, which include sperm and egg cells, and in a specific embodiment of the present invention, the reproductive cells are sperm.

[0038] The present invention proposes a detection method for sample DNA fragmentation based on a cascade amplification system of TdT and CRISPR / Cas12a system combined with a high-density SNA fluorescent probe; the detection linear range of the detection method is 1fM~10pM, and the detection sensitivity reaches 2.5fM. In addition, the method integrates TdT reaction and CRISPR / Cas12a detection in a single reaction tube, simplifies the operation process, reduces the risk of contamination and detection cost, ensures the consistency of results between different laboratories, and provides a standardized, cost-effective solution for sperm DNA damage detection, which is suitable for a wide range of clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The main principles and flow chart of sperm testing are shown;

[0040] Figure 2 Schematic diagram of the preparation of SNA probes;

[0041] Figure 3 Show the detection process of single tube reaction system;

[0042] Figure 4 The graph showing the relationship between the fluorescence signal intensity and the number of DNA breaks;

[0043] Figure 5 Shows the correlation analysis diagram between MDB and DFI;

[0044] Figure 6 Shows the comparison chart of normal sperm and weak sperm samples;

[0045] Figure 7 Shown are the ROC curves of MDB and DFI in distinguishing normal sperm from asthenozoosperm samples;

[0046] Figure 8 The feasibility experiment of real-time quantitative detection of DNA fragmentation in biological samples (DNA containing AP sites) by fluorescent biosensor is shown;

[0047] Fig. 9 The present invention shows the performance test of spherical nucleic acid (SNA) prepared by freeze compression method;

[0048] Fig.10 Screening of buffer solutions;

[0049] Fig.11 Shows the optimization of dATP concentration;

[0050] Fig.12 Shows the optimization of the ratio of Cas12a to crRNA;

[0051] Fig.13 Optimization of reaction time;

[0052] Fig.14 Optimization of reaction temperature. DETAILED DESCRIPTION

[0053] The present invention provides an efficient fluorescent sensor based on TdT / Cas12a cascade amplification and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0054] The present invention aims to provide a sperm DNA fragmentation detection method based on TdT / Cas12a cascade amplification system, overcoming the problems of low sensitivity, complex operation and poor quantitative accuracy in the prior art. The 3'-OH end of sperm DNA fragmentation is identified by TdT and polyadenylation is extended, and the CRISPR / Cas12a system is combined to identify and cut the fluorescent probe to achieve high-sensitivity quantitative detection of sperm DNA fragmentation. The present invention also introduces the average DNA break number (MDB) as a new quantitative analysis indicator, which provides more accurate and standardized test results compared to the traditional DNA fragmentation index (DFI), simplifies the operation process and significantly shortens the detection time, and is particularly suitable for clinical sperm quality assessment and male infertility diagnosis.

[0055] The steps of DNA fragmentation index (DFI) test include the following key steps: (1) Collect sperm samples and perform cell fixation and permeabilization treatment to permeabilize the sperm membrane to facilitate the entry of subsequent markers into the cell nucleus; (2) Perform TUNEL labeling and use terminal deoxynucleotidyl transferase (TdT) to add fluorescently labeled dUTP to the broken parts of sperm DNA; (3) Detect the intensity of fluorescent labeling by flow cytometry to reflect the DNA fragmentation of each sperm cell; (4) Calculate the percentage of DNA fragmentation in sperm (DFI) based on the fluorescence intensity, and evaluate sperm quality and DNA damage level by DFI value.

[0056] The difference between DFI and MDB of the present invention is:

[0057] DFI: It evaluates DNA damage by marking and detecting the proportion of DNA fragmentation in sperm (i.e. the percentage of fragmented sperm), but does not provide specific quantitative information on DNA damage. Sperm can only be divided into two categories: "normal" or "fragmented", and lacks quantitative analysis of the degree of DNA damage.

[0058] MDB: Directly measures the number of DNA break sites, reflecting the average number of breaks on each DNA chain, and can accurately quantify the degree of DNA damage. The MDB method provides the specific number of DNA break sites, rather than simply a classification result. Because the MDB method uses a cascade amplification reaction of TdT and Cas12a and combines it with a high-density SNA probe, its sensitivity is significantly improved, and it can detect extremely low levels of DNA damage, making it suitable for early detection.

[0059] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0060] Example 1 Preparation and application of efficient fluorescent sensor based on TdT / Cas12a cascade amplification

[0061] The preparation of an efficient fluorescent sensor based on TdT / Cas12a cascade amplification is used to detect the integrity of sperm DNA fragments, which further reflects the sperm quality. In the present invention, the detection principle and flow chart are as follows Figure 1 As shown, the 3′-OH end of sperm DNA is extended by TdT to generate a poly-A tail, and then the trans-cleavage activity of Cas12a is used to cleave the SNA reporter molecule to amplify the signal, generating a fluorescent signal. The intensity of the fluorescent signal is proportional to the number of DNA breakpoints. Therefore, by measuring the intensity of the fluorescent signal, the degree of DNA breakage can be quantitatively analyzed. Thus, highly sensitive sperm DNA breakpoint detection is achieved. The raw material sequence of the efficient fluorescent sensor based on TdT / Cas12a cascade amplification is shown in Table 1.

[0062] Table 1.

[0063] Name Sequence (5′→3′) Sequence number Hairpin reporter SHC6-ttttttttttgagacatgtctttatttgacatgtctc-FAM SEQ ID NO: 1 crRNA uaauuucuacuaaguguagauuuuuuuuuuuuuuuuuuuuuuu SEQ ID NO: 2

[0064] In the sequence table, base u of crRNA is represented by base t.

[0065] Hairpin reporter is a thiol-DNA probe, wherein SH is a thiol group, C6 is 6 carbon atoms (straight chain), and a fluorescent group is located at the other end of the thiol label, and the fluorescent group is FAM (Hairpin reporter thiol-DNA probe is synthesized by conventional methods).

[0066] The sulfhydryl group (SH) on the probe sequence was connected to the AuNP surface, and the SNA probe was successfully constructed on the AuNP. The poly-T sequence provides a spatial interval to maintain the activity of the functional probe region, while the functional nucleic acid sequence and FAM are used for the final fluorescence signal detection. When the target DNA (polyA tail formed at the end of the DNA break point by deoxynucleotidyl transferase using dATP as raw material) is recognized by crRNA, Cas12a in the detection system is activated, which indiscriminately cuts the functional segment of the SNA probe and releases a fluorescent signal, thereby realizing the detection of sperm DNA integrity.

[0067] The preparation and detection process of the efficient fluorescent sensor based on TdT / Cas12a cascade amplification is as follows:

[0068] 1. Preparation and performance testing of SNA probes

[0069] 1. Preparation of SNA probe

[0070] like Figure 2As shown in the figure, the preparation process of SNA fluorescent probes is shown. First, 45nm gold nanoparticles (AuNPs) are prepared by sodium citrate reduction method. Subsequently, thiol-DNA fluorescent probes are assembled onto the surface of gold nanoparticles by freeze compression method to form high-density nucleic acid-modified SNA probes. These SNA probes are used as detectors of fluorescent signals. The figure intuitively shows the preparation of AuNPs and their functionalization process.

[0071] The specific steps are as follows:

[0072] 1.1 Preparation of gold nanoparticles (AuNPs):

[0073] Add 1 mL of 1% gold chloride solution (HAuCl4·3H2O) to 100 mL of ultrapure water and heat to boiling in a three-necked flask. Quickly add 1 mL of freshly prepared 1% sodium citrate solution and keep stirring vigorously. The color of the solution will change from light yellow to wine red within a few minutes, indicating that gold nanoparticles have been successfully formed. Continue heating and stirring for 15 minutes, then stop heating and let the solution cool to room temperature. The final solution is stored in a dark place for later use.

[0074] 1.2 Thiol DNA modification:

[0075] AuNPs were functionalized with thiol-modified DNA (SEQ ID NO: 1). Hairpin reporter solution was mixed with AuNPs and the mixture was placed in a freezer for 15 minutes. The sample was frozen at -20°C for 1 hour and compressed by mechanical pressure (1 MPa) to form high-density spherical nucleic acids (SNA). After freeze compression, the sample was thawed and centrifuged at 10,000g and 4°C for 30 minutes to remove unbound DNA. The wash was repeated 3 times and resuspended in deionized water.

[0076] Methodological optimization: The selection and preparation of SNA reporter molecules and gold nanoparticles (AuNPs) were optimized to improve the detection performance. First, 45nm AuNPs were selected as the basic material, and AuNPs with uniform size and good stability were synthesized by sodium citrate reduction method, which provided a reliable basis for subsequent functionalization. AuNPs of this size have a large surface area, can achieve high-density DNA loading, and have good biocompatibility, thereby improving the stability of the SNA probe and the binding efficiency with the target DNA. During the functionalization process, the thiol (SH) in the thiol-DNA reporter molecule firmly binds to the gold atoms on the AuNP surface, allowing the DNA molecules to stably attach to the AuNP surface. At the same time, the density of DNA molecules on the AuNP surface was further increased by the freeze compression method to form a high-density SNA reporter molecule. This method not only enhances the stability of the probe, but also significantly improves the amplification effect of the fluorescence signal, making it have a significant advantage in detection sensitivity compared with traditional low-density SNA reporter molecules.

[0077] 2. Specific steps for sperm DNA fragmentation detection based on TdT and CRISPR / Cas12a:

[0078] 1. Sample preparation

[0079] Sperm DNA extraction: Use a sperm DNA extraction kit (from Shenzhen Baiyi Biotechnology Co., Ltd.) to extract DNA from semen samples according to the manufacturer's instructions. Ensure that the DNA extract does not contain protein and other impurities.

[0080] DNA breakage detection: In the extracted DNA sample, the 3'-OH end at the break provides a substrate for subsequent reactions.

[0081] 2. Composition of the reaction system:

[0082] DNA sample: DNA fragments containing 3'-OH termini;

[0083] Terminal deoxynucleotidyl transferase (TdT): used to catalyze the extension of the 3'-OH end, the concentration used is 1U / 60μL;

[0084] dATP: As the substrate for the extension reaction, the concentration used was 0.1 mM;

[0085] Mixed buffer: combines the optimal conditions of TdT and CRISPR / Cas12a (specifically Buffer 3, which is a mixture of TdT buffer and NEBuffer 2.1 in a certain proportion to ensure the compatibility of the two reaction components);

[0086] Other ingredients: Cas12a protein used at a concentration of 500nM, crRNA used at a concentration of 500nM, and spherical nucleic acid (SNA) used at a concentration of 100nM;

[0087] Reaction conditions:

[0088] Reaction temperature: 37°C.

[0089] Reaction time: 45 minutes (including the simultaneous time of TdT extension and Cas12a cutting).

[0090] The above detection steps specifically include TdT extension and simultaneous CRISPR / Cas12a cutting; TdT recognizes the 3'-OH end of DNA and catalyzes the gradual addition of dATP to extend and form a polyadenylic acid (polyA) tail chain. The extended polyA tail chain is specifically recognized by the designed crRNA, activating the trans cutting activity of CRISPR / Cas12a. The activated Cas12a cuts the SNA fluorescent probe, releasing a fluorescent signal, completing the entire detection process. Among them, the TdT extension reaction and CRISPR / Cas12a detection reaction are completed in one tube, which is called a single-tube reaction system; the detection process of the single-tube reaction system is as follows Figure 3 As shown;

[0091] Figure 3 It demonstrates how to detect sperm DNA fragmentation in a single-tube reaction system. The single-tube reaction system significantly simplifies the operation process by integrating all reaction steps in one reaction tube. There is no need to change the environment midway from sample processing to fluorescent signal detection, which reduces the risk of contamination and improves reaction efficiency. First, the reaction compatibility was optimized. Buffer3 buffer was used so that the TdT extension reaction and CRISPR / Cas12a cleavage reaction could be carried out simultaneously at 37°C, avoiding the complexity of multi-step operations. Secondly, the reaction conditions were optimized at a suitable temperature of 37°C, and 45 minutes was verified as the optimal reaction time through experiments, which not only ensured the generation of fluorescent signals, but also avoided nonspecific cleavage caused by too long reaction time. The reagent concentration was also finely adjusted. 1U / μL TdT enzyme ensured efficient extension of the 3'-OH end, 500nM Cas12a protein and crRNA ratio optimization ensured high sensitivity of the cleavage reaction, and 100nM FAM-labeled SNA probe was used to generate sufficient fluorescent signals to achieve quantitative detection of sperm DNA fragmentation. In summary, the design of the single-tube system not only improves the simplicity and automation potential of the reaction, but also makes the system particularly suitable for high-throughput clinical testing applications. The figure shows the entire process from sample addition to reaction system activation to fluorescence signal generation, highlighting the simplicity and rapidity of the reaction system.

[0092] 3. Quantitative Detection Process

[0093] The fluorescence signal was detected by a fluorescence spectrophotometer with the excitation wavelength set at 488 nm and the emission wavelength set at 515 nm.

[0094] Relationship between signal and number of breakpoints: Each sperm DNA breakpoint can trigger TdT extension and Cas12a trans-cutting, thereby releasing fluorescent signal. Therefore, the fluorescence signal intensity of the sample is proportional to the number of breakpoints in sperm DNA, such as Figure 4 As shown, with the increase of DNA breakpoints, the fluorescence signal intensity increases significantly, and the increase of fluorescence signal is proportional to the number of DNA breakpoints. It can be used for quantitative analysis of DNA fragmentation, and this system has high accuracy and sensitivity.

[0095] Calculate the average number of DNA breaks (MDB): By measuring the fluorescence intensity of the sample after the reaction, the number of break points (MDB) of each sperm DNA sample can be calculated to achieve quantitative analysis of the degree of DNA fragmentation.

[0096] The MDB calculation process is as follows:

[0097] (1) Fluorescence intensity measurement: After the reaction is completed, the fluorescence intensity of the sample is measured to obtain a fluorescent signal related to the amount of sperm DNA fragmentation.

[0098] (2) Standard curve: Use standard samples with known DNA breakpoint numbers to draw a standard curve of fluorescence intensity and breakpoint numbers. The standard curve is generated by different concentrations of DNA breakpoints, and its linear equation is:

[0099] FI=35.6·log(C target )+195.2

[0100] FI is fluorescence intensity;

[0101] C target is the breaking point concentration of the standard sample, the unit of the concentration is nM;

[0102] Linear correlation: The correlation coefficient is R 2 =0.9654, indicating that the linear fit is high and the standard curve can be used to accurately determine the breakpoint concentration of unknown samples.

[0103] Linear range: The standard curve is applicable to the target DNA concentration range from 1fM to 10pM.

[0104] (3) Calculation of the number of breakpoints in the sample: Substitute the fluorescence intensity of the sample into the standard curve equation to calculate the total concentration Y of the corresponding DNA breakpoints.

[0105] (4) Calculation of MDB: MDB represents the average number of breakpoints on each intact sperm DNA chain in the sample. The breakpoint concentration obtained by the fluorescence signal can be converted into the average number of breaks on each sperm DNA chain (×10 5 ), as the final MDB value;

[0106] The calculation formula of MDB is as follows:

[0107] MDB=Y×2.4375×10 5

[0108] By establishing a standard curve, the fluorescence signal intensity is converted into the quantitative result of DNA damage (MDB), and compared with the traditional DNA fragmentation index (DFI) to ensure the accuracy and effectiveness of the detection. Figure 5 As shown in A in FIG, MDB is highly positively correlated with DFI (R=0.744), indicating that the MDB parameter of the present invention is consistent with the traditional DFI method, further supporting the reliability of MDB as a quantitative analysis indicator of sperm DNA fragmentation; Figure 5 In B, MDB steps are simple and time-saving.

[0109] The results of normal sperm and weak sperm samples are as follows Figure 6 As shown. The difference in the mean DNA fragmentation number (MDB) between normal sperm and asthenozoosperm samples, through statistical analysis of the MDB of the two groups of samples, revealed significant differences in sperm DNA fragmentation. Normal sperm samples have lower MDB values, usually between 1.5 and 3.0, indicating that their DNA integrity is high; while the MDB values ​​of asthenozoosperm samples are significantly higher, usually exceeding 5.0, reflecting more severe DNA damage.

[0110] Statistical analysis showed that the MDB value of the asthenozoospermia group was significantly higher than that of the normal sperm group (P<0.05), and the difference was statistically significant. This indicates that the number of DNA breakpoints in asthenozoosperm samples increased significantly, which is closely related to their decreased fertility. By comparing the MDB values, normal sperm and asthenozoosperm can be clearly distinguished, providing an accurate quantitative indicator for sperm quality assessment. Compared with the traditional DNA fragmentation index (DFI), MDB performs better in sensitivity and specificity, can better reflect the degree of DNA fragmentation, has a high clinical application value, and is especially suitable for sperm DNA damage assessment in infertile patients.

[0111] The ROC curves of MDB and DFI in distinguishing normal sperm from weak sperm samples are shown in Figure 2. Figure 7As shown. The ROC curve shows the trade-off between sensitivity and specificity. The AUC value of MDB (0.8417) is higher than that of DFI (0.7917), indicating that the MDB parameter has a higher diagnostic performance in distinguishing the quality of sperm samples. This proves the superiority of the method of the present invention in clinical sperm quality assessment.

[0112] 4. Feasibility experiment of real-time quantitative detection of DNA fragmentation in biological samples (DNA containing AP sites) using fluorescent biosensors

[0113] (1) Ultrasonic treatment of DNA

[0114] Schematic diagram of ultrasonic treatment of DNA Figure 8 A in Figure 1. In order to simulate different degrees of sperm DNA fragmentation, the experiment used ultrasound to treat DNA samples containing AP sites. The ultrasound treatment settings were: 2 minutes of ultrasound, vigorous vibration, pause for 30 seconds, and repeat the cycle. This process can artificially introduce DNA fragmentation for subsequent detection experiments.

[0115] (2) Fluorescence response after different ultrasound cycle times

[0116] like Figure 8 B in the figure. After different ultrasonic treatment times, the degree of DNA fragmentation gradually increased. As the ultrasonic treatment time increased, the number of 3'-OH ends in the DNA fragments increased, resulting in a gradual increase in fluorescence intensity. The growth rate of the fluorescence signal was positively correlated with the number of DNA breaks, indicating that the sensor can sensitively detect the degree of DNA fragmentation.

[0117] (3) Schematic diagram of DNA containing AP site being cleaved by APE1

[0118] like Figure 8 C and D in the figure. The experiment selected DNA containing AP sites treated with APE1 enzyme. According to DNA sequence analysis, each DNA with AP sites was split into four fragments after APE1 enzyme treatment. This enzyme cleavage method was used to simulate the decomposition of DNA to further verify the ability of the sensor in detecting different degrees of DNA fragmentation.

[0119] (4) Fluorescence response after APE1 cleavage increases with incubation time

[0120] As the incubation time increases, the APE1 enzyme gradually breaks down the DNA more completely, producing more 3'-OH ends, leading to an increase in the fluorescence signal. The growth rate of the fluorescence signal accelerates with the extension of the incubation time, indicating that the detection system can quantitatively detect the terminal site content of DNA in biological samples in real time.

[0121] 3. Performance of SNA probes for detection of DNA fragmentation

[0122] (1) Sensitivity and detection limit ( Fig. 9 A~D)

[0123] Linear relationship between fluorescence intensity and target concentration: In Figure A, the SNA probe prepared by the freeze-compression method showed a significant increase in fluorescence intensity with increasing target concentration, indicating its extremely high sensitivity in detecting low-concentration targets.

[0124] Linear detection range: Fig. 9 Figure B shows the wide linear detection range of the system from 1fM to 10pM, and the linear relationship is FI = 35.6·log(C target )+195.2, and the correlation coefficient (R2=0.9654) is close to 1, indicating that the SNA probe has a strong linear fitting ability.

[0125] Detection limit: According to Fig. 9 C and D in the figure, the detection limit of the DNA fragmentation detection method with the SNA probe of the present invention reached 2.5fM, which is about 40 times higher than the traditional ssDNA probe (usually with a higher detection limit). This shows that the frozen compressed SNA is significantly better than the traditional fluorescent molecules in signal amplification and can produce reliable fluorescent signals at extremely low target concentrations. The method of evaluating sperm DNA integrity of the present invention is compared with other methods as shown in Table 1.

[0126] Table 1. Comparison of the method of the present invention for assessing sperm DNA integrity with other methods

[0127] Treatment Linear range Detection limit time source DNA Strand Displacement / TdT Enzyme 0.01~4nM 0.01nM 1.5h A Endonuclease IV and TdT enzyme 0.1~15nM 0.03nM 2h B Endonuclease IV / TdT enzyme 0.5~10nM 1.03nM 3h C Strand displacement / TdT enzyme 0.01~4nM 0.35nM 80min D TdT enzyme / Cas12a 1fM~10pM 2.5fM 45min The present invention

[0128] (A): B.Yan, H.Fan, H.Ji, S.Li, H.Hu, X.Gu, S.Jia, Y.Liu, J.Guo, Z.Yang, L.Zhou, Acta, 1280(2023)341821.

[0129] (B): B.Yan, W.Ye, J.Wang, S.Jia, X.Gu, H.Hu, W.Xiang, T.Wu, X.Xiao, Evaluation of Sperm DNA Integrity by Mean Number of Sperm DNA Breaks Rather Than SpermDNA Fragmentation Index, Clin Chem, 68 (2022) 540-549.

[0130] (C): Y. Zhou, J. Wang, L. Zhuo, C. Pei, S. Jia, J. Tian, ​​H. Wang, B. Yan, L. Ma, Fluorescent enzyme-based biosensor for sensitive analysis of DNA damage incryopreserved sperm, Cryobiology, 113(2023)104591.

[0131] (D): B.Yan, J.Wang, Y.Zhou, L.Pei, F.Zhang, B.Gao, H.Wang, The application of mean number of DNA breakpoints in sperm cryopreservation, Cryobiology, 116(2024)104937.

[0132] (2) Repeatability and stability

[0133] Repeatability experiment: Fig. 9 Figure E shows the coefficient of variation of the SNA probe in the same batch and different batches (1.99% to 2.72%), with small fluctuations in the data, showing good repeatability. This consistency is very important in actual testing because it ensures consistent results between different experiments.

[0134] Long-term stability: Fig. 9 The F in the figure shows that the fluorescence intensity of the SNA probe has almost no significant change after being stored at -20°C for 1 to 12 days, indicating that the probe remains stable under long-term storage conditions. This is better than some traditional fluorescent reporter molecules, which may be photobleached or degraded during storage, while the SNA generated by the freeze-compression method has better storage resistance.

[0135] Example 2 Optimization of efficient fluorescent sensor based on TdT / Cas12a cascade amplification

[0136] 1. Optimization of experimental conditions

[0137] 1. Effect of buffer conditions

[0138] First, the effects of different buffers on the fluorescence signal were compared, using TdT buffer (50mM potassium acetate, 20mMTris-acetic acid and 10mM magnesium acetate, pH 7.9 at 25°C), NEBuffer 2.1 (50mM NaCl, 10mM Tris-HCl, 10mM MgCl2 and 100μg / ml recombinant albumin, pH 7.9 at 25°C) and a mixed solution of the two (Buffer3). The experimental results showed that ( Fig.10 ), Buffer 3 (i.e., a mixed solution of TdT buffer NEBuffer 2.1) produced the highest signal-to-noise ratio, so Buffer 3 was selected as the optimal buffer in subsequent experiments. This optimization ensures that TdT extension and Cas12a cutting work efficiently and synergistically in the same system.

[0139] 2. Effect of dATP concentration

[0140] In the extension reaction catalyzed by TdT, the concentration of dATP directly affects the length of the poly-A tail, which in turn affects the cutting efficiency of Cas12a. Fig.11 ), a dATP concentration of 0.1mM works best, a higher dATP concentration may lead to over-extension, affecting the recognition and cutting of Cas12a, while a lower concentration may lead to insufficient poly-A tail and fail to effectively activate Cas12a.

[0141] 3. Ratio of Cas12a to crRNA

[0142] In order to optimize the binding efficiency of Cas12a and crRNA, the experiment compared the fluorescence signals under different ratios ( Fig.12 ), and found that the signal generated by the ratio of 1:1 was the best. This ratio ensures the best match between Cas12a and crRNA, which helps to efficiently activate the trans-cleavage activity of Cas12a during the reaction.

[0143] 4. Optimization of response time

[0144] The experiment determined that 45 minutes was the optimal reaction time by measuring the fluorescence signal at different reaction times ( Fig.13 ). At this time point, the fluorescence signal reaches a stable state and the reaction efficiency is the highest. This optimization ensures the rapidity of the detection method while ensuring the completeness of the reaction.

[0145] 5. Optimization of reaction temperature

[0146] As the temperature increases, the fluorescence signal gradually increases, reaching a maximum at 37°C and then decreasing slightly at 42°C ( Fig.14 ). The signal-to-noise ratio reached the highest level at 37°C, indicating that 37°C is the optimal temperature for this reaction, at which the efficiency of Cas12a cleavage and generation of fluorescent signals is highest.

[0147] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fluorescence sensing system, characterized in that: include: Deoxynucleotidyl transferase, Cas12a, crRNA and SNA fluorescent probe; The nucleotide sequence of the SNA fluorescent probe is shown in SEQ ID NO: 1; The nucleotide sequence of the crRNA is shown in SEQ ID NO:

2.

2. The fluorescence sensing system according to claim 1, characterized in that: The fluorescent sensing system also includes dATP and Buffer 3; The Buffer 3 comprises potassium acetate, Tris-acetic acid, magnesium acetate, NaCl, Tris-HCl, MgCl2 and recombinant albumin.

3. The fluorescence sensing system according to claim 2, characterized in that: The Buffer 3 comprises: 20mM-40mM potassium acetate, 10mM-15mM Tris-acetic acid, 3mM-9mM magnesium acetate, 122mM-20mM NaCl, 1mM-5mM Tris-HCl, 1mM-5mM MgCl2 and 25μg / ml-40μg / ml recombinant albumin.

4. The fluorescence sensing system according to any one of claims 1 to 3, characterized in that: The concentration of the deoxynucleotidyl transferase is 0.010U / μL to 0.030U / μL; The concentration of Cas12a is 400nM to 600nM; The ratio of the concentration of Cas12a to the concentration of crRNA is 10:1 to 1:2; The SNA fluorescent probe is used at a concentration of 50nM to 150nM; The dATP is used at a concentration of 0.01 mM to 0.5 mM.

5. Use of the fluorescent sensing system according to any one of claims 1 to 4 in detecting DNA fragmentation.

6. A method for detecting DNA fragmentation for non-diagnostic purposes, characterized in that: The method comprises using the fluorescent sensing system described in any one of claims 1 to 4 to detect a sample to obtain the DNA fragmentation status.

7. The method according to claim 6, characterized in that The DNA fragmentation profile includes the average number of break points on the DNA chain.

8. The method according to claim 7, characterized in that The steps include: Step 1, reacting the sample, deoxynucleotidyl transferase, dATP, Cas12a, crRNA and SNA fluorescent probe in buffer 3 and detecting the fluorescent signal; determining the total concentration Y of the DNA breakpoint of the sample according to the fluorescent signal; Step 2: Calculate the average number of breakpoints MDB on each complete sperm DNA chain in the sample according to the following formula: CIS=Y×2.4375×10 5 。 9. The method according to claim 8, characterized in that The reaction conditions are 25°C to 42°C, 30min to 60min; The method for determining the total concentration Y of the DNA breakpoints of the sample based on the fluorescence signal is a standard curve method.

10. The method according to any one of claims 7 to 9, characterized in that: The sample includes blood, tissue, cells or cell extracts.

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