Detection method of base complementary pairing acting force

Abstract

The invention discloses a method for detecting base complementary pairing acting force. The method comprises the following steps: (A) marking a single-stranded DNA probe on the surface of diamond; (B) modifying a paramagnetic ion labeled single-stranded DNA probe on the surface of the substrate; (C) forming a'sandwich structure 'through complementary pairing of bases, and fixing the diamond on the substrate; and (D) diamond quantum signal changes are collected through an optical microscope system, and an acting force field is calculated. The detection method disclosed by the invention can be used for detecting the complementary base pairing force in real time in the nucleic acid detection process, provides a combination mode of quantum image information and molecular mechanics information, improves the specificity and precision in the detection process, and has a wide biological application prospect.

Description

Technical Field

[0001] The present invention relates to a method for detecting DNA or RNA, and in particular to a method for detecting base complementary pairing force. Background Art

[0002] The importance of base complementary pairing force detection is becoming increasingly prominent, especially in the context of many challenges facing the current biological research and application fields. On the one hand, with the rapid development of gene editing technology, the requirements for base pairing accuracy have increased unprecedentedly, and any tiny mismatch may lead to unpredictable consequences, such as gene mutations and disease occurrence. On the other hand, the research on complex diseases such as cancer and hereditary diseases urgently needs more accurate molecular diagnostic methods, and abnormal base complementary pairing is one of the important molecular markers of these diseases. However, the existing detection technology still has problems such as insufficient sensitivity, low specificity, and complex operation in practical applications, which makes it difficult to meet the needs of fast, accurate, and high-throughput detection.

[0003] For example, in the prior art, the patent with application number CN201910106779.8 and name “Multimodal biomechanical microscope and measurement method” can measure the cell traction force in real time and realize multimodal imaging of traditional image information and mechanical information, but cannot simultaneously realize imaging detection of base complementary pairing force. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a method for detecting base complementary pairing forces, which can convert base complementary pairing forces into detectable magnetic changes, provide a way to combine quantum image information with molecular mechanics information, and improve the specificity and accuracy of the detection process.

[0005] Technical solution: The method for detecting the base complementary pairing force of the present invention comprises the following steps:

[0006] (A) Single-stranded DNA probes are labeled on the diamond surface;

[0007] (B) Single-stranded DNA probes labeled with paramagnetic ions are modified on the substrate surface;

[0008] (C) The diamond is fixed on the substrate by forming a "sandwich structure" through complementary base pairing;

[0009] (D) The changes in diamond quantum signals are collected through an optical microscope system and the force field is calculated.

[0010] Wherein, in step (A), the diamond is a nanodiamond containing a nitrogen vacancy color center, denoted as FNDs; the average particle size of the FNDs is about 100-150 nm; and the surface of the FNDs is modified with carboxyl groups.

[0011] The specific process of step (A) is as follows: mixing the surface carboxylated diamond with a crosslinking agent to fully activate the carboxyl groups on the surface of the diamond; washing after activation, adding the miRNA-21 probe with a 5'-end modified amino group to the washed product, and reacting to generate amide to obtain the FND-NH2 probe.

[0012] Wherein, the cross-linking agent is N-hydroxysuccinimide (NHS) or 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

[0013] The specific process of step (B) is as follows: pre-treating the substrate for activation, then adding it to a silane coupling agent for grafting reaction; adding digoxin antibody, and modifying the grafted substrate by strain-promoted azide-alkyne cycloaddition reaction; then adding a miRNA-21 capture probe modified with digoxin at the 3' end, so that the substrate is coupled with the miRNA-21 capture probe modified with digoxin at the 3' end; adding a paramagnetic metal salt solution for reaction, washing after the reaction, and obtaining a substrate with paramagnetic metal ions and miRNA-21 capture probe coupled on the surface. Wherein, the paramagnetic metal is preferably at least one of Gd, Cr, lanthanide or actinide; the paramagnetic metal salt solution is preferably a Gd salt solution; and the Gd salt is preferably GdCl3·6H2O. The silane coupling agent is 3-aminopropyltrimethoxysilane.

[0014] Wherein, in step (C), the "sandwich structure" is obtained by mixing the nanoparticles obtained in steps (A) and (B) in a buffer solution and then adding miRNA-21 for incubation and capture.

[0015] Wherein, in step (C), the components of the buffer include: 2×SSC, 9.5-10.5 mM pH=7.4-8.0 Tris-HCl, 4.5-5.5 mM MgCl2, 4.5-5.5 mM KCl, 0.5%-1.5% commercially available skim milk, 0.5-1.5 mM CTAB, and 0.5%-1.5% trehalose.

[0016] Wherein, in step (D), the diamond quantum signal collected by the optical microscope system is the relaxation time (T1) of FNDs; and the calculation of the force field is assisted by a computer software program.

[0017] Wherein, in the step (D), the optical microscope system is used to obtain T1 images of FNDs before and after forming a "sandwich structure" with the SiO2 substrate; the optical microscope system is used to obtain T1 images of the FNDs during the PBS rinse process after forming a "sandwich structure" with the SiO2 substrate.

[0018] Wherein, in the step (D), a worm chain model is used to describe the relationship between the force applied to the FNDs and the T1 value.

[0019] Beneficial effects: Compared with the prior art, the present invention achieves the following significant effects:

[0020] (1) The present invention establishes an experimental method for converting base complementary pairing forces into detectable magnetic changes, which can detect base complementary pairing forces in real time during nucleic acid detection, and provides a way to combine quantum image information with molecular mechanics information, thereby improving the specificity and accuracy of the detection process and having broad prospects for biological applications. (2) Diamonds containing NV color centers (FNDs) have stable fluorophores. Compared with traditional fluorescent dyes, quantum dots and other fluorophores, FNDs can overcome the data instability caused by photobleaching, light flickering, etc., and help to accurately quantify the base complementary pairing forces formed during nucleic acid detection. (3) The detection accuracy of the method of the present invention can reach 1 piconewton, so the forces between single base pairs can be sensed, which helps to screen mismatched base pairs that appear during the detection process and improve the accuracy and specificity of nucleic acid detection. (4) FNDs and paramagnetic Gd 3+ The ions all have good biocompatibility and can achieve same-scale, long-term coupled observation of mechanical signals in biochemical environments, maintain the original state of the samples, and provide a way to combine quantum image information with molecular mechanics information, which has broad prospects for biological applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the method for detecting the base complementary pairing force of the present invention. DETAILED DESCRIPTION

[0022] The present invention is described in further detail below.

[0023] like Figure 1 As shown, the device used in the method includes: an objective lens 8, a laser 9, a beam splitter 12, a filter 13, and a camera 14. The objective lens 8 is used to irradiate the sample. An inclined beam splitter 12 is provided above the objective lens, a filter 13 is provided above the beam splitter 12, and a camera 14 is provided above the filter 13. The laser 9 emits a 532nm laser 10 that irradiates the objective lens through the beam splitter 12. The red emission light 11 of the objective lens passes through the filter 13 and irradiates the camera 14. Figure 1 It can be seen that the sample includes a SiO2 substrate 1, a copper wire 2 arranged on the SiO2 substrate 1 for transmitting 2.87 GHz microwaves, and a paramagnetic Gd 3+Ion 3, single-stranded DNA probe modified with digoxigenin at the 3' end 4, nucleic acid chain to be tested 5, single-stranded DNA probe modified with amino group (NH2) at the 5' end, FNDs 7 containing NV color center.

[0024] 1. Conjugation of FNDs and miRNA-21 detection probe, the specific steps are as follows:

[0025] Take 100 μg of 100 nm surface carboxylated FND, record it as FND-COOH, add 2 mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), 2 mg N-hydroxysuccinimide (NHS), dilute to 1 mL, mix thoroughly and place in a shaker at room temperature for 1 hour to fully activate the carboxyl groups on the surface of FNDs, and wash three times at 4 ° C and 20000 g centrifugal force to remove free salts. Then add the miRNA21 detection probe with 5' end modified amino group (NH2), mix the two thoroughly and place in a shaker at room temperature for 3 hours to generate amides to obtain FND-NH2 probe; wherein, the miRNA21 detection probe with 5' end modified amino group (NH2) is commercially available. The labeled DNA probes used in the present invention below are all commercially available.

[0026] 2. Coupling paramagnetic Gd on the surface of SiO2 substrate 3+ ions and miRNA-21 capture probe, the specific steps are:

[0027] (1) First, the surface of the SiO2 substrate was activated and pretreated with piranha solution, and then it was added into 1M 3-aminopropyltrimethoxysilane (APTMS) solution to graft it onto the surface of the SiO2 substrate;

[0028] (2) adding 10 μg / mL of digoxin antibody and modifying the SiO2 substrate obtained in step (1) through strain-promoted azide-alkyne cycloaddition (SPAAC) reaction;

[0029] (3) adding a miRNA-21 capture probe modified with digoxigenin at the 3' end, so that the SiO2 substrate obtained in step (2) captures the miRNA-21 capture probe modified with digoxigenin at the 3' end;

[0030] (4) Dissolve GdCl3·6H2O in water to form a paramagnetic Gd 3+ The solution is mixed with the SiO2 substrate obtained in step (3) to load the SiO2 substrate with paramagnetic Gd 3+ ions; after sufficient washing to remove free ions, surface-coupled paramagnetic Gd 3+ ions and SiO2 substrate of miRNA-21 capture probe.

[0031] 3. miRNA-21 forms a "sandwich" structure with the DNA probe. The specific steps are:

[0032] (1) 10 μL, 100 μg / μL of the FNDs coupled with the miRNA-21 detection probe obtained in step 1 and the surface-coupled paramagnetic Gd obtained in step 2 were added. 3+ ions mixed with the SiO2 substrate of the miRNA-21 capture probe, so that the FNDs coupled with the miRNA-21 detection probe were loaded onto the surface-coupled paramagnetic Gd 3+ ions and the SiO2 substrate surface of the miRNA-21 capture probe;

[0033] (2) adding 100 μL of pH 7.4 buffer, the components of which include: 2×SSC, 10 mM pH 7.4 Tris-HCl, 5 mM MgCl2, 5 mM KCl, 1% commercial skim milk, 1 mM CTAB, and 1% trehalose;

[0034] (3) Add 10 μL of 1 nM miRNA-21, mix thoroughly and incubate at room temperature for 3 hours; then rinse with 10 mM PBS buffer (pH = 7.4), blow dry with nitrogen to obtain a "sandwich structure" base complementary pairing sample for subsequent detection.

[0035] 4. Fluorescence detection of miRNA-21, the specific steps are as follows:

[0036] The sample to be tested prepared in step 3 is placed under the objective lens of a Raman / fluorescence spectrometer with a confocal system, 532nm excitation light is used, and 680nm fluorescence signal is collected. The minimum detection limit can reach 0.23fM.

[0037] 5. Detection of relaxation time T1 of miRNA-21, the specific steps are as follows:

[0038] The sample prepared in step 3 and the free FNDs were placed under the objective lens of a Raman / fluorescence spectrometer with a confocal system. The 532nm continuous excitation light was used to collect the change of relaxation time T1, and the paramagnetic Gd 3+ ions reduce the relaxation time T1 of FNDs by 2-8 times.

[0039] 6. Relaxation time T1 imaging of miRNA-21, the specific steps are:

[0040] First, the sample to be tested prepared in step three is subjected to 100 μm×100 μm imaging detection, and then the sample is rinsed with 10 mM PBS buffer with pH=7.4, and imaging detection is performed in the area during the rinsing process. During the rinsing process, the base complementary pairing chain will be stretched to the longest; then, the sample surface is cleaned with concentrated sulfuric acid, and after sufficient deionized water washing, imaging detection is performed again in the area, and the relaxation time T1 imaging changes in the three processes are compared.

[0041] 7. Quantify the relationship between base complementary pairing force and relaxation time T1 imaging. The specific steps are as follows:

[0042] By using the traditional worm-like chain (WLC) model, we further expand its application scope and construct a mechanical response model that can describe the nucleic acid chain under external tensile force by introducing the external force component along the z-axis. This extended model not only retains the original WLC's accurate description of the rigidity and flexibility of the chain segment, but also takes into account the chain extension effect caused by external force. In this way, the model intuitively reflects the dynamic relationship between external force and nucleic acid chain displacement. The specific process is:

[0043] 1. Determine model parameters:

[0044] Persistence Length, L p )The rigidity of the nucleic acid chain determines the average bending length of the chain in its natural state.

[0045] Total chain length (Contour Length, L c ): Maximum length of the chain when fully extended.

[0046] 2. Select an applicable entropy elasticity model:

[0047] Under smaller external forces, the chain exhibits more entropy-driven elastic behavior. This behavior can be described using a simplified entropy elastic theory.

[0048] 3. Establish the entropy elastic model equation:

[0049] For small stretching forces, the stretching of nucleic acid chains can be described using the entropic elastic model equation of the following form:

[0050]

[0051] Where f is the applied force, k B T is the product of temperature and the Boltzmann constant, L p is the persistence length, L c is the total chain length and z is the extended length of the chain.

[0052] 4. Perform numerical simulation:

[0053] According to the experimental or application requirements, the initial conditions of the simulation are set, such as temperature, force range, etc., and then the entropy elastic model equations are solved using the Euler method.

[0054] 5. Result analysis:

[0055] Analyze force-extension curves to explore the entropy changes and mechanical properties of nucleic acid chains. Pay attention to the stretching of the chain under different forces.

Claims

1. A method for detecting base complementary pairing force, characterized in that: The following steps are involved: (A) Single-stranded DNA probes are labeled on the diamond surface; (B) Single-stranded DNA probes labeled with paramagnetic ions are modified on the substrate surface; (C) The diamond is fixed on the substrate by forming a "sandwich structure" through complementary base pairing; (D) The changes in diamond quantum signals are collected through an optical microscope system and the force field is calculated.

2. The method for detecting base complementary pairing force according to claim 1, characterized in that: In step (A), the diamond is a nanodiamond containing a nitrogen vacancy color center, denoted as FNDs; the average particle size of the FNDs is about 100-150 nm; and the surface of the FNDs is modified with carboxyl groups.

3. The method for detecting base complementary pairing force according to claim 1, characterized in that: The specific process of step (A) is as follows: mixing the surface carboxylated diamond with a crosslinking agent to fully activate the carboxyl groups on the diamond surface; washing after activation, adding the miRNA-21 probe with a modified amino group at the 5' end to the washed product, and reacting to generate amide to obtain the FND-NH2 probe.

4. The method for detecting base complementary pairing force according to claim 1, characterized in that: The specific process of step (B) is as follows: pre-treating the substrate for activation, and then adding it to a silane coupling agent for grafting reaction; adding digoxin antibody, and modifying the grafted substrate through a strain-promoted azide-alkyne cycloaddition reaction; then adding a miRNA-21 capture probe modified with digoxin at the 3' end, so that the substrate is coupled with the miRNA-21 capture probe modified with digoxin at the 3' end; adding a paramagnetic metal salt solution for reaction, and washing after the reaction is completed to obtain a substrate with paramagnetic metal ions and miRNA-21 capture probes coupled on the surface.

5. The method for detecting base complementary pairing force according to claim 1, characterized in that: In step (C), the "sandwich structure" is obtained by mixing the nanoparticles obtained in steps (A) and (B) in a buffer solution and then adding miRNA-21 for incubation and capture.

6. The method for detecting base complementary pairing force according to claim 3, characterized in that: The cross-linking agent is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide.

7. The method for detecting base complementary pairing force according to claim 4, characterized in that: The crosslinking agent is 3-aminopropyltrimethoxysilane.

8. The method for detecting base complementary pairing force according to claim 1, characterized in that: In step (D), the diamond quantum signal collected by the optical microscope system is the relaxation time T1 of the diamond; the calculation of the force field is assisted by a computer software program.

9. The method for detecting base complementary pairing force according to claim 1, characterized in that: In the step (D), the optical microscope system is used to obtain T1 images of the diamond before and after forming a "sandwich structure" with the substrate; the optical microscope system is used to obtain T1 images of the diamond during the PBS washing process after forming a "sandwich structure" with the substrate.

10. The method for detecting base complementary pairing force according to claim 1, characterized in that: In the step (D), a worm chain model is used to describe the relationship between the force applied to the diamond and the T1 value.

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