Functionalized acupuncture needle and surface modification method and application thereof

Abstract

The invention discloses a functionalized acupuncture needle and a surface modification method and application thereof, and the surface modification method comprises the following three steps: 1) threading the surface of a common acupuncture needle to obtain a threaded acupuncture needle; 2) modifying the surface of the threaded acupuncture needle with polydopamine to obtain a polydopamine-coated threaded acupuncture needle; and 3) modifying nucleic acid binding protein on the thread acupuncture needle wrapped by the polydopamine to obtain the functionalized acupuncture needle. The width of a threaded groove formed in the surface of the threaded acupuncture needle is 1-300 microns; and the depth of the thread groove is 10 nm to 500 nm. The surface-modified acupuncture needle has remarkable advantages. The kit is good in biocompatibility, does not bleeding when the free nucleic acid of the lymph node cells is extracted, reduces the diffusion risk, and can be used for various detections. The nucleic acid capturing efficiency is improved through threading treatment, and high-specificity adsorption is achieved through the functional coating. And the whole-flow process is adaptive to industrialization and clinical transformation, a kit can be prepared, and an efficient tool is provided for early tumor screening.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical devices, and relates to a functionalized acupuncture needle, a surface modification method thereof, and an application thereof. Background Art

[0002] Cell-free nucleic acids include cell-free DNA and cell-free RNA. Cell-free nucleic acids have strong application prospects in the fields of tumor precision diagnosis, tumor prognosis monitoring, and detection of disease-related bacteria, viruses and other microorganisms. Cell-free nucleic acids exist in body fluids such as blood, urine, and lymph fluid, and carry the genetic information of tumor tissues and pathogenic microorganisms. Therefore, they can be used as detection markers for liquid biopsy. Current research has verified that circulating tumor DNA in cell-free nucleic acids has good application prospects in the early diagnosis and prognosis monitoring of tumors, and has the characteristics of accuracy, safety, and minimally invasive. However, the sample volume of cell-free nucleic acids in body fluids is small and the half-life is short. Therefore, cell-free nucleic acids have not been widely used in clinical tests at present.

[0003] Currently, blood samples are the biological samples widely used in clinical liquid biopsies. However, the low content and fast metabolism of cell-free nucleic acids in blood make the detection results of cell-free nucleic acids based on blood unstable and difficult to standardize. As another circulatory system of the human body, the lymphatic system is closer to the lesion in space and is the primary route for metastasis of many tumors. Therefore, the lymphatic system is expected to become a substitute for blood and a source of cell-free nucleic acids for liquid biopsy. Currently, the main difficulty in liquid biopsy based on lymph fluid is the lack of a minimally invasive / non-invasive method for extracting cell-free nucleic acids from lymph fluid. Therefore, the present invention plans to develop a minimally invasive / non-invasive method for extracting cell-free nucleic acids from lymph fluid.

[0004] Acupuncture therapy is an important part of traditional Chinese medicine. Among them, "needle" refers to "acupuncture", which is a method of treating diseases by stimulating acupoints with various needles. Currently, in clinical Chinese medicine, acupuncture therapy can be used to relieve discomfort caused by various diseases and conditions, such as nausea and vomiting caused by chemotherapy and postoperative, toothache, migraine and other painful discomforts. Patent CN119258284A discloses a nano-needle and its preparation method and application. The nano-needle includes an acupuncture needle with a host molecule modified on its surface. The host molecule includes a cyclic molecule capable of forming an inclusion complex structure. This patent modifies a cyclic molecule (such as cyclodextrin, etc.) capable of forming an inclusion complex structure on the surface of the acupuncture needle, which can form a cavity and endow the acupuncture needle with the ability to load and release functional molecules, effectively realizing the dual functions of acupuncture and drug delivery.

[0005] The widespread use of disposable acupuncture needles makes acupuncture treatment non-invasive or minimally invasive, with extremely low infection risks. The needle body length of commonly used acupuncture needles ranges from 13 mm (half an inch) to 75 mm (three inches), so there is a possibility of piercing the superficial lymph nodes under the skin. Therefore, acupuncture needles can be good carriers for sampling cell-free nucleic acids in lymph nodes, especially inflamed and swollen lymph nodes. However, currently, the surface of stainless steel acupuncture needles is smooth, and the modified coating is prone to peeling off, lacking good surface modification technology. Therefore, they cannot yet be used as sampling tools for cell-free nucleic acids in the clinical lymphatic system. Summary of the Invention

[0006] Based on the above-mentioned disadvantages existing in the prior art, the purpose of this application is to provide a functionalized acupuncture needle, its surface modification method and application, so that the surface-modified functionalized acupuncture needle can be used to capture cell-free nucleic acids (cell-free DNA and cell-free RNA) in the lymphatic system, and further applied to tumor detection, tumor prognosis monitoring and the detection of disease-related bacteria, viruses and other microorganisms.

[0007] One of the technical solutions of the present invention provides a method for surface modification of a functionalized acupuncture needle, including the following three steps:

[0008] 1) Thread the surface of a common acupuncture needle to obtain a threaded acupuncture needle;

[0009] 2) Modify polydopamine on the surface of the threaded acupuncture needle to obtain a polydopamine-coated threaded acupuncture needle;

[0010] 3) Modify nucleic acid-binding protein on the polydopamine-coated threaded acupuncture needle to obtain a functionalized acupuncture needle.

[0011] Further, the threading of the surface of the acupuncture needle in step 1) means forming a threaded groove on the surface of the acupuncture needle by using a metal etching method; the metal etching method is selected from any one of numerical control lathe cutting, laser etching or manual etching.

[0012] Further, the width of the threaded groove formed on the surface of the threaded acupuncture needle in step 1) is 1 μm to 300 μm, preferably 1 μm, 30 μm, 100 μm. The depth of the formed threaded groove is 10 nm to 500 nm, preferably 30 nm, 100 nm, 500 nm.

[0013] In some specific embodiments of the present invention, in step 1), the method for surface modification of a functionalized acupuncture needle is prepared by using a manual etching method, and the steps are as follows: Pad the tip of a common stainless steel acupuncture needle on a flat and clean tabletop, and press the acupuncture needle with a craft knife; then hold the handle of the acupuncture needle and pull the acupuncture needle backward, rotating the acupuncture needle while pulling to form a spiral groove at the tip of the acupuncture needle.

[0014] Furthermore, the surface of the threaded acupuncture needle in step 2) is modified with polydopamine by immersing the threaded acupuncture needle in a dopamine solution. In the dopamine solution, the concentration of dopamine is 1 mg / mL–10 mg / mL; the thickness of the obtained polydopamine layer is 200 nm to 1 μm, preferably 200 nm, 300 nm, 500 nm, 1 μm.

[0015] In some specific embodiments of the present invention, the steps of modifying the surface of the threaded acupuncture needle with polydopamine in step 2) are as follows: The threaded acupuncture needle prepared in step 1) is successively immersed in ultrapure water, ethanol, and ultrapure water, and ultrasonically cleaned for 3 to 5 minutes respectively to obtain a surface-cleaned threaded acupuncture needle; then the above-mentioned cleaned threaded acupuncture needle is immersed in a dopamine hydrochloride solution, and after reacting at room temperature for 16-24 hours (preferably 24 hours), the reacted threaded acupuncture needle is ultrasonically cleaned in PBS for 15-30 s (preferably 15 s), and rinsed three times with clean PBS to obtain a polydopamine-coated threaded acupuncture needle; in this step, 8-12 mM Tris-HCl with pH = 8.4-8.6 is used as a buffer in the dopamine hydrochloride solution, preferably 10 mM Tris-HCl with pH = 8.5.

[0016] Furthermore, the nucleic acid-binding protein in step 3) is a protein molecule having the function of binding nucleic acids (DNA and / or RNA), preferably any one of histone or Sul12n protein. The concentration of the nucleic acid-binding protein is 0.1 ng / mL - 1 mg / mL. The specific operation steps for modifying the nucleic acid-binding protein on the polydopamine-coated threaded acupuncture needle are as follows: The polydopamine-coated threaded acupuncture needle obtained in step 2) is placed in a solution containing the nucleic acid-binding protein and reacted overnight at room temperature; then it is rinsed three times with PBS to obtain the functionalized acupuncture needle; in this step, PBS is used as a buffer for the solution of the nucleic acid-binding protein.

[0017] Furthermore, the Sul12n protein is a DNA-binding protein with a relative molecular weight of approximately 12 kD, and its protein sequence number in NCBI is SiRe 2004. In some specific embodiments of the present application, the Sul12n protein is extracted from Saccharolobus islandicus REY15A, and the extraction steps refer to the literature Ding N, Chen Y, Chu Y, Zhong C, Huang L, Zhang Z. Lysine Methylation Modulates the Interaction of Archaeal Chromatin Protein Cren7 With DNA. Front Microbiol. 2022 Mar 3;13:837737. doi: 10.3389 / fmicb.2022.837737.

[0018] Polydopamine, as a biomimetic self-adhesive polymer, can easily deposit on various materials to form a coating. During the formation of polydopamine, dopaquinone can react with the hydroxyl or oxide layer on the metal surface to form a Schiff base bond, increasing the stability of the coating; polydopamine has abundant dopaquinone and phenolic hydroxyl groups (-OH), which can form covalent bonds with the amino groups (-NH2) and sulfhydryl groups (-SH) on the surface of nucleic acid-binding proteins, and polydopamine has strong adhesion, which also helps the nucleic acid-binding protein to bind to it; the structure of acupuncture needle-polydopamine-nucleic acid-binding protein is used because the polydopamine coating can fix the nucleic acid-binding protein on the acupuncture needle in a relatively stable form, and the acupuncture needle has strong mechanical properties and can bring the nucleic acid-binding protein into the subcutaneous lymphatic tissue of the human body in a minimally invasive manner to capture free nucleic acids of lymphatic system cells.

[0019] The second technical solution of the present invention provides a functionalized acupuncture needle prepared by the above technical solution.

[0020] The third technical solution of the present invention provides the application of the above functionalized acupuncture needle in the preparation of a kit for in vitro detection of tumors.

[0021] Furthermore, in the kit for in vitro detection of tumors, the functionalized acupuncture needle is used to capture free nucleic acids of cells; the kit also includes tumor detection reagents.

[0022] Furthermore, the kit is used in the fields of tumor diagnosis, tumor prognosis monitoring, and pathogenic microorganism detection, etc.

[0023] Further, the positions for capturing cell-free nucleic acids include at least any one of blood, lymphoid tissue, and tumor tissue. The lymphoid tissue is the lymph nodes near the tumor tissue, preferably the inflamed and enlarged lymph nodes; the tumor tissue is a superficial tumor, including any one of breast cancer, skin cancer, and head and neck cancer.

[0024] Further, the method of using the functionalized acupuncture needle (i.e., the capturing method) is: inserting the functionalized acupuncture needle into the target position, leaving it for 5 - 30 minutes and then pulling it out; the leaving time is preferably 5 minutes, 15 minutes, or 30 minutes.

[0025] Further, the use for tumor diagnosis and tumor prognosis monitoring means eluting the nucleic acids captured by the functionalized acupuncture needle and using biological methods to analyze tumor-related DNA gene mutations and gene copy number variations; the use for detecting pathogenic microorganisms means eluting the nucleic acids captured by the functionalized acupuncture needle and using biological methods to identify the types of microorganisms such as bacteria and viruses and analyze the population ratios.

[0026] Even further, the biological methods include at least any one of qPCR, RT-qPCR, digital PCR, and gene sequencing, but are not limited to these methods.

[0027] Further, the elution of the nucleic acids captured by the functionalized acupuncture needle is specifically achieved by using proteinase K or trypsin to digest proteins, releasing the captured nucleic acids into the PBS buffer, and then using a nucleic acid purification kit to isolate and extract the nucleic acids.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The surface-modified acupuncture needle provided by the present invention has good biocompatibility and can conveniently and quickly extract cell-free nucleic acids from lymph nodes. During the process of extracting cell-free nucleic acids from lymph nodes, the entire operation process can be achieved without bleeding, greatly reducing the risk of the spread of cancer cells and pathogenic microorganisms in conventional biopsies. The extracted cell-free nucleic acids from lymph nodes can be used for tumor detection, tumor treatment prognosis monitoring, pathogenic microorganism detection, etc.

[0030] (2) The surface structure optimization significantly improves the nucleic acid capture efficiency. The threading treatment of the present invention forms micron- to nanometer-scale grooves on the surface of the acupuncture needle, greatly increasing the effective specific surface area and providing physical anchor sites for the uniform loading of the subsequent polydopamine coating. Combining the strong adhesion of polydopamine, the threaded structure can prevent the coating from falling off and ensure the stable fixation of nucleic acid-binding proteins.

[0031] (3) The functionalized coating system enables highly specific nucleic acid adsorption. The polydopamine intermediate layer not only enhances surface biocompatibility, but its rich phenolic hydroxyl and amino groups can be directionally coupled with nucleic acid-binding proteins such as histones (e.g., Sul12n protein) to form a three-level composite structure of "thread-PDA-protein". This design realizes the efficient capture of nucleic acids through the synergistic effect of physical adsorption (thread grooves) and chemical binding (PDA / protein).

[0032] (4) The whole process technology is adapted to industrial production and clinical transformation. The technical solution can use numerical control lathe cutting or laser etching for thread processing, with precise and controllable process parameters. Combined with the self-polymerization reaction of polydopamine, large-scale production can be achieved without complex equipment. In addition, the functionalized acupuncture needles can be directly integrated into the standard lymph node puncture process, and a kit (including PCR primers and sequencing reagents) can be made to realize "sampling-detection" integration, greatly shortening the detection cycle and providing an efficient tool for early tumor screening. Description of the Drawings

[0033] Figure 1 It is the bright-field photo of the acupuncture needle with threads on the surface in Example 1 of the present invention;

[0034] Figure 2 It is the scanning electron microscope photo of the acupuncture needle with threads on the surface in Example 1 of the present invention;

[0035] Figure 3 It is the qPCR experimental result diagram in Example 1 of the present invention, used to verify the ability of the functionalized acupuncture needle prepared in Example 1 to capture double-stranded DNA;

[0036] Figure 4 It is the bright-field photo of the functionalized acupuncture needle inserted into the lymph node removed clinically in Example 2 of the present invention;

[0037] Figure 5 It is the detected tumor-related mutant gene (A) and the hematoxylin-eosin staining result (B) of the same lymph node tissue sample in Example 2 of the present invention, scale bar = 50 μM;

[0038] Figure 6 It is the qPCR experimental result diagram in Example 3 and Comparative Examples 1-2 of the present invention, used to verify the ability of the functionalized acupuncture needle prepared in Example 3 to capture two kinds of double-stranded DNA; the amount of double-stranded DNA captured by an acupuncture needle only processed by etching (Needle, Comparative Example 1), an acupuncture needle only wrapped with polydopamine (PDA@Needle, Comparative Example 2), and a Sul12n-functionalized acupuncture needle (Sul12n@PDA@Needle) from a 0.2 mL 1 ng / mL double-stranded DNA solution respectively;

[0039] Figure 7This is the result graph of the RT-qPCR experiment in Example 3 of the present invention, which is used to verify the ability of the functionalized acupuncture needle prepared in Example 3 to capture a single-stranded RNA. Detailed implementation mode

[0040] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made. These all belong to the protection scope of the present invention.

[0041] There is no particular limitation on the source of all raw materials of the present invention, and those purchased on the market or prepared according to the conventional methods well-known to those skilled in the art are all acceptable.

[0042] The concentration of the PBS buffer solution used in the embodiments of the present invention is: 1×PBS, and the pH is 7.4.

[0043] Example 1 A manually etched functionalized acupuncture needle and its function verification

[0044] (1) Preparation steps:

[0045] 1) Pad the tip of the ordinary stainless steel acupuncture needle on a flat and clean tabletop, and press the acupuncture needle with a craft knife. Then hold the handle of the acupuncture needle and pull the acupuncture needle backward, rotating the acupuncture needle while pulling, to form a spiral groove at the front end of the acupuncture needle. The bright-field picture of the acupuncture needle with a spiral groove is shown in the appendix Figure 1 , and the scanning electron microscope picture is shown in the appendix Figure 2 . It can be seen that spiral scratches have appeared on the surface of the acupuncture needle, and a threaded acupuncture needle is obtained.

[0046] 2) Immerse the above-prepared threaded acupuncture needle in 400 μL of ultrapure water, ultrasonically clean for 3 minutes, then immerse in 400 μL of ethanol, ultrasonically clean for 3 minutes, and then immerse in 400 μL of ultrapure water and ultrasonically clean for 3 minutes to obtain a surface-cleaned threaded acupuncture needle. Then immerse the above-cleaned threaded acupuncture needle in 400 μL of 6 mg / mL dopamine hydrochloride solution (10 mM Tris-HCl with pH = 8.5 as the buffer solution), react at room temperature for 24 hours, then place the reacted threaded acupuncture needle in 400 μL of PBS and ultrasonically clean for 15 s, and rinse three times with clean PBS to obtain a polydopamine-coated threaded acupuncture needle.

[0047] (3) Place the above-mentioned dopamine-coated spiral acupuncture needles in 400 μL of 1 mg / mL histone solution (PBS as the buffer) and react overnight at room temperature. After rinsing three times with PBS, the functionalized acupuncture needles capable of capturing lymph node circulating tumor DNA are obtained.

[0048] (2) Verify its function using synthetic double-stranded DNA:

[0049] (1) Place the above-mentioned functionalized acupuncture needles capable of capturing lymph node circulating tumor DNA into 200 μL of PBS solution containing synthetic double-stranded DNA with a total concentration of 1 ng / mL (there are 3 types of double-stranded DNA in the same solution: GAPDH + GAPDH-com; ACTB + ACTB-com; TP53 + TP53-com, and the sequence information is shown in SEQ.ID.NO.1 - 6 in Table 1). After standing and reacting at room temperature for 30 min, take out the functionalized acupuncture needles, and rinse the functionalized acupuncture needles that have captured double-stranded DNA once with 500 μL of PBS. Then, immerse the functionalized acupuncture needles that have captured double-stranded DNA into 200 μL of 100 μg / mL proteinase K (a serine protease with broad cleavage activity, commonly used in nucleic acid extraction to digest proteins to remove protein impurities bound to nucleic acids and obtain pure nucleic acid samples) solution (PBS as the buffer), react at 56 °C for 1.5 hours, and react at 95 °C for 10 minutes. Finally, use the GeneJET PCR Purification Kit (purchased from Thermo Scientific TM ) to remove proteinase K from the reaction solution, and redissolve the double-stranded DNA in 20 μL of buffer.

[0050] (2) Verify the double-stranded DNA captured by the functionalized acupuncture needles through qPCR experiments. (The qPCR reaction system is: 5 μL of 2x SGExcel Universal SYBR qPCR Mix, 1 μL of 10 μM Forward Primer (see sequences SEQ.ID.NO.7, SEQ.ID.NO.9, SEQ.ID.NO.11), 1 μL of 10 μM Reverse Primer (see sequences SEQ.ID.NO.8, SEQ.ID.NO.10, SEQ.ID.NO.12), 1 μL of Template DNA (see sequences SEQ.ID.NO.1 - 6), 2 μL of ddH2O. The qPCR reaction conditions are: 95 °C, 3 min; 95 °C, 10 s; 57 °C, 30 s; 72 °C, 30 s. Amplify for 40 cycles.) The results of the qPCR experiment are shown in the appendix Figure 3 , and the experimental results show that the functionalized acupuncture needles provided in this example successfully captured the three added double-stranded DNA fragments.

[0051] Table 1 Information on the synthetic DNA sequences used in Example 1

[0052]

[0053]

[0054] In the table, GAPDH is the partial sense strand sequence of the GAPDH gene; GAPDH-com is the complementary strand sequence of the aforementioned GAPDH. ACTB is the partial sense strand sequence of the ACTB gene; ACTB-com is the complementary strand sequence of the aforementioned ACTB; TP53 is the partial sense strand sequence of the P53 gene; TP53-com is the complementary strand sequence of the aforementioned TP53. Sequences 7-12 are used as primers in subsequent qPCR.

[0055] Example 2 A manually etched functionalized acupuncture needle and its application

[0056] (1) Preparation steps:

[0057] 1) Pad the tip of a common stainless steel acupuncture needle on a flat and clean tabletop, and press the acupuncture needle with a craft knife. Then hold the handle of the acupuncture needle and pull the acupuncture needle backward, rotating the acupuncture needle while pulling to make a spiral groove at the front end of the acupuncture needle.

[0058] 2) Immerse the threaded acupuncture needle prepared above in 400 μL of ultrapure water and ultrasonically clean for 3 minutes, then immerse it in 400 μL of ethanol and ultrasonically clean for 3 minutes, and then immerse it in 400 μL of ultrapure water and ultrasonically clean for 3 minutes to obtain a surface-cleaned threaded acupuncture needle. Then soak the above-cleaned threaded acupuncture needle in 400 μL of 6 mg / mL dopamine hydrochloride solution (10 mM Tris-HCl with pH = 8.5 as the buffer) and react at room temperature for 24 hours. After that, place the reacted threaded acupuncture needle in 400 μL of PBS and ultrasonically clean for 15 s, and rinse it three times with clean PBS to obtain a polydopamine-coated threaded acupuncture needle.

[0059] 3) Place the above polydopamine-coated threaded acupuncture needle in 400 μL of 1 mg / mL histone solution (PBS as the buffer) and react at room temperature overnight. After rinsing three times with PBS, the required functionalized acupuncture needle capable of capturing lymph node circulating tumor DNA is obtained.

[0060] (2) Application in lymph nodes

[0061] 1) As attached Figure 4As shown, two functionalized acupuncture needles capable of capturing circulating tumor DNA prepared in the above Example 1 and Example 2 were simultaneously inserted into the same lymph node removed from a clinical head and neck cancer surgery. After standing at room temperature for 30 min, the functionalized acupuncture needles were taken out. Then, the functionalized acupuncture needles capturing double-stranded DNA were immersed in 200 μL of protease K solution at 100 μg / mL (PBS as the buffer), reacted at 56 °C for 1.5 hours, and reacted at 95 °C for 10 minutes. Finally, the above reaction solution was ultrasonically vibrated for 1 minute, and the protease K in the reaction solution was removed using the GeneJET PCR Purification Kit (purchased from Thermo Scientific TM ) and the obtained double-stranded DNA was redissolved in 20 μL of buffer solution.

[0062] 2) The circulating tumor DNA in the lymph nodes captured by the functionalized acupuncture needles was verified to be 25.4 ng by a fluorometer nucleic acid quantifier ( Life Technologies).

[0063] 3) The cell-free DNA in the captured lymph nodes was analyzed by next-generation sequencing. By comparing with the COSMIC database (a well-known and widely used database in the fields of biology and medicine, which will not be elaborated here), it was found that tumor-related mutations occurred at the gene loci of KMT2D, FAT1, NOTCH1, and EGFR in the cell-free DNA of the collected clinical samples (these loci are closely related to the occurrence and development of cancer and can therefore be used as sites for cancer diagnosis) ( Figure 5 as shown in A). The experimental results prove that the new method provided by this application can be used for tumor detection of cell-free DNA derived from clinical lymph nodes. On the other hand, the staining results of the clinical hematoxylin-eosin staining method ( Figure 5 as shown in B) also indicate that the lymph node of this clinical sample is a metastatic lymph node of head and neck cancer. The conclusions obtained by the developed method and the clinical hematoxylin-eosin staining method are consistent, verifying the reliability of the developed method.

[0064] The hematoxylin-eosin staining method described in this example refers to the following literature: Hematoxylin and EosinStaining of Tissue and Cell Sections.Cold Spring Harbor Protocols 2008(5):pdb.prot4986.

[0065] Comparative Example 1 An acupuncture needle only subjected to etching processing

[0066] Preparation steps:

[0067] 1) A spiral groove was made at the front end of the ordinary stainless steel acupuncture needle using a numerically controlled lathe;

[0068] 2) Immerse the prepared threaded acupuncture needles in 400 μL of ultrapure water and ultrasonically clean for 3 minutes, then immerse in 400 μL of ethanol and ultrasonically clean for 3 minutes, and then immerse in 400 μL of ultrapure water and ultrasonically clean for 3 minutes to obtain surface-clean threaded acupuncture needles, thus obtaining a functionalized acupuncture needle that only undergoes etching processing, denoted as the Needle group.

[0069] Comparative Example 2 A polydopamine-coated acupuncture needle

[0070] Preparation steps:

[0071] 1) Use a CNC lathe to make a spiral groove at the front end of a common stainless steel acupuncture needle.

[0072] 2) Immerse the prepared threaded acupuncture needles in 400 μL of ultrapure water and ultrasonically clean for 3 minutes, then immerse in 400 μL of ethanol and ultrasonically clean for 3 minutes, and then immerse in 400 μL of ultrapure water and ultrasonically clean for 3 minutes to obtain surface-clean threaded acupuncture needles.

[0073] 3) Then immerse the above-cleaned threaded acupuncture needles in 400 μL of 6 mg / mL dopamine hydrochloride solution (10 mM Tris-HCl with pH = 8.5 as the buffer solution) and react at room temperature for 24 hours. After the reaction, place the threaded acupuncture needles in 400 μL of PBS and ultrasonically clean for 15 s, and rinse three times with clean PBS to obtain polydopamine-coated threaded acupuncture needles, denoted as the PDA@Needle group.

[0074] Example 3 A Sul12n-functionalized acupuncture needle and its function verification

[0075] (1) Preparation steps:

[0076] 1) Use a CNC lathe to make a spiral groove at the front end of a common stainless steel acupuncture needle.

[0077] 2) Immerse the prepared threaded acupuncture needles in 400 μL of ultrapure water and ultrasonically clean for 3 minutes, then immerse in 400 μL of ethanol and ultrasonically clean for 3 minutes, and then immerse in 400 μL of ultrapure water and ultrasonically clean for 3 minutes to obtain surface-clean threaded acupuncture needles. Then immerse the above-cleaned threaded acupuncture needles in 400 μL of 6 mg / mL dopamine hydrochloride solution (10 mM Tris-HCl with pH = 8.5 as the buffer solution) and react at room temperature for 24 hours. After the reaction, place the threaded acupuncture needles in 400 μL of PBS and ultrasonically clean for 15 s, and rinse three times with clean PBS to obtain polydopamine-coated threaded acupuncture needles.

[0078] (3) Place the above poly-dopamine-coated spiral acupuncture needles in 400 μL of 0.1 mg / mL Sul12n protein solution (with PBS as the buffer) and react overnight at room temperature. After rinsing three times with PBS, the desired functionalized acupuncture needles capable of capturing circulating free nucleic acids (DNA and RNA) in lymph nodes are obtained.

[0079] (2) Verify its function using synthetic double-stranded DNA:

[0080] (1) Place the above functionalized acupuncture needles capable of capturing circulating free nucleic acids in lymph nodes into 200 μL of PBS solution containing synthetic double-stranded DNA fragments (GAPDH + GAPDH-com, a total of 1 ng / mL, and the sequences are shown in SEQ.ID.NO.1 - 2). After standing at room temperature for 30 min, take out the functionalized acupuncture needles and rinse the functionalized acupuncture needles capturing double-stranded DNA once with 500 μL of PBS. Then, immerse the functionalized acupuncture needles capturing double-stranded DNA into 200 μL of 100 μg / mL proteinase K solution (with PBS as the buffer), react at 56 °C for 1.5 hours, and react at 95 °C for 10 minutes. Finally, use the GeneJET PCR purification kit (purchased from Thermo Scientific TM ) to remove proteinase K from the reaction solution, and redissolve the obtained double-stranded DNA in 20 μL of buffer.

[0081] (2) Verify the double-stranded DNA captured by the functionalized acupuncture needles through qPCR experiments. (The qPCR reaction conditions are: 95 °C, 3 min; 95 °C, 10 s; 57 °C, 30 s; 72 °C, 30 s. Amplify for 40 cycles.) The results of the qPCR experiment are shown in the appendix Figure 6 . The experimental results show that the functionalized acupuncture needles successfully captured the added GAPDH double-stranded DNA fragments. Moreover, compared with the non-functionalized acupuncture needles, the absolute amount of double-stranded DNA extracted by the functionalized acupuncture needles is the highest, with an average value in the range of approximately 100 - 150 pg. And statistical analysis shows that there is a significant difference between the Su12n@PDA@Needle group and the PDA@Needle group, indicating that the functionalization modification has statistical significance for improving the DNA capture ability.

[0082] (3) Verify its function using synthetic RNA:

[0083] 1) Place the above-mentioned polydopamine-coated spiral acupuncture needles into 400 μL of Sul12n protein solution at 0.125 μg / mL or 400 μL at 0.25 μg / mL or 400 μL at 0.5 μg / mL (PBS as the buffer) and react overnight at room temperature. After rinsing three times with PBS, the functionalized acupuncture needles capable of capturing circulating free nucleic acids (DNA and RNA) in lymph nodes are obtained. Place the above-mentioned functionalized acupuncture needles capable of capturing circulating free nucleic acids in lymph nodes into 200 μL of PBS solution containing synthetic single-stranded RNA fragments (1 ng / mL, the sequence is shown as SEQ.ID.NO.13 in Table 2). After standing at room temperature for 30 min, take out the functionalized acupuncture needles and rinse the functionalized acupuncture needles capturing single-stranded RNA once with 500 μL of PBS. Then, immerse the functionalized acupuncture needles capturing single-stranded RNA into 200 μL of proteinase K solution at 100 μg / mL (PBS as the buffer), react at 56 °C for 1.5 hours, and react at 95 °C for 10 minutes. Finally, use a column-type RNA rapid concentration and purification kit (purchased from Sangon Biotech, Shanghai) to remove proteinase K from the reaction solution, and redissolve the obtained single-stranded RNA in 20 μL of buffer.

[0084] 2) Verify the single-stranded RNA captured by the functionalized acupuncture needles through RT-qPCR experiments. (The reverse transcription reaction system is: 1 μL of Template RNA (the sequence is shown as SEQ.ID.NO.13 in Table 2), 1 μL of 10 μM GAPDH Forward Primer (the sequence information is shown as SEQ.ID.NO.7 in Table 1), 3.5 μL of RNase free ddH2O, 2 μL of 5×Reaction Buffer, 0.5 μL of RNase Inhibitor, 1 μL of 10 mM dNTP Mix, 1 μL of AMV RT. The reaction conditions for reverse transcription are: 42 °C, 45 min; 85 °C, 5 min. The qPCR reaction conditions are: 95 °C, 3 min; 95 °C, 10 s; 57 °C, 30 s; 72 °C, 30 s. Amplify for 40 cycles.) The results of the RT-qPCR experiments are shown in the appendix Figure 7 , and the experimental results show that the functionalized acupuncture needles successfully captured the added single-stranded RNA fragments. With the increase in the concentration of Sul12n, the absolute amount of the extracted RNA sequence also increased, and the concentration range of added Sul12n (50 ng - 200 ng) did not show a downward trend in the RNA extraction amount. Therefore, it is speculated that the peak value may appear at a higher concentration.

[0085] Table 2 Synthetic nucleic acid sequence information used in Example 3

[0086]

[0087] The above description of the embodiments is provided to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A method for surface modification of a functionalized acupuncture needle, characterized in that, It includes the following three steps: 1) Thread the surface of a common acupuncture needle to obtain a threaded acupuncture needle; 2) Modify the surface of the threaded acupuncture needle with polydopamine to obtain a threaded acupuncture needle wrapped with polydopamine; 3) Modify the nucleic acid-binding protein on the threaded acupuncture needle wrapped with polydopamine to obtain a functionalized acupuncture needle.

2. The surface modification method of a functionalized acupuncture needle according to claim 1, wherein The threading of the surface of the acupuncture needle in step 1) means forming a threaded groove on the surface of the acupuncture needle by means of metal etching; the metal etching method is selected from any one of numerical control lathe cutting, laser etching or manual etching.

3. The surface modification method of a functionalized acupuncture needle according to claim 2, characterized in that, In step 1), the steps of the manual etching are as follows: Pad the tip of the common acupuncture needle on a flat and clean tabletop, and press the acupuncture needle with a knife; then hold the handle of the acupuncture needle and pull the acupuncture needle backward, rotating the acupuncture needle while pulling, to form a spiral groove at the tip of the acupuncture needle.

4. A method for surface modification of a functionalized acupuncture needle according to claim 1, characterized in that, The width of the threaded groove formed on the surface of the threaded acupuncture needle in step 1) is 1 μm to 300 μm; the depth is 10 nm to 500 nm.

5. A method for surface modification of a functionalized acupuncture needle according to claim 1, characterized in that, In step 2), the modification of the surface of the threaded acupuncture needle with polydopamine is obtained by immersing the threaded acupuncture needle in a dopamine solution. In the dopamine solution, the concentration of dopamine is 1 mg / mL–10 mg / mL; the thickness of the polydopamine layer obtained is 200 nm to 1 μm.

6. A method for surface modification of a functionalized acupuncture needle according to claim 1, characterized in that, The steps of modifying the surface of the threaded acupuncture needle with polydopamine in step 2) are as follows: Immerse the threaded acupuncture needle prepared in step 1) in ultrapure water, ethanol, and ultrapure water in sequence, and perform ultrasonic cleaning respectively to obtain a threaded acupuncture needle with a clean surface; then immerse the above-mentioned clean threaded acupuncture needle in a dopamine hydrochloride solution, react at room temperature, place the reacted threaded acupuncture needle in PBS for ultrasonic cleaning, and rinse with clean PBS to obtain a threaded acupuncture needle wrapped with polydopamine; in this step, 8–12 mM Tris-HCl with pH = 8.4–8.6 is used as a buffer in the dopamine hydrochloride solution.

7. A method for surface modification of a functionalized acupuncture needle according to claim 1, wherein, The nucleic acid-binding protein in step 3) is a protein molecule with the function of binding nucleic acids; the nucleic acid is DNA and / or RNA; the nucleic acid-binding protein is selected from any one of histone or Sul12n protein; the concentration of the nucleic acid-binding protein is 0.1 ng / mL to 1 mg / mL.

8. A method for surface modification of a functionalized acupuncture needle according to claim 1, characterized in that, The specific operation steps of modifying the nucleic acid-binding protein on the threaded acupuncture needle wrapped with polydopamine in step 3) are as follows: Place the threaded acupuncture needle wrapped with polydopamine obtained in step 2) in a solution containing the nucleic acid-binding protein and react at room temperature; then rinse with PBS to obtain the functionalized acupuncture needle; in this step, PBS is used as a buffer in the solution of the nucleic acid-binding protein.

9. A functionalized acupuncture needle prepared by the method according to any one of claims 1 to 8.

10. Use of the functionalized acupuncture needle according to claim 9 in the preparation of a kit for in vitro detection of tumors, characterized in that, In a kit for in vitro detection of tumors, the functionalized acupuncture needle is used to capture cell-free nucleic acids; the kit also includes a tumor detection reagent.

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