High-sensitivity embryo chromatin analysis method

Abstract

The invention relates to the field of cell genetic engineering, and discloses a high-sensitivity embryo chromatin analysis method which comprises the following steps: S1, puncturing an embryo zona pellucida through a micromanipulation instrument, sucking 5-8 trophoblast cells, and immersing a sample into 4 DEG C chromatin stabilizing liquid containing 1mM of ethylenediamine tetraacetic acid and bovine serum albumin within 8 seconds after the sample is obtained; s2, processing the sample by adopting a gradient fixation method; s3, sequentially carrying out low-intensity ultrasonication and restriction enzyme combined treatment; s4, carrying out specific labeling by using a double-labeling probe system; s5, implementing two-stage signal amplification in the micro-fluidic chip; and S6, carrying out three-dimensional space conformation analysis by adopting a super-resolution microscope. A silicon nitride microscopic needle penetrates through the zona pellucida at a puncture angle of 75-85 degrees, and the suction speed is controlled to be 0.5 mu L / s in combination with a PID closed loop, so that the DNA fragmentation rate can be reduced to 2.1 + / -0.3% (the proportion of 5kb fragments is 98.5%), and the embryo survival rate is gt; the H3K9 acetylation modification level is improved by 5.3 times, and the chromatin accessibility is improved.

Description

Technical Field

[0001] This invention relates to the field of cell genetic engineering, specifically to a highly sensitive method for analyzing embryonic chromatin. Background Technology

[0002] Cell genetic engineering is a biotechnology field based on the principles of genetics and cell biology. It involves the targeted modification or recombination of the genetic material of cells through gene editing, transgenic technology, cell fusion, chromosome manipulation, and other means to alter the genetic information of cells, regulate their biological characteristics, or endow them with new functions. Its core lies in the precise manipulation of genetic elements within cells to achieve targeted regulation of organism traits, metabolic pathways, or developmental processes. It is widely used in gene therapy, disease model construction, biopharmaceuticals, crop genetic improvement, and industrial microbial optimization. Embryo chromatin analysis is an important part of cellular genetic engineering.

[0003] Existing embryonic chromatin analysis uses needles with an outer diameter >15μm, a fixed puncture angle and speed for sample collection, and a single concentration of fixative (such as 1% paraformaldehyde), which leads to an excessively high sample damage rate and excessive cross-linking, reducing chromatin accessibility and affecting the accuracy of subsequent analysis. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a highly sensitive embryonic chromatin analysis method, which solves the problems of excessively high embryonic damage rate and reduced chromatin accessibility caused by the use of needles with an outer diameter >15μm for sample collection and the use of single-concentration fixatives in existing embryonic chromatin analysis.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a highly sensitive method for embryonic chromatin analysis, comprising the following steps:

[0006] S1. Using a micromanipulator, puncture the zona pellucida of the embryo and aspirate 5-8 trophoblast cells. Within 8 seconds of obtaining the sample, immerse it in a chromatin stabilizing solution containing 1 mM EDTA and bovine serum albumin at 4°C.

[0007] S2. Samples were treated using a gradient fixation method: in the first stage, the samples were fixed with paraformaldehyde at 4°C for 5 minutes, and in the second stage, the samples were fixed with paraformaldehyde at 25°C for 3 minutes, followed by washing with phosphate buffer 3 times.

[0008] S3. Perform low-intensity ultrasonic disruption and restriction endonuclease combination treatment sequentially;

[0009] S4. Use a dual-label probe system for specific labeling. The probe contains a fluorescent label and a biotin-modified group. After purification by high performance liquid chromatography, the labeling efficiency is verified to be ≥95%.

[0010] S5. Implement two-stage signal amplification in the microfluidic chip, with a chip channel size of 50×100μm and a temperature control accuracy of ±0.3℃;

[0011] S6. Three-dimensional spatial conformation analysis was performed using super-resolution microscopy, and the chromatin spatial model was reconstructed by calculating the cross-correlation function of fluorescence signals.

[0012] The above technical solution involves: S1 stage: transferring 5-8 feeder cells to 4℃ chromatin stabilizing solution (containing 1mM EDTA and bovine serum albumin) within 8 seconds to inhibit nuclease activity and maintain chromatin structure, with a DNA fragmentation rate of <3%; S2 stage: using a gradient fixation method, treating with paraformaldehyde at 4℃ for 5 minutes to reduce epitope masking, followed by treatment at 25℃ for 3 minutes to improve antigen stability; washing three times with phosphate buffer to remove residual cross-linking agent; S3 stage: 0.5W / cm² 2 Sonication disrupted and loosened chromatin, followed by EcoRI / HindIII double enzyme digestion to cleave the boundaries of topologically associated domains, achieving an opening efficiency of 89±5%. In stage S4, a 5'-Cy3 / 3'-biotin dual-labeled probe was used; biotin modification provided an anchoring point for signal amplification. In stage S5, signal amplification was performed on a 50×100μm microfluidic chip: streptavidin-horseradish peroxidase catalyzed tyramine deposition, and fluorescent antibody coupled the signal. Temperature control at ±0.3℃ improved the signal-to-noise ratio by 40 times. In stage S6, multicolor fluorescent point clouds were acquired using super-resolution microscopy, and 30nm chromatin fibers were reconstructed using a cross-correlation function algorithm and a Beads-on-a-string model, achieving a resolution of 5kb and a sample detection rate >91%. CRISPR validation confirmed a chromatin loop reconstruction accuracy of 94.3%.

[0013] Preferably, in S1, the micromanipulator needle is made of silicon nitride, with a diameter of 8±0.5 micrometers, the puncture angle is controlled between 75-85 degrees, the aspiration speed is 0.5 microliters / second, and after the sample is obtained, a stabilizing solution containing 10 micromoles of histone deacetylase inhibitor is added.

[0014] Preferably, in step S2, a low-intensity ultrasonic disruptor is used to process the sample, with parameters set to a power density of 3-4 W / cm³. 2 The pulse duration is 1 second per pulse, with a 2-second interval, and the total processing time is 2 minutes.

[0015] Preferably, the restriction endonuclease combination in S3 comprises:

[0016] MseI: 10.5 ± 0.5 units / µL, recognizing site TTAA.

[0017] NcoI: 15.2 ± 0.3 units / µL, recognition site C^CATGG.

[0018] HinfI: 8.1 ± 0.2 units / µL, recognizing site G^ANTC.

[0019] The enzyme digestion reaction buffer was a pH 7.4 tris(hydroxymethyl)aminomethane hydrochloride buffer containing 5 mmol magnesium chloride and 50 mmol sodium chloride, followed by reaction at 37 °C for 45 ± 2 minutes.

[0020] Preferably, the dual-labeled probe in S4 meets the following requirements: length 20±1 base pairs, no off-target binding verified by sequence alignment tools, guanine-cytosine content 45-55%, melting temperature 68±2℃, containing 5 phosphate thioester modifications, spaced 2-3 nucleotides apart, cyanine 5 fluorescent label located at the 3' end, biotin linked to the 5' end through a 10-carbon spacer arm, working concentration 50 nanomolar, and hybridization temperature 55±0.5℃.

[0021] Preferably, the signal amplification in S5 includes:

[0022] First-level scale-up: Streptavidin-modified 20±2 nm gold nanoparticles with a surface coating rate of ≥80%, incubated at 37°C for 45 minutes;

[0023] Secondary amplification: The catalytic hairpin assembly system contained 10 micromoles of hairpin probe H1, 5 millimoles of magnesium ions, and 0.1% Tween-20. Amplification was performed at 37°C for 60 minutes to produce a dendritic DNA structure.

[0024] Three-stage amplification: A second antibody labeled with quantum dots was introduced, with a particle size of 8 nanometers and an emission wavelength of 625 nanometers, which bound at 4°C for 30 minutes.

[0025] Preferably, the microfluidic chip adopts a polydimethylsiloxane-glass composite structure, the channel surface is treated with 0.1% poloxamer 407, the flow rate control adopts a pressure-driven mode with an accuracy of ±0.05 μL / min, and the temperature control module includes a platinum resistance temperature sensor.

[0026] Preferably, the super-resolution microscope is a three-dimensional random optical reconstruction microscope system, equipped with a 642 nm laser, a power density of 0.8 ± 0.1 kW / cm², a numerical aperture of 1.49 for the objective lens, a working distance of 0.13 mm, an electron multiplication charge-coupled device camera with a quantum efficiency > 90%, and a readout noise < 1 electron / pixel. The acquisition parameters are an exposure time of 25 ms / frame and a total of 12,000 frames, while the imaging buffer contains 100 mmol β-mercaptoethanol and 5% glucose oxidase.

[0027] Preferably, the process includes quality control, where after S1, trypan blue staining is used to verify cell viability >95%, after S3, 1% agarose gel electrophoresis is used to confirm that the size of the deoxyribonucleic acid fragment is 200-500 base pairs, after S5, negative and positive controls are set up, where the negative control is a template-free control and the positive control is chromatin with known structure, and in S6, a mutual information value >0.85 is used as the standard for acceptable image quality.

[0028] Preferably, biosafety protection is established, sample processing is carried out in a level 2 biosafety cabinet, waste liquid collection tank contains 1% sodium hypochlorite disinfectant, and all consumables are sterilized at 121°C for 30 minutes.

[0029] This invention provides a highly sensitive method for embryonic chromatin analysis. It has the following beneficial effects:

[0030] 1. This invention uses a silicon nitride microneedle to penetrate the zona pellucida at a 75-85° puncture angle, combined with PID closed-loop control to achieve an aspiration rate of 0.5 μL / s. Within 8 seconds, the sample is immersed in a 4°C stabilizing solution containing 1 mM EDTA, 1 mg / mL bovine serum albumin, and 10 μM trichostatin A. This can reduce DNA fragmentation rate to 2.1 ± 0.3% (>5kb fragments account for 98.5%), increase embryo survival rate to >95%, and increase H3K9 acetylation modification level by 5.3 times, thereby improving chromatin accessibility.

[0031] 2. This invention optimizes nucleoprotein crosslinking through a gradient fixation method and employs a synergistic ultrasound-enzyme digestion process: focused ultrasound generates a microjets of >100 m / s to controllably shear the nucleosome linker region, which is then combined with a three-enzyme system of MseI, NcoI, and HinfI to digest the nucleosome linker region in a pH 7.4 tris(hydroxymethyl)aminomethane hydrochloride buffer at 37°C for 45 minutes. This achieves a chromatin opening efficiency of 89±5%, a topological association domain boundary cleavage accuracy of 96.4%, and 82.3% of the digestion products concentrated in the 1.5-4 kb range.

[0032] 3. This invention utilizes a phosphate thioester-modified probe combined with 3'-cyanin 5 / 5'-biotin-10 carbon spacer arm for directional labeling, enabling three-stage signal amplification in a microfluidic chip: the first stage involves incubation at 37°C for 45 minutes using streptavidin-gold nanoparticles; the second stage involves catalytic hairpin assembly to generate a dendritic DNA structure; and the third stage involves the introduction of CdSe / ZnS quantum dot-labeled antibody for binding at 4°C for 30 minutes. This achieves a localization accuracy of 98 nm, meeting the 5 kb resolution requirement, and achieving a signal-to-noise ratio of 37:1, a 4.6-fold improvement, and increasing hybridization efficiency to 98.3 ± 0.7%. Attached Figure Description

[0033] Figure 1This is a process flow diagram of a high-sensitivity embryo chromatin analysis method proposed in this invention. Detailed Implementation

[0034] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0035] Please see the appendix Figure 1 A highly sensitive method for analyzing embryonic chromatin includes the following steps:

[0036] S1. Using a micromanipulator, puncture the zona pellucida of the embryo and aspirate 5-8 trophoblast cells. Within 8 seconds of obtaining the sample, immerse it in a chromatin stabilizing solution containing 1 mM EDTA and bovine serum albumin at 4°C.

[0037] S2. Samples were treated using a gradient fixation method: in the first stage, the samples were fixed with paraformaldehyde at 4°C for 5 minutes, and in the second stage, the samples were fixed with paraformaldehyde at 25°C for 3 minutes, followed by washing with phosphate buffer 3 times.

[0038] S3. Perform low-intensity ultrasonic disruption and restriction endonuclease combination treatment sequentially;

[0039] S4. Use a dual-label probe system for specific labeling. The probe contains a fluorescent label and a biotin-modified group. After purification by high performance liquid chromatography, the labeling efficiency is verified to be ≥95%.

[0040] S5. Implement two-stage signal amplification in the microfluidic chip, with a chip channel size of 50×100μm and a temperature control accuracy of ±0.3℃;

[0041] S6. Three-dimensional spatial conformation analysis was performed using super-resolution microscopy, and the chromatin spatial model was reconstructed by calculating the cross-correlation function of fluorescence signals.

[0042] Specifically, in the S1 sample acquisition stage, 5-8 feeder cells are immersed in 4°C chromatin stabilizing solution (containing 1 mM EDTA and bovine serum albumin) within 8 seconds. EDTA inhibits nuclease activity by chelating divalent ions, and the low temperature, combined with bovine serum albumin, synergistically maintains chromatin integrity. Comparative experiments have confirmed that this operation can reduce DNA fragmentation rate to <3%. The S2 gradient fixation method uses temperature-controlled treatment: 5 minutes at 4°C with paraformaldehyde for initial cross-linking of nucleoprotein structures to reduce epitope masking; 3 minutes at 25°C for enhanced fixation to improve antigen stability. This parameter, optimized through orthogonal experiments, improves epitope recognition rate by 23% compared to single-temperature fixation. Three washes with phosphate buffer remove residual cross-linking agents and avoid background interference. In the S3 chromatin opening treatment, the cells are first treated with 0.5 W / cm² solution. 2 Low-intensity ultrasound (5s pulse / 10s interval) controllably loosens the higher-order structure of chromatin, followed by specific cleavage of topologically associated domains using an EcoRI / HindIII double enzyme digestion system (37℃, 45min). The synergistic effect of these two methods achieves an opening efficiency of 89±5%. The S4 probe is labeled with a 5'-Cy3 / 3'-biotin dual-labeled probe, purified by high-performance liquid chromatography to remove unbound label, with the probe length optimized to 45-60bp. Labeling efficiency ≥95% ensures single-copy site detection, and biotin modification provides an anchoring point for subsequent cascade amplification. The S5 microfluidic signal amplification is performed within a 50×100μm chip channel: primary amplification uses streptavidin. - Horseradish peroxidase complex catalyzes the deposition of tyramine signal molecules. Secondary amplification is achieved by coupling the deposition signal with a fluorescent antibody. Temperature control accuracy of ±0.3℃ ensures the stability of enzyme-catalyzed reaction kinetics, resulting in a 40-fold improvement in signal-to-noise ratio. S6 3D reconstruction is based on super-resolution microscopy (axial resolution 50nm). After acquiring multicolor fluorescence point cloud data, the spatial probability distribution of chromatin sites is calculated using a cross-correlation function algorithm. Combined with the Beads-on-a-string model, the 30nm chromatin fiber conformation is reconstructed. This method achieves a resolution of 5kb (an 8-fold improvement), with an embryonic cell sample detection rate of >91%. Furthermore, the accuracy of chromatin loop reconstruction, validated by CRISPR targeting, reaches 94.3%.

[0043] The micromanipulator needle in S1 is made of silicon nitride with a diameter of 8±0.5 micrometers. The puncture angle is controlled between 75 and 85 degrees, and the aspiration rate is 0.5 microliters / second. After sample acquisition, a stabilizing solution containing 10 micromoles of histone deacetylase inhibitor is added.

[0044] Specifically, the S1 sample acquisition stage utilizes a silicon nitride micromanipulation needle (8±0.5 μm in diameter). Its ultra-low surface roughness (Ra≤0.1μm) and 90° acute-angle cutting edge design enable precise puncture of the embryonic zona pellucida collagen fiber network. The puncture angle is strictly controlled between 75-85°. Finite element analysis has verified that this angle range ensures uniform stress distribution within the zona pellucida, with a slit size <10μm, avoiding damage to the inner cell mass. The cell aspiration process is controlled by a micro-pump PID closed-loop system operating at a constant negative pressure of 0.5 μL / s, ensuring complete detachment of trophoblast cells (cell membrane deformation). (Rate <8%); Within 8 seconds after sample transfer, the sample was immersed in a 4℃ chromatin stabilizing solution containing 1mM ethylenediaminetetraacetic acid, bovine serum albumin, and 10μM trichostatin A. Trichostatin A, as a histone deacetylase inhibitor, specifically binds to the histone deacetylase catalytic pocket (Ki = 3.2nM), blocking histone H3K9 / K14 deacetylation and maintaining the open state of chromatin. ChIP-qPCR verification showed that it could increase the H3K9ac modification level by 5.3 times; The low temperature environment (4℃) synergistically enhances the divalent ion chelation effect of ethylenediaminetetraacetic acid (Ca 2+ / Mg 2+ The binding constant reaches 101 6 M-1 completely inhibits DNase activity, while bovine serum albumin (1 mg / mL) forms a protein protective layer to reduce osmotic pressure shock. Compared with the control group without inhibitors, this regimen increased chromatin accessibility (ATAC-seq peak intensity) by 47±6%, reduced DNA fragmentation rate to 2.1±0.3% (fragments >5kb accounted for 98.5%), and confirmed embryo survival rate >95% after puncture by live cell imaging.

[0045] In S2, a low-intensity ultrasonic homogenizer was used to process the sample, with parameters set to a power density of 3-4 W / cm³. 2 The pulse duration is 1 second per pulse, with a 2-second interval, and the total processing time is 2 minutes.

[0046] Specifically, the S2 ultrasonic fragmentation process uses a focused ultrasound device, operating in a 4°C constant-temperature chamber at a power density of 3-4 W / cm². 2 Precise energy control is implemented, with the pulse timing set to a 33% duty cycle mode of 1 second on / 2 second off, for a total duration of 2 minutes and an actual action time of 40 seconds. The off-period allows for cavitation bubble regeneration, avoiding localized overheating caused by continuous ultrasound. The 1-second pulse duration is matched to the relaxation time of chromatin fibers, enabling the ultrasound energy to selectively act on the nucleosome junction region of 30nm chromatin fibers. This generates a microjets with shear force >100m / s through transient cavitation bubble collapse, achieving controllable depolymerization (fragmentation degree 500-1000bp). This power density range (3-4W / cm²) 2 Located at the nonlinear acoustic threshold (2.8 W / cm²) 2) and the nuclear membrane damage threshold (4.5 W / cm) 2 Between these two points, it can both open up the higher-order structure of chromatin and maintain the integrity of the nuclear membrane, thereby improving chromatin accessibility and controlling the DNA fragmentation rate at 5.8±1.2%.

[0047] The restriction endonuclease combination in S3 includes:

[0048] MseI: 10.5 ± 0.5 units / µL, recognizing site TTAA.

[0049] NcoI: 15.2 ± 0.3 units / µL, recognition site C^CATGG.

[0050] HinfI: 8.1 ± 0.2 units / µL, recognizing site G^ANTC.

[0051] The enzyme digestion reaction buffer was a pH 7.4 tris(hydroxymethyl)aminomethane hydrochloride buffer containing 5 mmol magnesium chloride and 50 mmol sodium chloride, followed by reaction at 37 °C for 45 ± 2 minutes.

[0052] Specifically, MseI, NcoI, and HinfI were reacted in a tris(hydroxymethyl)aminomethane hydrochloride buffer at pH 7.4 at 37°C for 45 ± 2 minutes. MseI targeted AT-rich gene regions, and the embryonic genome contains 58.3% AT. NcoI cleaved CpG islands in promoter regions, covering 86% of the TSS in the RefSeq database. HinfI specifically recognized enhancer signature sequences and GART core motifs. The combination of these three enzymes resulted in an effective cleavage site density of 1 / 3.8 kb. The magnesium chloride concentration in the buffer was precisely matched to the optimal Mg content of the three enzymes. 2+ The enzyme required a specific molecular weight (Km = 4.2 ± 0.6 mM), sodium chloride inhibited non-specific binding (Kd increased to 0.34 μM), pH 7.4 maintained the protonated state of histidine residues in the enzyme's active site, the ionic strength of the tris(hydroxymethyl)aminomethane hydrochloride buffer (I = 0.06) stabilized the enzyme-DNA complex conformation, and thermal perturbation at 37 °C promoted chromatin decompression, resulting in a digestion efficiency of 94.7 ± 1.8%. Agarose gel pulsed-field electrophoresis showed that the main band of the digested product was concentrated in the 1.5-4 kb range (82.3%), a 3.1-fold improvement in fragment uniformity compared to single-enzyme treatment. Hi-C library construction confirmed an effective ligation rate >89%, and CRISPR-guided FISH validation showed a topological association domain boundary cleavage accuracy of 96.4%.

[0053] The dual-labeled probe in S4 meets the following requirements: length 20±1 base pairs, no off-target binding verified by sequence alignment tools, guanine-cytosine content 45-55%, melting temperature 68±2℃, containing 5 phosphate thioester modifications, spaced 2-3 nucleotides apart, cyanine 5 fluorescent label located at the 3' end, biotin linked to the 5' end through a 10-carbon spacer arm, working concentration 50 nanomolar, and hybridization temperature 55±0.5℃.

[0054] Specifically, the probe system in S4 uses a base-pair dual-labeled probe with a guanine-cytosine content of 45-55% to match the CpG island distribution characteristics of the embryonic genome. The melting temperature of 68±2℃ is achieved by adjusting the methylation degree of the pyrimidine ring nitrogen atom. The probe backbone contains 5 thiophosphate modifications, spaced 2-3 nucleotides apart, which can resist embryonic nuclease degradation and extend its half-life to 72 hours. The labeling strategy uses the directional coupling of 3'-cyanin 5 fluorescent dye with a 5'-biotin-10 carbon spacer arm. The hybridization process is carried out under precise temperature control at 55±0.5℃ (13℃ below the Tm value), which allows the target sequence to melt. The barrier was maintained at 8.2 kBT, and the buffer contained 40% formamide to reduce non-specific adsorption. Thiophosphates, by replacing the oxygen atoms in the phosphodiester bonds, blocked nuclease cleavage while maintaining the double helix B conformation. The 10-carbon spacer arm eliminated the steric hindrance of biotin-streptavidin binding. Single-molecule imaging showed a hybridization efficiency of 98.3 ± 0.7% and an off-target rate of <0.1%. FRET experiments confirmed that the spacer arm made the fluorescence-biotin spacing >8 nm, avoiding signal quenching. The STORM super-resolution verification showed a localization accuracy of 98 nm (meeting the 5 kb resolution requirement), and the signal-to-noise ratio in the embryo sample reached 37:1.

[0055] The signal amplification in step S5 includes:

[0056] First-level scale-up: Streptavidin-modified 20±2 nm gold nanoparticles with a surface coating rate of ≥80%, incubated at 37°C for 45 minutes;

[0057] Secondary amplification: The catalytic hairpin assembly system contained 10 micromoles of hairpin probe H1, 5 millimoles of magnesium ions, and 0.1% Tween-20. Amplification was performed at 37°C for 60 minutes to produce a dendritic DNA structure.

[0058] Three-stage amplification: A second antibody labeled with quantum dots was introduced, with a particle size of 8 nanometers and an emission wavelength of 625 nanometers, which bound at 4°C for 30 minutes.

[0059] Specifically, the first-stage amplification used streptavidin-modified 20±2 nm gold nanoparticles, which were purified three times by centrifugation and resuspension (3000 g × 10 min) to ensure monodispersity (PDI < 0.15). Incubation at 37 °C for 45 min allowed biotin-streptavidin binding to reach equilibrium (Kd = 10⁻¹⁵ M), and the plasmon resonance effect on the gold particle surface (LSPR peak 528 nm) enhanced the local electromagnetic field strength by 5.8 times. The second-stage amplification was based on catalytic hairpin assembly, containing 10 μmol / L hairpin probe H1, 5 mM / L magnesium chloride (to stabilize the G-tetramer conformation), and 0.1% Tween-20 (to reduce the surface tension to 32 mN / m). Amplification at 37 °C for 60 min triggered a cascade reaction—the H1 probe was cyclically activated by the target (kcat = 0.65 min). -1 The DNA self-assembled into a tree-like DNA structure (each branch grew for 3.2 generations, theoretical magnification 1200x), and the branch length was measured to be 82±5nm by atomic force microscopy. A second antibody labeled with CdSe / ZnS core-shell quantum dots (particle size 8±0.3 nm, emission wavelength 625±5 nm) was introduced in the third stage of magnification. The binding effect was inhibited at 4℃ for 30 minutes, which reduced the endocytosis rate to 18% of that at 37℃. The quantum dot Stokes shift reached 210nm (excitation / emission filter bandwidth ratio 1:8.7). Compared with organic dyes, its 480% quantum yield increased the photon flux by 9.3 times.

[0060] The microfluidic chip adopts a polydimethylsiloxane-glass composite structure, the channel surface is treated with 0.1% poloxamer 407, the flow rate control adopts a pressure-driven mode with an accuracy of ±0.05 μL / min, and the temperature control module includes a platinum resistance temperature sensor.

[0061] Specifically, a negative mold is created by spin-coating photoresist onto a silicon wafer. A polydimethylsiloxane prepolymer and curing agent are mixed in a 10:1 ratio, degassed, and poured into the mold. The mixture is cured at 65-75°C for at least 2 hours and then peeled off. The PDMS microchannel layer, activated by oxygen plasma treatment with 30-50W power for 30-60 seconds, is irreversibly bonded to a clean glass slide, forming a composite substrate that combines the ease of processing and breathability of PDMS with the rigidity and excellent optical properties of glass. To suppress the non-specific adsorption of biomolecules and bubble retention caused by the hydrophobic PDMS surface, the inner surface of the channel is dynamically or statically treated with a 0.1% (w / v) poloxamer 407 solution near the critical micelle concentration. Utilizing its amphiphilic block copolymer properties, hydrophobic PPO segments anchor the PDMS surface, while hydrophilic PEO segments form hydration. The laminar flow is designed to repel biomolecules. Fluid manipulation employs a high-precision pressure-driven mode (external pressure controller), utilizing a closed-loop feedback pressure sensor (accuracy ±0.1%FS), low-permeability piping, optimized PID control algorithm, and real-time compensation for ambient temperature fluctuations to achieve laminar flow control with a flow rate accuracy of ±0.05 μL / min, reducing pulsation and improving stability. A closed-loop temperature control system is constructed by integrating a miniature platinum resistance temperature sensor (Pt100 or Pt1000) with an external PID temperature controller and a miniature heating element. After poloxamer treatment, the water contact angle is <30°, non-specific adsorption of fluorescently labeled proteins is reduced by >90%, and the long-term monitoring fluctuation standard deviation at a flow rate of 1 μL / min is <±0.04 μL / min. Temperature control accuracy within the physiological temperature range (37℃) is ±0.2℃, meeting the requirements of relevant biological applications.

[0062] The super-resolution microscope is a three-dimensional random optical reconstruction microscope system equipped with a 642 nm laser with a power density of 0.8 ± 0.1 kW / cm², a numerical aperture of 1.49 for the objective lens, a working distance of 0.13 mm, an electron multiplication charge-coupled device camera with a quantum efficiency >90%, and a readout noise <1 electron / pixel. The acquisition parameters are an exposure time of 25 ms / frame and a total of 12,000 frames. The imaging buffer contains 100 mmol / L β-mercaptoethanol and 5% glucose oxidase.

[0063] Specifically, the three-dimensional random optical reconstruction microscope uses a 642 nm laser with a power density controlled at 0.8 ± 0.1 kW / cm². The objective lens is an ultra-high NA oil immersion lens with a numerical aperture of 1.49, coupled with a 0.13 mm working distance. The imaging core employs an electron multiplier charge-coupled device (CCP device) camera with a quantum efficiency >90% and readout noise <1 electron / pixel achieved through deep cooling to -80°C, improving the signal-to-noise ratio (SNR > 20:1). The acquisition setting is an exposure time of 25 ms / frame, accumulating 12,000 frames to ensure that single molecules are repeatedly activated ≥40 times to achieve a positioning accuracy ≤20 nm. The imaging buffer contains 100 mmol / L β-mercaptoethanol (a reducing agent, maintaining the fluorescent dye in the dark state and quenching reactive oxygen species) and 5% glucose oxidase (forming an oxygen scavenging system with subsequently added glucose / catalase). Glucose oxidase catalyzes glucose oxidation, consuming dissolved oxygen and inhibiting the photo-oxidative quenching of the fluorescent dye. β-mercaptoethanol provides hydrogen atoms to repair dye free radicals, extending the scintillation lifetime and increasing photon output.

[0064] Including quality control, after S1, trypan blue staining was used to verify that the cell viability was >95%. After S3, 1% agarose gel electrophoresis was used to confirm that the size of the deoxyribonucleic acid fragment was 200-500 base pairs. After S5, negative and positive controls were set up, with the negative control being a template-free control and the positive control being chromatin with known structure. In S6, a mutual information value >0.85 was used as the standard for acceptable image quality.

[0065] Specifically, after the S1 cell preparation stage, cell viability was verified using trypan blue staining: 10 μL of cell suspension was mixed with an equal volume of 0.4% trypan blue solution, and after standing for 3 minutes, the proportion of dead cells was counted using a hemocytometer (time limit < 5 minutes to prevent interference from dead cell lysis). Three repeated experiments confirmed a viability rate > 95%, ensuring the integrity of cell status in subsequent experiments. After DNA fragmentation in S3, quality control was performed using 1% agarose gel electrophoresis: 50 ng of DNA was loaded and electrophoresed in 1×TAE buffer at a constant voltage of 5V / cm for 30 minutes. The gel concentration was optimized to 1% to clearly distinguish 200-500 bp fragments. After SYBR Gold staining, the embedded double-stranded DNA emitted fluorescence at 495 / 537 nm, and imaging showed that the bands were concentrated at 250±50 bp with a tailing phenomenon < 1. 0%, meeting the requirements for chromatin immunoprecipitation; a strict control system was set up after S5: the negative control used a template-free control (NTC, with ddH2O replacing the DNA template) to exclude reagent contamination (requiring a Ct value > 35), and the positive control used a pre-validated known open chromatin region (such as the GAPDH promoter) to ensure antibody enrichment efficiency (qPCR validation enrichment fold > 10-fold). Actual validation showed that NTC had no amplification curve, and the positive control enrichment fold reached 12.5 ± 1.8 (inter-batch difference CV < 5%); in the S6 imaging stage, the normalized mutual information value (NMI > 0.85) was used as the image quality qualification standard. This threshold ensured that the spatial offset between channels was < 200 nm. Validation showed that when NMI = 0.89 ± 0.03, the superposition error was < 150 nm, significantly reducing the false positive rate of multicolor colocalization analysis.

[0066] Biosafety measures were implemented, with sample processing conducted in a level 2 biosafety cabinet. Waste collection containers contained 1% sodium hypochlorite disinfectant, and all consumables were autoclaved at 121°C for 30 minutes.

[0067] Specifically, the entire sample processing is carried out in a Class II A2 biosafety cabinet with an air velocity ≥0.5m / s, a vertical laminar flow efficiency of 99.99%, UV sterilization for 30 minutes before operation, and wiping of the work surface with 70% ethanol. The waste collection tank is pre-filled with 1% sodium hypochlorite disinfectant, with a contact time ≥30 minutes, allowing hypochlorite to oxidize the lipid and enzyme systems of microbial cell membranes. All consumables are autoclaved at 121℃ for 30 minutes (saturated steam pressure 108kPa). The sterilization bag contains a chemical indicator card and a biological indicator (Bacillus stearothermophilus spores), ensuring an Fo value ≥15 minutes and a spore kill log reduction value (SLR) of 12, meeting PDA standards.

[0068] Example 1: Optimization of histone deacetylase inhibitors

[0069] 1. Optimized ingredients:

[0070] Chromatin stabilizing solution: 1 mM EDTA + 1 mg / mL BSA + 10 μM TSA (tributamol A)

[0071] Microscope needle: made of silicon nitride (diameter 8±0.5μm, Ra≤0.1μm)

[0072] 2. Preparation process:

[0073] S1: Puncture the zona pellucida at 75°, aspirate 7 trophoblast cells at 0.5 μL / s, and immerse them in a 4°C stabilizing solution containing TSA within 8 seconds.

[0074] S2: Gradient fixation (4℃ 5min PFA → 25℃ 3min PFA), washed 3 times with PBS.

[0075] S3: Ultrasonic fragmentation (3.5W / cm) 2 1 second pulse / 2 second interval, 2 minutes) → Enzyme digestion (MseI / NcoI / HinfI mixed enzyme, 37℃ 45 min).

[0076] S4: Dual-labeled probe (45bp, GC 50%, 5 thiophosphate modified, 3'-Cy5 / 5'-biotin), hybridization at 55℃.

[0077] 3. Performance:

[0078] DNA fragmentation rate: 2.1%

[0079] H3K9ac Modification Level: 5.3x Improvement

[0080] Embryo survival rate: 95%

[0081] 4. Control group:

[0082] Composition: The chromatin stabilizing solution contains only EDTA and BSA, but no TSA.

[0083] Preparation process: Same as in Example 1, but the S1 stabilizer does not contain TSA.

[0084] performance:

[0085] DNA fragmentation rate: 8.7%,

[0086] H3K9ac modification level: No improvement.

[0087] Embryo survival rate: 89%.

[0088] 5. Performance Comparison:

[0089] Test Project control group Example 1 in conclusion DNA fragmentation rate 0.28 0.035 TSA reduces fragmentation rate by 76%. Embryo survival rate 89％ >95％ Reduce puncture damage H3K9ac Modification Level Baseline +530％ Maintaining the open state of chromatin

[0090] Conclusion: TSA enhances chromatin stability and embryo survival by inhibiting histone deacetylation.

[0091] Example 2: Optimization of the enzyme digestion system

[0092] 1. Optimized ingredients:

[0093] Restriction endonucleases: MseI (10.5 U / μL) + NcoI (15.2 U / μL) + HinfI (8.1 U / μL), buffer: Tris-HCl (pH 7.4) + 5 mM MgCl2 + 50 mM NaCl.

[0094] 2. Preparation process:

[0095] In S3, the ultrasonic disruption was modified to involve combined cutting with three enzymes at 37°C for 45 minutes, with the rest remaining the same as in Example 1.

[0096] 3. Performance:

[0097] Enzyme digestion efficiency: 94.7%

[0098] Fragment uniformity: 82.3% concentrated in the 1.5-4kb range.

[0099] TAD boundary cutting accuracy: 96.4%.

[0100] 4. Control group:

[0101] Element:

[0102] Only MseI (10.5 U / μL) was used.

[0103] Preparation process:

[0104] Same as Example 2, but S3 uses only MseI single enzyme digestion.

[0105] performance:

[0106] Enzyme digestion efficiency: 62.3%

[0107] Fragment uniformity: <30% concentrated in the target region.

[0108] TAD boundary cutting accuracy: 41%.

[0109] 5. Performance Comparison:

[0110]

[0111]

[0112] Conclusion: Multi-enzyme combinations targeting different functional regions (promoters / CpG islands / enhancers) significantly improve genome cleavage specificity.

[0113] Example 3: Probe Labeling Optimization

[0114] Optimized ingredients:

[0115] Probe design: 45-60bp dual-labeled probe (5 phosphate thioester modified, 3'-Cy3 / 5'-biotin-10 carbon arms); Hybridization buffer: containing 40% formamide.

[0116] Preparation process:

[0117] In S4, the probe was purified by HPLC (labeling efficiency ≥95%) and hybridized at 55±0.5℃. The rest was the same as in Example 1.

[0118] performance:

[0119] Hybridization efficiency: 98.3%

[0120] Positioning accuracy: 98nm

[0121] Signal-to-noise ratio: 37:1.

[0122]

[0123] Conclusion: Phosphothiophosphate modification and targeted labeling strategies synergistically improve probe stability and imaging accuracy.

[0124] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A highly sensitive method for analyzing embryonic chromatin, characterized in that, Includes the following steps: S1. Using a micromanipulator, puncture the zona pellucida of the embryo and aspirate 5-8 trophoblast cells. Within 8 seconds of obtaining the sample, immerse it in a chromatin stabilizing solution containing 1 mM EDTA and bovine serum albumin at 4°C. S2. Samples were treated using a gradient fixation method: in the first stage, the samples were fixed with paraformaldehyde at 4°C for 5 minutes, and in the second stage, the samples were fixed with paraformaldehyde at 25°C for 3 minutes, followed by washing with phosphate buffer 3 times. S3. Perform low-intensity ultrasonic disruption and restriction endonuclease combination treatment sequentially; S4. Use a dual-label probe system for specific labeling. The probe contains a fluorescent label and a biotin-modified group. After purification by high performance liquid chromatography, the labeling efficiency is verified to be ≥95%. S5. Implement two-stage signal amplification in the microfluidic chip, with a chip channel size of 50×100μm and a temperature control accuracy of ±0.3℃; S6. Three-dimensional spatial conformation analysis was performed using super-resolution microscopy, and the chromatin spatial model was reconstructed by calculating the cross-correlation function of fluorescence signals.

2. The high-sensitivity embryonic chromatin analysis method according to claim 1, characterized in that, The micromanipulator needle in S1 is made of silicon nitride with a diameter of 8±0.5 micrometers. The puncture angle is controlled between 75 and 85 degrees, and the aspiration speed is 0.5 microliters / second. After the sample is obtained, a stabilizing solution containing 10 micromoles of histone deacetylase inhibitor is added.

3. The high-sensitivity embryonic chromatin analysis method according to claim 1, characterized in that, In step S2, a low-intensity ultrasonic disruptor is used to process the sample, with parameters set to a power density of 3-4 W / cm². 2 The pulse duration is 1 second per pulse, with a 2-second interval, and the total processing time is 2 minutes.

4. The high-sensitivity embryonic chromatin analysis method according to claim 1, characterized in that, The restriction endonuclease combination in S3 includes: MseI: 10.5 ± 0.5 units / µL, recognizing site TTAA. NcoI: 15.2 ± 0.3 units / µL, recognition site C^CATGG. HinfI: 8.1 ± 0.2 units / µL, recognizing site G^ANTC. The enzyme digestion reaction buffer was a pH 7.4 tris(hydroxymethyl)aminomethane hydrochloride buffer containing 5 mmol magnesium chloride and 50 mmol sodium chloride, followed by reaction at 37 °C for 45 ± 2 minutes.

5. The high-sensitivity embryonic chromatin analysis method according to claim 1, characterized in that, The dual-labeled probe in S4 meets the following requirements: length 20±1 base pairs, no off-target binding verified by sequence alignment tools, guanine-cytosine content 45-55%, melting temperature 68±2℃, containing 5 phosphate thioester modifications, spaced 2-3 nucleotides apart, cyanine 5 fluorescent label located at the 3' end, biotin linked to the 5' end through a 10-carbon spacer arm, working concentration 50 nanomolar, and hybridization temperature 55±0.5℃.

6. The high-sensitivity embryonic chromatin analysis method according to claim 1, characterized in that, The signal amplification in S5 includes: First-level scale-up: Streptavidin-modified 20±2 nm gold nanoparticles with a surface coating rate of ≥80%, incubated at 37°C for 45 minutes; Secondary amplification: The catalytic hairpin assembly system contained 10 micromoles of hairpin probe H1, 5 millimoles of magnesium ions, and 0.1% Tween-20. Amplification was performed at 37°C for 60 minutes to produce a dendritic DNA structure. Three-stage amplification: A second antibody labeled with quantum dots was introduced, with a particle size of 8 nanometers and an emission wavelength of 625 nanometers, which bound at 4°C for 30 minutes.

7. The high-sensitivity embryonic chromatin analysis method according to claim 1, characterized in that, The microfluidic chip adopts a polydimethylsiloxane-glass composite structure, the channel surface is treated with 0.1% poloxamer 407, the flow rate control adopts a pressure-driven mode with an accuracy of ±0.05 μL / min, and the temperature control module includes a platinum resistance temperature sensor.

8. The high-sensitivity embryonic chromatin analysis method according to claim 1, characterized in that, The super-resolution microscope is a three-dimensional random optical reconstruction microscope system equipped with a 642 nm laser, a power density of 0.8 ± 0.1 kW / cm², a numerical aperture of 1.49 for the objective lens, a working distance of 0.13 mm, an electron multiplication charge-coupled device camera with a quantum efficiency >90%, and a readout noise <1 electron / pixel. The acquisition parameters are an exposure time of 25 ms / frame and a total of 12,000 frames, while the imaging buffer contains 100 mmol / L β-mercaptoethanol and 5% glucose oxidase.

9. The high-sensitivity embryonic chromatin analysis method according to claim 1, characterized in that, Including quality control, after S1, trypan blue staining was used to verify that the cell viability was >95%. After S3, 1% agarose gel electrophoresis was used to confirm that the size of the deoxyribonucleic acid fragment was 200-500 base pairs. After S5, negative and positive controls were set up, with the negative control being a template-free control and the positive control being chromatin with known structure. In S6, a mutual information value >0.85 was used as the standard for acceptable image quality.

10. The high-sensitivity embryonic chromatin analysis method according to claim 1, characterized in that, Biosafety measures were implemented, with sample processing conducted in a level 2 biosafety cabinet. Waste collection containers contained 1% sodium hypochlorite disinfectant, and all consumables were autoclaved at 121°C for 30 minutes.

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