Infrared-nanopore coupled system for detecting ctDNA methylation in early stage of cancer

Abstract

The present invention relates to the field of biological detection technology, and in particular to an infrared-nanopore combined system for detecting early ctDNA methylation in cancer, comprising: a denaturation module, used for heating and heating to denature ctDNA into single-stranded ctDNA; a hybridization module, used for mixing hybridization probe DNA and single-stranded ctDNA to form a new double-stranded structure; a detection pool, used for detecting double-stranded DNA molecules after hybridization, comprising a nanopore, an infrared emission source and a carrier layer for placing DNA molecules to be detected; an irradiation module, which emits an infrared band of 2870 or 2960cm through an infrared emission source ‑1 The infrared irradiation is used to irradiate the nanopore and the DNA molecule to be tested, and the infrared light energy is transferred to the nanopore and the DNA molecule to be tested; the acquisition module is used to collect data on the changes in electrical signals caused by the DNA molecule to be tested passing through the nanopore. The present invention aims to induce specific vibrations of methyl groups in the narrow space of the nanopore through infrared radiation, thereby achieving faster, simpler, more sensitive and specific ctDNA methylation detection.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to an infrared-nanopore combined system for detecting early stage ctDNA methylation of cancer. Background Art

[0002] Circulating tumor DNA (ctDNA) in the blood is a fragmented DNA that carries cancer-specific genetic and epigenetic information. Because its methylation pattern is consistent with its cell or tissue origin, ctDNA methylation can be used as a biomarker for early cancer screening and prediction.

[0003] Although the commonly used DNA methylation detection systems have their own advantages, they still have shortcomings. For example, the bisulfite in the bisulfite sequencing method is destructive to trace ctDNA and is not suitable for early cancer screening; the enzyme used in the methylation-sensitive restriction endonuclease method has the phenomenon of incomplete digestion and false positives; the droplet digital PCR based on PCR technology is expensive and time-consuming. Therefore, the detection of low-abundance ctDNA methylation is still facing challenges, and it is necessary to design a detection system with higher sensitivity and specificity to achieve accurate detection of trace ctDNA methylation. Summary of the invention

[0004] To solve the above problems, the present invention provides an infrared-nanopore combined system for detecting ctDNA methylation in the early stage of cancer, which aims to induce specific vibration of methyl groups in the narrow space of the nanopore through infrared radiation, thereby achieving faster, simpler, more sensitive and specific ctDNA methylation detection.

[0005] In order to achieve the above object, the technical scheme of the present invention is as follows: an infrared-nanopore combined system for detecting early ctDNA methylation in cancer, comprising:

[0006] Denaturation module, used for heating and denaturing ctDNA into single-stranded ctDNA;

[0007] A hybridization module, used to mix hybridization probe DNA and single-stranded ctDNA to form a new double-stranded structure;

[0008] A detection pool, used to detect double-stranded DNA molecules after hybridization, comprising a nanopore, an infrared emission source, and a carrier layer for placing the DNA molecules to be detected;

[0009] The irradiation module emits infrared waves of 2870 or 2960 cm through an infrared emission source. -1 The infrared radiation is used to illuminate the nanopore and the DNA molecule to be tested, and the infrared light energy is transferred to the nanopore and the DNA molecule to be tested;

[0010] An acquisition module, used to acquire the current amplitude and signal duration of the electrical signal caused by the ctDNA molecules to be tested passing through the nanopore;

[0011] The data analysis module is used to calculate and analyze the change data of the electrical signals and signal duration of each fragment in the ctDNA molecules of the patient to be tested, and output the test results.

[0012] The technical principle of the above scheme is as follows: the system includes a denaturation module to heat and denature the ctDNA into a single strand, and a hybridization module to hybridize the patient's ctDNA single strand with the probe DNA to form a double strand. Under the infrared irradiation of the irradiation module, the methyl group vibrates in the nanopore to produce an electrical signal change, and the data is recorded by the sensitive detection area and analyzed by the data analysis module.

[0013] The above scheme has the following beneficial effects:

[0014] 1. This scheme transmits infrared light energy to the nanopore and the DNA molecule to be tested in the form of radiation emitted by the radiation module, causing specific groups in the DNA molecule to vibrate and increasing the frequency of interaction with the nanopore, so that the methyl groups in the ctDNA produce characteristic signals and achieve the purpose of accurate detection of low-abundance ctDNA methylation.

[0015] 2. This scheme uses nanopores to identify and detect methyl-modified ctDNA. It has the characteristics of no need for labeling, no need for amplification, and real-time detection. It can detect low-abundance substances through nanoscale channels and achieve the effect of accurate detection of trace ctDNA methylation.

[0016] 3. By emitting infrared band at 2870 or 2960cm -1 Infrared irradiation achieves specific molecular vibrations, causing the methyl groups to produce different stretching vibration forms. By generating obvious and distinguishable characteristic signals, it not only improves the resolution of nanopore detection and reduces the concentration requirement for ctDNA methylation detection, but also expands the specific detection range, providing a new way to draw sub-nanometer scale structural change detection maps.

[0017] Furthermore, the denaturation module has a temperature control function, and can control the heating temperature at 90 to 95°C.

[0018] In step 1 of the infrared-nanopore combined system for detecting ctDNA methylation in early cancer, the probe DNA is a probe DNA that is not completely complementary to the ctDNA, and when the DNA double strand after hybridization of the probe with the ctDNA is in a base-inverted state when the methylated cytosine is in a base-inverted state.

[0019] Beneficial effects: By designing the temperature to be controlled within 90 to 95°C during the heating denaturation process, it can not only effectively destroy the double-stranded structure of ctDNA, but also help maintain the relative stability of DNA molecules and reduce the risk of thermal decomposition damage to DNA molecules.

[0020] Furthermore, the probe DNA is a probe DNA that is not completely complementary to the ctDNA, and the probe DNA is used to make the methylated cytosine in the ctDNA present a base-inverted state after hybridization.

[0021] Beneficial effects: By designing and using incompletely complementary probe DNA, the hybridized DNA double-stranded DNA presents a base inverted state at the methylated cytosine, increasing the probability of interaction between the DNA molecule to be tested and the nanopore, and improving the recognition resolution of methylation modification.

[0022] Furthermore, the concentration of the probe DNA was adjusted to 50 to 100 nmol / ml.

[0023] Beneficial effects: By limiting the hybridization concentration of the probe DNA, it is beneficial to the specific binding of the probe DNA to the ctDNA single strand, reducing nonspecific hybridization with other DNAs and improving the accuracy of subsequent detection.

[0024] Furthermore, the hybridization module has a temperature control function, which can control the hybridization environment temperature at 50 to 70°C.

[0025] Beneficial effects: By controlling the hybridization temperature, the hybridization reaction can be carried out efficiently at an appropriate temperature, thereby improving the specific binding between the probe and ctDNA.

[0026] Furthermore, the hybridization module also has a pH control function, which can control the pH value of the reaction solution within a range of 7 to 7.5.

[0027] Beneficial effects: By controlling the pH value of the hybridization environment to 7 to 7.5, a neutral or weakly alkaline environment is created, which enhances the stability of the DNA molecules after hybridization, while also improving the hybridization efficiency and shortening the time required for hybridization.

[0028] Furthermore, the hybridization reaction time is controlled within 1 to 1.5 hours.

[0029] Beneficial effects: Controlling the hybridization time to 1 to 1.5 hours is conducive to the full combination of probe DNA and ctDNA target sequence, reducing the risk of nonspecific binding and shortening the detection cycle.

[0030] Furthermore, the acquisition module includes a sensitive detection area, which is located on the nanopore and is used to identify the contact between the nanopore and the molecule and generate an electrical signal for detection.

[0031] Beneficial effects: By setting a sensitive detection area on the nanopore, the passage of molecules can be monitored in real time and the electrical signal data can be accurately recorded, providing a reliable basis for subsequent data analysis and result judgment.

[0032] Further, the current amplitude of the electrical signal collected by the acquisition module includes Ib current amplitude and Ic current amplitude, the Ib current amplitude is used to reflect the current amplitude threshold of the unmethylated ctDNA of the patient to be tested, and the Ic current amplitude is used to reflect the current amplitude caused by the methyl group of the methylated ctDNA of the patient to be tested;

[0033] The analysis process of the data analysis module is to analyze whether there is an abnormal Ic value in the electric signal current amplitude of each fragment of the ctDNA of the patient to be tested, and to make a judgment based on the duration of the electric signal. When the Ic value in the electric signal current amplitude of a certain fragment of the ctDNA of the patient to be tested is greater than the Ic value in the electric signal current amplitude of other fragments of the ctDNA of the patient to be tested, and the duration of the electric signal of the fragment is greater than the duration of the electric signal of other fragments, a "positive detection" signal mark is output; otherwise, a "negative detection" signal mark is output in other cases.

[0034] Beneficial effects: Due to the different characteristics of ctDNA molecules of different patients to be tested and the different concentrations of ctDNA during detection, it is difficult to analyze the electrical signal detection results by setting standard values. The present invention measures and distinguishes the Ib current amplitude and the Ic current amplitude through the acquisition module, which can effectively distinguish the unmethylated and methylated parts in the ctDNA of the patient to be tested, and combines and compares the electrical signal current amplitude and duration of different ctDNA fragments, which can improve the accuracy of identifying abnormally methylated ctDNA fragments and reduce the risk of errors in the detection results.

[0035] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a schematic diagram of the module operation flow of methylation detection in an embodiment of the infrared-nanopore combined system for detecting early ctDNA methylation in cancer of the present invention;

[0037] Figure 2 This is a schematic diagram of methylation detection in an embodiment of an infrared-nanopore combined system for detecting early ctDNA methylation in cancer according to the present invention;

[0038] Figure 3 This is a schematic diagram of a DNA molecule to be detected after hybridization is completed in an embodiment of an infrared-nanopore combined system for detecting early-stage ctDNA methylation of cancer according to the present invention;

[0039] Figure 4This is a schematic diagram of the stretching vibration of the methyl group in an embodiment of the infrared-nanopore combined system for detecting early ctDNA methylation in cancer of the present invention. DETAILED DESCRIPTION

[0040] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0042] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0043] The following is further described in detail through specific implementation methods:

[0044] Embodiment 1:

[0045] As attached Figure 1 , Figure 2 , Figure 3 and Figure 4 Shown: Infrared-nanopore combined system for detecting early ctDNA methylation in cancer, including:

[0046] The denaturation module is used to control the heating temperature at 90 to 95°C, and heat the ctDNA to denature it into single-stranded ctDNA. Since ctDNA originates from DNA fragments resulting from cell apoptosis, necrosis, or active release into body fluids, these fragments may be more susceptible to thermal damage. Therefore, the temperature and time need to be more finely controlled during the heating process to reduce the risk of unnecessary degradation of DNA. The temperature range of 90 to 95°C is sufficient to destroy the hydrogen bonds between the double strands of ctDNA, separating them into single strands, while maintaining the relative stability of the DNA molecules.

[0047] The hybridization module is used to mix the hybridization probe DNA and the single-stranded ctDNA to form a new double-stranded structure.

[0048] The detection pool is used to detect double-stranded DNA molecules after hybridization, including a nanopore, an infrared emission source and a carrier layer for placing the DNA molecules to be tested; the nanopore is used to detect whether there are methylation modification characteristics in the DNA molecules to be tested. Due to the small size of the nanopore, the detection concentration requirement is reduced, thereby achieving more precise detection of the molecules to be tested and improving detection accuracy.

[0049] The irradiation module emits infrared waves of 2870 or 2960 cm through an infrared emission source. -1 The infrared radiation is used to irradiate the nanopore and the DNA molecule to be tested, and the infrared light energy is transferred to the nanopore and the DNA molecule to be tested. When the infrared radiation wave number is 2870cm -1 When the infrared radiation wave number is 2960cm -1 When the DNA is cleaved, the methyl group undergoes an asymmetric stretching vibration state, which is conducive to more rapid detection of the vibration information of the DNA molecule. In addition, the detection efficiency can be improved through the vibration as an identification indicator.

[0050] The acquisition module includes a sensitive detection area, the sensitive detection area is located on the nanopore, and the acquisition module collects the current amplitude and signal duration of the electrical signal generated for detection when the nanopore contacts the molecule to be tested through the sensitive detection area. The current amplitude includes Ib current amplitude and Ic current amplitude. The Ib current amplitude is used to reflect the current amplitude threshold of the unmethylated ctDNA of the patient to be tested, and the Ic current amplitude is used to reflect the current amplitude caused by the methyl group of the methylated ctDNA of the patient to be tested. Then, the current amplitude and signal duration of the electrical signal of each fragment are transmitted to the data analysis module;

[0051] The data analysis module receives the data collected by the acquisition module, analyzes whether there are abnormal Ic values ​​in the electric signal current amplitudes of each fragment of the ctDNA of the patient to be tested, and makes a judgment based on the duration of the electric signal. When the Ic value in the electric signal current amplitude of a certain fragment of the ctDNA of the patient to be tested is greater than the Ic value in the electric signal current amplitude of other fragments of the ctDNA of the patient to be tested, and the duration of the electric signal of the fragment is greater than the duration of the electric signal of other fragments, a signal mark of "positive detection" is output. Otherwise, a signal mark of "negative detection" is output in other cases. Since the order and length of ctDNA of different patients are different, the electric signal current amplitudes collected in the sensitive detection area are difficult to be uniformly compared and detected. By collecting the Ic current amplitude for specific identification of the methylation group of the ctDNA of the patient to be tested, it is beneficial to identify whether the patient's ctDNA is methylated, thereby improving the accuracy and reliability of the detection.

[0052] During the test, ctDNA samples are first extracted from the body of the person to be tested, and the ctDNA is denatured from double-stranded ctDNA to single-stranded ctDNA through the denaturation module. After the denaturation is completed, it is transferred to the hybridization module, and the single-stranded probe DNA is hybridized with the single-stranded ctDNA to form a double-stranded DNA molecule to be tested. At this time, the DNA molecule to be tested is transferred to the carrier layer of the detection pool, and the infrared emission source of the irradiation module is started to emit 2870 or 2960cm -1 The infrared radiation is irradiated on the DNA to be tested. The energy received by the infrared radiation will escape in the direction of the nanopore. When passing through the nanopore, it will come into contact with the nanopore. The sensitive detection area on the nanopore detects the electrical signal generated by the mutual contact, collects the electrical signal and divides it into Ib current amplitude and Ic current amplitude. The data collected by the sensitive detection area is transmitted to the data analysis module for analysis and judgment, and finally the test result is output.

[0053] Embodiment 2:

[0054] As attached Figure 3 As shown, the difference from Example 1 is that the hybridization module controls the hybridization environment temperature at 50 to 70°C; since the hybridization reaction is a temperature-dependent process, the hybridization reaction can be carried out rapidly and efficiently at a suitable temperature, and the hybridization temperature is set at 50 to 70°C, which is lower than the denaturation temperature, to reduce the thermal decomposition of ctDNA due to excessively high temperature, which is beneficial to the specific binding between the probe and ctDNA.

[0055] The pH value of the reaction solution is controlled at 7 to 7.5. Since a too low pH value of the hybridization reaction will cause acid hydrolysis of the DNA molecules, maintaining the pH value within the range of 7 to 7.5 to create a neutral or weak alkaline environment can increase the stability of the DNA molecules after hybridization. In addition, the weak alkaline environment can also increase the hybridization efficiency.

[0056] Use probe DNA that is not completely complementary to ctDNA; since hybridization reaction is a temperature-dependent process, the hybridization reaction can proceed rapidly and efficiently at a suitable temperature. Set the hybridization temperature between 50 and 70°C, which is lower than the denaturation temperature, to reduce thermal decomposition of ctDNA due to excessively high temperature, which is beneficial to the specific binding between the probe and ctDNA.

[0057] In this environment, mixed hybridization of the probe DNA and single-stranded ctDNA to form a new double-stranded structure is carried out, and the hybridization reaction time is controlled within 1 to 1.5 hours. Based on the specificity of the DNA molecules after hybridization, if the hybridization time is too short, it may lead to insufficient binding between the probe DNA and the target sequence, thereby reducing the hybridization efficiency and increasing the risk of nonspecific binding. By limiting the hybridization time, the probability of nonspecific binding can be reduced and the detection cycle can be shortened.

[0058] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. An infrared-nanopore combined system for detecting early ctDNA methylation in cancer, characterized in that: include: Denaturation module, used for heating and denaturing ctDNA into single-stranded ctDNA; A hybridization module, used to mix hybridization probe DNA and single-stranded ctDNA to form a new double-stranded structure; A detection pool, used to detect double-stranded DNA molecules after hybridization, comprising a nanopore, an infrared emission source, and a carrier layer for placing the DNA molecules to be detected; The irradiation module emits infrared waves of 2870 or 2960 cm through an infrared emission source. -1 The infrared radiation is used to illuminate the nanopore and the DNA molecule to be tested, and the infrared light energy is transferred to the nanopore and the DNA molecule to be tested; An acquisition module, used to acquire the current amplitude and signal duration of the electrical signal caused by the ctDNA molecules to be tested passing through the nanopore; The data analysis module is used to calculate and analyze the change data of the electrical signals and signal duration of each fragment in the ctDNA molecules of the patient to be tested, and output the test results.

2. The infrared-nanopore combined system for detecting early ctDNA methylation in cancer according to claim 1, characterized in that: The denaturation module has a temperature control function that can control the heating temperature at 90 to 95°C.

3. The infrared-nanopore combined system for detecting early ctDNA methylation in cancer according to claim 2, characterized in that: The probe DNA is not completely complementary to the ctDNA, and is used to make the methylated cytosine in the ctDNA present a base-inverted state after hybridization.

4. The infrared-nanopore combined system for detecting early ctDNA methylation in cancer according to claim 3, characterized in that: The concentration of the probe DNA was adjusted to 50 to 100 nmol / ml.

5. The infrared-nanopore combined system for detecting early ctDNA methylation in cancer according to claim 4, characterized in that: The hybridization module has a temperature control function, which can control the hybridization environment temperature at 50 to 70°C.

6. The infrared-nanopore combined system for detecting early ctDNA methylation in cancer according to claim 5, characterized in that: The hybridization module also has a pH control function, which can control the pH value of the reaction solution between 7 and 7.

5.

7. The infrared-nanopore combined system for detecting early ctDNA methylation in cancer according to claim 6, characterized in that: The hybridization reaction time is controlled within 1 to 1.5 h.

8. The infrared-nanopore combined system for detecting early ctDNA methylation in cancer according to claim 7, characterized in that: The acquisition module includes a sensitive detection area, which is located on the nanopore. The sensitive detection area is used to identify the contact between the nanopore and the molecule and generate an electrical signal for detection.

9. The infrared-nanopore combined system for detecting early ctDNA methylation in cancer according to claim 8, characterized in that: The current amplitude of the electrical signal collected by the acquisition module includes the Ib current amplitude and the Ic current amplitude. The Ib current amplitude is used to reflect the current amplitude threshold of the unmethylated ctDNA of the patient to be tested, and the Ic current amplitude is used to reflect the current amplitude caused by the methyl group of the methylated ctDNA of the patient to be tested. The analysis process of the data analysis module is to analyze whether there is an abnormal Ic value in the electric signal current amplitude of each fragment of the ctDNA of the patient to be tested, and to make a judgment based on the duration of the electric signal. When the Ic value in the electric signal current amplitude of a certain fragment of the ctDNA of the patient to be tested is greater than the Ic value in the electric signal current amplitude of other fragments of the ctDNA of the patient to be tested, and the duration of the electric signal of the fragment is greater than the duration of the electric signal of other fragments, a "positive detection" signal mark is output; otherwise, a "negative detection" signal mark is output in other cases.

Images