WO2023178704A1 - Sonde polypeptidique, son procédé de préparation et son utilisation - Google Patents

Sonde polypeptidique, son procédé de préparation et son utilisation Download PDF

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Publication number
WO2023178704A1
WO2023178704A1 PCT/CN2022/083202 CN2022083202W WO2023178704A1 WO 2023178704 A1 WO2023178704 A1 WO 2023178704A1 CN 2022083202 W CN2022083202 W CN 2022083202W WO 2023178704 A1 WO2023178704 A1 WO 2023178704A1
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Prior art keywords
resin
polypeptide probe
tentagel
reaction
substrate
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PCT/CN2022/083202
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English (en)
Chinese (zh)
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赵子健
丁丁
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京东方科技集团股份有限公司
北京京东方技术开发有限公司
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Priority to CN202280000558.2A priority Critical patent/CN117136193A/zh
Priority to PCT/CN2022/083202 priority patent/WO2023178704A1/fr
Publication of WO2023178704A1 publication Critical patent/WO2023178704A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection

Definitions

  • the present disclosure relates to the field of biotechnology, and in particular to a polypeptide probe and its preparation method and application.
  • Malignant lymphoma has extremely high malignant degree and fatality rate, and has become one of the main hot spots in medical research in recent years.
  • diffuse large B-cell lymphoma is the most common type of non-Hodgkin lymphoma and is a malignant lymphoma.
  • hematopathologists rely on sampling for pathological analysis to arrive at a correct diagnosis of diffuse large B-cell lymphoma.
  • sampling for pathological analysis is difficult and often occurs in the middle and late stages of the disease, resulting in patients not being able to receive timely and effective treatment.
  • a polypeptide probe including at least two hydrophobic amino acid residues.
  • the number of hydrophobic amino acid residues accounts for one quarter to two thirds of the number of amino acid residues of the polypeptide probe.
  • the amino acid includes at least one of His, Gly, Cys, Leu, Asn, Arg, Trp, Phe, Asp, Ser, and Tyr.
  • the number of amino acid residues is 5-15.
  • sequence of amino acid residues includes at least one of SEQ ID NO. 1 ⁇ SEQ ID NO. 15.
  • sequence of the amino acid residues of the polypeptide probe is SEQ ID NO. 6.
  • the sequence of the amino acid residues of the polypeptide probe includes at least one of SEQ ID NO. 1 to SEQ ID NO. 15.
  • a method for preparing a polypeptide probe includes:
  • S1 includes S11.
  • S2 includes S22.
  • S3 includes S33.
  • S4 includes S44.
  • S5 includes S55.
  • S6 includes S66.
  • S7 includes S77.
  • S8 includes S88.
  • kits including the polypeptide probe as described above.
  • the kit further includes a surfactant, an anti-enzymatic agent, and a fluorescent stain for cells.
  • the volume percentage of the surfactant is 0.5% to 5%
  • the molar concentration of the anti-enzymatic agent is 0.5mmol/L to 10mmol/L
  • the molar concentration of the cell fluorescent stain is 0.5mmol/L ⁇ 50mmol/L.
  • the surfactant includes at least one of polyoxyethylene sorbitan monolaurate, sodium lauryl sulfate, and fatty acid sorbitan. And/or; the anti-enzymatic agent includes ethylenediaminetetraacetic acid. and/or; the cytofluorescent staining agent includes rhodamine B.
  • a chip including a substrate and a polypeptide probe as described above, and the polypeptide probe is disposed on the substrate.
  • a chip preparation method including:
  • a polypeptide probe is coupled to one side of the substrate, and the polypeptide probe is as described above.
  • the polypeptide probe coupled to one side of the substrate includes:
  • forming the substrate includes:
  • An adhesion layer is formed on the substrate side.
  • a passivation layer is formed on a side of the adhesion layer away from the substrate, and the passivation layer is patterned.
  • a gold film is plated and etched on the side of the passivation layer away from the substrate.
  • Polypeptide probes were coupled to the gold film.
  • forming a passivation layer on a side of the adhesion layer away from the substrate includes forming a first insulating layer and a second insulating layer.
  • the first insulating layer is formed on a side of the adhesive layer away from the substrate.
  • the second insulating layer is formed on a side of the second insulating layer away from the substrate.
  • an application of the chip as described above in detecting diffuse large B-cell lymphoma is provided.
  • Figures 1 to 2 are flow charts of methods for preparing polypeptide probes according to some embodiments
  • Figure 3 is a structural diagram of the principle of magnetic bead primary screening according to some embodiments.
  • FIGS. 4A to 4B are flow charts of chip preparation methods according to some embodiments.
  • Figures 5A to 5E are cross-sectional structural diagrams corresponding to each step in the chip preparation method according to some embodiments.
  • Figures 6A to 6O are time and response curves of polypeptide probes under different CD138 concentration conditions according to some embodiments.
  • Figures 7A to 7B are flow cytometry characterization diagrams according to some embodiments.
  • Figure 8 is an electron microscope image of magnetic beads bound to cells according to some embodiments.
  • first and second are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Therefore, features defined as “first” and “second” may explicitly or implicitly include one or more of these features. In the description of the embodiments of the present disclosure, unless otherwise specified, "plurality" means two or more.
  • At least one of A, B and C has the same meaning as “at least one of A, B or C” and includes the following combinations of A, B and C: A only, B only, C only, A and B The combination of A and C, the combination of B and C, and the combination of A, B and C.
  • a and/or B includes the following three combinations: A only, B only, and a combination of A and B.
  • the term “if” is optionally interpreted to mean “when” or “in response to” or “in response to determining” or “in response to detecting,” depending on the context.
  • the phrase “if it is determined" or “if [stated condition or event] is detected” is optionally interpreted to mean “when it is determined" or “in response to the determination" or “on detection of [stated condition or event]” or “in response to detection of [stated condition or event]”.
  • DNA deoxyribonucleic acid, deoxyribonucleic acid
  • dAMP adenine deoxynucleotide
  • TMP thymine deoxynucleotide
  • CMP cytosine deoxyribonucleotide
  • dGMP guanine deoxyribonucleotide
  • test kit is a box used to contain chemical reagents for testing chemical components, drug residues, virus types, etc.
  • the box can also be a tube or other container.
  • His is the abbreviation of Histidine (histidine), with a molecular weight of 155.141 and a basic amino acid.
  • Gly is the abbreviation of Glycine.
  • the molecular weight is 75.052 and it belongs to the aliphatic class.
  • Cys the abbreviation of Cysteine (cysteine), has a molecular weight of 121.145 and belongs to the sulfur-containing category.
  • Leu the abbreviation of Leucine (leucine)
  • Leucine has a molecular weight of 131.16 and belongs to the aliphatic class.
  • Arg the abbreviation of Arginine, has a molecular weight of 174.188 and belongs to the basic amino acids.
  • Trp the abbreviation of Tryptophan (tryptophan)
  • Trptophan has a molecular weight of 204.213 and belongs to the aromatic class.
  • Asparagine has a molecular weight of 132.104 and belongs to amides.
  • Phenylalanine the abbreviation of Phenylalanine (phenylalanine)
  • Phenylalanine has a molecular weight of 165.177 and belongs to the aromatic category.
  • Asp the abbreviation of Aspartic acid, has a molecular weight of 133.089 and belongs to the basic amino acids.
  • Ser the abbreviation of Serine, has a molecular weight of 105.078 and belongs to the hydroxyl group.
  • Tyr is the abbreviation of Tyrosine (tyrosine), its molecular weight is 181.176, and it belongs to the aromatic class.
  • HBTU O-benzotriazole-tetramethylurea hexafluorophosphate
  • PyBop is benzotriazol-1-yl-oxytripyrrolidinyl phosphorus hexafluorophosphate, with the molecular formula C 18 H 28 F 6 N 6 OP 2 .
  • CD138 is a member of the syndecan family of four transmembrane proteins and is capable of binding to heparin sulfate and chondroitin sulfate molecules. Its main function is to control cell growth and differentiation and maintain cell adhesion and migration. Expression of CD138 antibodies in human hematopoietic cells is restricted to plasma cells in normal bone marrow, and early B cell precursors in human bone marrow are CD138 negative. CD138 may help distinguish viable myeloma cells from apoptotic cells and is also expressed on endothelial cells, fibroblasts, keratinocytes, and normal hepatocytes.
  • CD138 is helpful in the diagnosis of plasmacytoma, but some non-lymphoid hematopoietic tumors (such as squamous cell carcinoma, myoepithelioma, medullary thyroid carcinoma, melanoma) may have plasmacytoid morphology and may also be CD138 positive. In addition, CD138 exhibits different expression levels with the development process of lymphocytes, and is highly expressed on the surface of tumor cells, especially circulating tumor cells, in patients with diffuse large B.
  • non-lymphoid hematopoietic tumors such as squamous cell carcinoma, myoepithelioma, medullary thyroid carcinoma, melanoma
  • CD138 exhibits different expression levels with the development process of lymphocytes, and is highly expressed on the surface of tumor cells, especially circulating tumor cells, in patients with diffuse large B.
  • Hanks solution is mainly used for rinsing tissue blocks, rinsing cells, and preparing other reagents during cell culture.
  • Hanks solution is the most commonly used inorganic salt solution and balanced salt solution (BSS) in biomedical experiments, referred to as H.
  • Hanks solution is mainly used to prepare culture media, diluents and cell cleaning solutions.
  • PBS buffer is the most widely used buffer in biochemical research. Its main components include Na 2 HPO 4 , KH 2 PO 4 , NaCl and KCl. It is generally used as a solvent to dissolve protective reagents.
  • PBST buffer is PBS buffer added with polyoxyethylene sorbitan monolaurate (Tween 20).
  • NHL is the abbreviation of non-Hodgkin’s lymphoma.
  • Tentagel-NH 2 resin is a PEG and PS resin used as a carrier for solid phase synthesis.
  • Common blood tumors mainly include various types of leukemia, multiple myeloma and malignant lymphoma.
  • malignant lymphoma has extremely high malignancy and fatality rate, and has become one of the main research hotspots in the medical community in recent years.
  • Diffuse large B-cell lymphoma is the most common type of NHL and accounts for the majority of cases of clinically "aggressive" or "intermediate-high malignant" lymphoma.
  • hematopathologists rely on appropriate biopsy and evidence of B-cell immunophenotype to arrive at the correct diagnosis of diffuse large B-cell lymphoma.
  • CD138 expresses different levels with the development process of lymphocytes, and is highly expressed on the surface of tumor cells, especially circulating tumor cells, in patients with diffuse large B. That is to say, in the early stage of the onset of diffuse large B-cell lymphoma, whether you have diffuse large B-cell lymphoma can be determined based on the expression level of CD138.
  • detection sensitivity there is no effective early screening method to obtain a correct diagnosis of diffuse large B-cell lymphoma in the early stages of the onset of diffuse large B-cell lymphoma.
  • the onset of diffuse large B-cell lymphoma is already in the In the middle and late stages, patients cannot receive timely and effective treatment.
  • the polypeptide probe includes at least two hydrophobic amino acid residues. Wherein, the number of hydrophobic amino acid residues accounts for one quarter to two thirds of the number of amino acid residues of the polypeptide probe.
  • CD138 When using peptide probes to detect diffuse large B-cell lymphoma, CD138 is expressed at different levels with the development process of lymphocytes and is highly expressed on the surface of tumor cells, especially circulating tumor cells, in patients with diffuse large B cells.
  • CD138 is a proteoglycan, and its core protein is a type I transmembrane protein with a single-chain structure. It has an N-terminal functional region, a hydrophobic transmembrane segment and a short intracellular C-terminal region, so it can be divided into three regions, namely, extracellular region, transmembrane region and cytoplasmic region. The extracellular region contains five serine-glycine repeats.
  • the transmembrane region consists of approximately 25 amino acids, and the cytoplasmic region consists of approximately 30 amino acids. Due to the presence of hydrophobic and hydrophilic amino acid residues in the peptide probe, it binds to CD138 during detection to show high affinity.
  • the amino acid includes at least one of His, Gly, Cys, Leu, Asn, Arg, Trp, Phe, Asp, Ser, and Tyr.
  • the number of amino acid residues is 5 to 15, such as 5, 6, 8, 10 or 15. This disclosure uses 6 for explanation.
  • the sequence of amino acid residues includes at least one of SEQ ID NO. 1 ⁇ SEQ ID NO. 15.
  • the sequences of the amino acid residues of SEQ ID NO.1 to SEQ ID NO.15 are shown in Table 1 below:
  • polypeptide probes provided by some embodiments of the present disclosure have the characteristics of good biocompatibility, convenient and fast synthesis, strong affinity, strong specificity, strong penetrating power, and fast clearance speed.
  • sequence of the amino acid residues of the polypeptide probe is the sequence of the amino acid residues shown in SEQ ID NO. 6 in Table 1.
  • some embodiments of the present disclosure also provide a method for preparing a polypeptide probe, including S1 to S8.
  • S1 includes S11.
  • the above-mentioned removal of protecting groups and removal of Fmoc protecting groups from Tentagel-NH 2 resin is carried out in a removing agent.
  • the removing agent can be at least one of hexahydropyridine and alkali solution, such as alkali solution. It includes at least one of sodium hydroxide and potassium hydroxide, to which the disclosure is not specifically limited.
  • HBTU is used as a peptide coupling reagent. According to actual needs, the peptide coupling reagent can also be PyBop, which is not specifically limited in this disclosure.
  • S2 includes S22.
  • the Fmoc protecting group is removed after mixing the Tentagel-NH 2 resin in a removing agent.
  • the removing agent can be at least one of hexahydropyridine and alkali solution.
  • the alkali solution includes hydroxide. At least one of sodium and potassium hydroxide, which is not specifically limited in this disclosure.
  • HBTU is used as a peptide coupling reagent. According to actual needs, the peptide coupling reagent can also be PyBop, which is not specifically limited in this disclosure.
  • S3 includes S33.
  • the Fmoc protecting group is removed after mixing the Tentagel-NH 2 resin in a removing agent.
  • the removing agent can be at least one of hexahydropyridine and alkali solution.
  • the alkali solution includes hydroxide. At least one of sodium and potassium hydroxide, which is not specifically limited in this disclosure.
  • HBTU is used as a peptide coupling reagent. According to actual needs, the peptide coupling reagent can also be PyBop, which is not specifically limited in this disclosure.
  • S4 includes S44.
  • the Fmoc protecting group is removed in a removing agent after mixing the Tentagel-NH 2 resin.
  • the removing agent can be at least one of hexahydropyridine and alkali solution, such as alkali solution. It includes at least one of sodium hydroxide and potassium hydroxide, to which the disclosure is not specifically limited.
  • HBTU is used as a peptide coupling reagent. According to actual needs, the peptide coupling reagent can also be PyBop, which is not specifically limited in this disclosure.
  • S5 includes S55.
  • the Fmoc protecting group is removed after mixing the Tentagel-NH 2 resin in a removing agent.
  • the removing agent can be at least one of hexahydropyridine and alkali solution.
  • the alkali solution includes hydroxide. At least one of sodium and potassium hydroxide, which is not specifically limited in this disclosure.
  • HBTU is used as a peptide coupling reagent. According to actual needs, the peptide coupling reagent can also be PyBop, which is not specifically limited in this disclosure.
  • S6 includes S66.
  • the Fmoc protecting group is removed after mixing the Tentagel-NH 2 resin in a removing agent.
  • the removing agent can be at least one of hexahydropyridine and alkali solution.
  • the alkali solution includes hydroxide. At least one of sodium and potassium hydroxide, which is not specifically limited in this disclosure.
  • HBTU is used as a peptide coupling reagent. According to actual needs, the peptide coupling reagent can also be PyBop, which is not specifically limited in this disclosure.
  • S7 includes S77.
  • the above solvent may be water and/or methanol.
  • S8 Cleave the dry resin obtained in S7 and perform preliminary screening to obtain a polypeptide probe.
  • the sequence of the amino acid residues of the polypeptide probe includes at least one of SEQ ID NO.1 ⁇ SEQ ID NO.15.
  • S8 includes S88.
  • the above-mentioned cleavage agent may be an acidic solution.
  • the acidic solution may be a hydrogen fluoride solution, and the mass fraction of the hydrogen fluoride solution is 95%.
  • the principle process of the initial screening of magnetic beads in S88 is shown in Figure 3.
  • CD138 is attached to the surface of magnetic beads 30, and the free peptide probe 20 can be screened out by combining CD138 with magnetic beads 30.
  • the polypeptide probe 20 with weak or no binding will not bind.
  • the magnetic field generated by the magnet 40 will enrich the bound ones, and finally obtain the sequence of amino acid residues such as SEQ ID NO.1 ⁇ SEQ ID NO.
  • a kit including the polypeptide probe 20 as above.
  • This kit can be used to detect diffuse large B-cell lymphoma for non-disease diagnosis and/or non-treatment purposes, and is easy to operate.
  • the kit further includes a surfactant, an anti-enzyme cleavage agent, a cytofluorescent stain, and a PBS buffer.
  • surfactant is used for magnetic bead binding and capture to prevent cell aggregation and at the same time improve the binding efficiency of the polypeptide probe 20 and the cell surface antigen.
  • Anti-enzymatic agents effectively achieve anti-enzymatic action.
  • Cell fluorescent stains are used to fluorescently stain test sample cells to facilitate detection and analysis.
  • PBS buffer provides a detection environment for the detection of diffuse large B cells, ensuring that biologically active substances maintain their most complete characteristics, so as to facilitate the detection of sample cells.
  • the pH value of the PBS buffer can be 7.1 to 7.6.
  • the pH value is 7.1, 7.2, 7.3, 7.4, 7.5 or 7.6, which is not specifically limited in the embodiments of the present disclosure.
  • the volume percentage of the surfactant is 0.5% to 5%.
  • the volume percentage of the surfactant is 0.5%, 1%, 2%, 4% or 5%. This disclosure does not apply to this. Make specific limitations.
  • the molar concentration of the anti-enzymatic agent is 0.5mmol/L to 10mmol/L.
  • the molar concentration of the anti-enzymatic agent is 0.5mmol/L, 1mmol/L, 3mmol/L, 8mmol/L. L or 10mmol/L, this disclosure does not specifically limit this.
  • the molar concentration of the cytofluorescent stain is 0.5mmol/L to 50mmol/L, for example, 0.5mmol/L, 5mmol/L, 10mmol/L, 20mmol/L, 40mmol/L or 50mmol/L. L, this disclosure does not specifically limit this.
  • the surfactant includes at least one of polyoxyethylene sorbitan monolaurate, sodium lauryl sulfate, and fatty acid sorbitan; exemplarily, the surfactant includes polyoxyethylene sorbitan monolaurate. Sorbitan monolaurate, embodiments of the present disclosure are not limited thereto.
  • the anti-enzymatic agent includes ethylenediaminetetraacetic acid, to which embodiments of the present disclosure are not limited.
  • the cytofluorescent staining agent includes rhodamine B, to which embodiments of the present disclosure are not limited.
  • FIGS. 5A to 5E are cross-sectional structural diagrams corresponding to each step in the chip preparation method according to some embodiments. It should be understood that the steps shown in Figures 4A-4B are not exclusive and other steps may be performed before, after, or between any of the steps shown. Furthermore, some of the steps may be performed simultaneously or in a different order than that shown in FIGS. 4A-4B.
  • the preparation method of the chip in some embodiments will be described below with reference to Figures 4A to 4B and Figures 5A to 5E.
  • some embodiments of the present disclosure provide a chip 100, which includes a substrate 10 and the above polypeptide probe 20.
  • the polypeptide probe 20 is disposed on the substrate 10.
  • the chip 100 is used for differential diagnosis of diffuse large B-cell lymphoma.
  • the chip can be a microfluidic chip.
  • the microfluidic chip has the characteristics of high throughput, fast analysis speed, low contamination, and required Small sample size, cheap, safe and other advantages
  • combining peptide probes with microfluidic chips can effectively improve the sensitivity and timeliness of detection, as well as reduce the complexity and cost of operations.
  • some embodiments of the present disclosure also provide a method for preparing a chip 100 , including S100 to S200.
  • the matrix 10 serves as the carrier of the polypeptide probe 20.
  • the matrix 10 serves as an information processing center for the polypeptide probe 20 to detect sample cells, making the analysis and detection speed fast.
  • polypeptide probe 20 is coupled to the substrate 10.
  • coupling the polypeptide probe 20 to one side of the substrate 10 includes dissolving the polypeptide probe 20 in a solvent to obtain a solution.
  • the solvent may be ddH 2 O, but the disclosure is not limited thereto.
  • the dissolving solutions are respectively placed on the substrate 10, and incubated and blocked, thereby coupling the polypeptide probe 20 to the substrate 10.
  • S100 and S200 include: S110 to S140.
  • the adhesive layer 12 is formed on the substrate 11 side.
  • the substrate 11 may be glass
  • the adhesion layer 12 may be formed using a sol-gel method, a nanoparticle spin coating method, or a sputtering process.
  • the adhesion layer 12 is formed using a nanoparticle spin coating method.
  • the adhesion layer 12 is an optical glue with a thickness of 100nm to 800nm.
  • the thickness is 100nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm or 800nm. According to actual needs and process selection, the embodiment of the present disclosure does not Limited to this.
  • the formed passivation layer 13 includes forming a first insulating layer 131 and a second insulating layer 132.
  • the first insulating layer 131 may be a silicon dioxide layer
  • the second insulating layer 132 may be a silicon nitride layer, and the embodiment of the present disclosure is not limited thereto.
  • the first insulating layer 131 can be deposited on the side of the adhesion layer 12 away from the substrate 11 by inkjet printing or magnetron sputtering as needed.
  • the second insulating layer 132 can be deposited on the side of the first insulating layer 131 away from the substrate 11 by inkjet printing or magnetron sputtering as needed.
  • the thickness of the first insulating layer 131 is 1000 Angstroms.
  • the thickness of the second insulating layer 132 is 2000 Angstroms.
  • the thickness of the first insulating layer 131 and the second insulating layer 132 can be selected according to actual needs and processes, and this disclosure does not specifically limit this.
  • the patterning process includes photolithography, patterning and etching.
  • the glue coating process is spin coating at a pressure of 30kPa and a rotation speed of 300rpm for 10s, baking at 90°C for 120s; exposure after two repetitions, and development for 100s. Bake hard at 230°C for 30 minutes, and then perform inductively coupled ion etching.
  • FIG. 5C and FIG. 5D after the passivation layer 13 is patterned, a plurality of spaced passivation portions 130 are formed on the adhesion layer 12 . For example, a plurality of spaced passivation portions 130 are formed along the rows and columns.
  • Directional array arrangement is possible to form a plurality of spaced passivation portions 130 .
  • a gold film portion 14 is formed on the passivation portion 130 through gold plating and etching.
  • the orthographic projection of the gold film portion 14 in the direction perpendicular to the adhesion layer 12 is located in the passivation portion. Within the 130 area.
  • the gold film part 14 and the polypeptide probe 20 are connected together through a gold-sulfur bond, thereby forming a high-throughput array chip.
  • a high-throughput microarray chip can also be formed.
  • it for the detection of diffuse large B cells, it has the characteristics of high throughput, fast analysis speed, and low pollution, which improves the sensitivity and timeliness of detection.
  • Step (1) Weigh 150 mg of Tentagel-NH 2 and immerse it in hexahydropyridine to remove the FMOC protecting group and fully swell. Then wash the Tentagel-NH 2 resin three times with N, N-dimethylformamide and add reaction tube, and add 180 mg of His, Gly, Cys and HBTU three times respectively. After the reaction is completed, add hexahydropyridine to remove the FMOC protecting group again.
  • Step (2) Divide the Tentagel-NH 2 resin after the reaction in step (1) into three parts and add them to three reaction tubes respectively. Add 60 mg of Cys, Leu, Arg and HBTU to each reaction tube. Perform coupling. After the coupling is completed, mix the Tentagel-NH 2 resin in the three reaction tubes and immerse it in hexahydropyridine to remove the FMOC protecting group.
  • Step (3) Divide the Tentagel-NH 2 resin after the reaction in step (2) into three parts and add them to three reaction tubes respectively. Add 60 mg of Trp, Leu, Asn and HBTU is coupled. After the coupling is completed, mix the Tentagel-NH 2 resin in the three reaction tubes and immerse it in hexahydropyridine to remove the FMOC protecting group.
  • Step (4) Divide the Tentagel-NH 2 resin after the reaction in step (3) into three parts, and add them to three reaction tubes respectively. Add 45 mg of Arg, Trp, Phe and HBTU is coupled. After the coupling is completed, mix the Tentagel-NH 2 resin in the three reaction tubes and immerse it in hexahydropyridine to remove the FMOC protecting group.
  • Step (5) divide the Tentagel-NH 2 resin after the reaction in step (4) into four parts, and add them to four reaction tubes respectively, and add 36 mg of Gly, Phe, Leu, Asp, Ser and HBTU are coupled. After the coupling is completed, mix the Tentagel-NH 2 resin in the four reaction tubes and immerse it in hexahydropyridine to remove the FMOC protecting group.
  • Step (6) Divide the Tentagel-NH 2 resin after the reaction in step (5) into five equal parts, and add them to five reaction tubes respectively. Add 36 mg of Phe, Ser, Leu, Asp, Tyr and HBTU are coupled. After the coupling is completed, mix the Tentagel-NH 2 resin in the five reaction tubes and immerse it in hexahydropyridine to remove the FMOC protecting group.
  • Step (7) wash the Tentagel-NH 2 resin after the reaction in step (6) with water and methanol to achieve solvent replacement, shrink the resin at the same time, and remove the water and methanol by vacuuming to obtain a polypeptide-loaded resin. Dry the resin.
  • Step (8) Cleave the polypeptide-loaded dry resin obtained in step (7) in a volume fraction of hydrogen fluoride to cleave the polypeptide from the resin and simultaneously remove the side chain protecting group to obtain a probe library.
  • CD138 is connected to magnetic beads modified with carboxyl groups at the end. After being fully washed with PBS, Mix the modified magnetic beads and the probe library and interact for 30 minutes, and use the magnetic field to enrich the bound probes to obtain a polypeptide probe with the amino acid residue sequence shown in SEQ ID NO.1 to SEQ ID NO.15. Needle.
  • the preparation method of the polypeptide probe further includes: step (9) to step (11).
  • Step (9) Dissolve the polypeptide probes obtained in step (8) into ddH 2 O and prepare a solution with a concentration of 100 ⁇ g/mL.
  • Step (10) Spot the solution obtained in step (9) on the surface of a bare gold chip. Repeat three spots for each solution. After incubating at 4°C for 12 hours, infiltrate the bare gold chip into 5% skim milk. Sealed at 4°C for 12 hours, washed with 10X PBS 1X PBS ultrapure water, and dried with nitrogen.
  • Step (11) install the bare gold chip processed in step (10) on the SPRi instrument, measure the SPRi angle and adjust it to the best optical position, select relevant detection points in the detection area, including sample points and blank points, and set the experimental flow rate is 2 ⁇ L/s; use PBS as buffer solution to flow into the flow cell until the baseline is stable, and then detect CD138 with concentrations of 50nmol/L, 100nmol/L, 200nmol/L, 400nmol/L and 800nmol/L.
  • the binding time is 300 seconds
  • the dissociation time is 300 seconds
  • phosphoric acid is introduced between each concentration for regeneration.
  • the detection results are the time and response curves shown in Figure 6A and Figure 6O.
  • Step (a) gradient concentration cell sample configuration: positive cells and negative cells are used as the test group, where the positive cells are diffuse large B cells, the negative cells are HeLa cells, and the number ratio of positive cells to negative cells is 1: 10. Mix and repeat pipetting to mix.
  • Step (b) magnetic bead coupling In PBS solution, 1 mg magnetic beads and 10 ⁇ g peptide probe were gently shaken at room temperature for 1 hour. The magnetic beads were then washed 5 times with PBS buffer with a volume fraction of 0.01% bovine serum albumin, left to stand for 30 s each time, and finally resuspended in PBS solution to obtain a magnetic bead suspension with a concentration of 15 mg/mL.
  • Step (c) positive cell enrichment: Mix the positive cells thoroughly with the magnetic bead suspension, shake by hand in an ice box at 4°C for 30 minutes, and then wash three times with PBS (containing 0.1% BSA and 2mmol/LEDTA) buffer. , resuspended in PBS.
  • PBS containing 0.1% BSA and 2mmol/LEDTA
  • Step (d) specificity analysis: Use a magnetic field to separate the captured cells, quantify the protein expression through flow cytometry, and calculate the positive ratio.
  • Step (1) microarray patterning: clean the glass, then spin-coat a layer of optical glue to form an adhesion layer 12 with a thickness of 100 nm to 800 nm, and then deposit a silicon dioxide layer with a thickness of 1000 angstroms and a silicon dioxide layer with a thickness of 2000 angstroms.
  • the angstrom silicon nitride layer is protected and patterned by photolithography with glue coating.
  • the glue coating parameters are 30kPa ⁇ 300rpm*10s, baked at 90°C for 120s; exposed after two repetitions, developed for 100s, and hard baked at 230°C for 30min. , and then perform inductively coupled ion etching.
  • the thickness of the adhesion layer 12 can be 100nm, 200nm, 400nm, 600nm or 800nm, and different thicknesses can be selected according to actual needs, which is not specifically limited in this disclosure.
  • the thickness of the silicon dioxide layer and the silicon nitride layer can also be selected according to actual needs, and the present disclosure does not specifically limit this.
  • Step (2) bare gold array construction: At 240°C, gold is evaporated on the silicon nitride layer to form a gold film with a thickness of 300 nm, and then reactive ion etching is performed.
  • Step (3) couple the polypeptide probe 20 to the surface of the gold film.
  • Some embodiments of the present disclosure provide a method for detecting diffuse large B-cell lymphoma, the steps of which are as follows:
  • Sample extraction Use a disposable anticoagulated vacuum tube to collect 2 mL to 5 mL of human venous blood, store it at room temperature, and use ethylenediaminetetraacetic acid or heparin for anticoagulation.
  • Sample processing Add 2mL of lymphocyte separation solution to a sterile plastic centrifuge tube, add 2mL of PBS buffer to the vacuum tube containing 2mL of blood sample, and mix well. Then slowly add the mixed blood sample PBS mixture into the centrifuge tube containing the lymphocyte separation solution, so that the blood sample is located on the upper layer of the extraction solution as much as possible, and then centrifuge at 20°C and 1500 rpm for 15 minutes.
  • the centrifuge tube is divided into 4 layers, from top to bottom: plasma, mononuclear cells, granular leukocytes, and red blood cells. Use a capillary to extend into the mononuclear cell layer (located at the interface between the cell separation solution and the plasma), and gently remove it along the tube wall. Aspirate all cells. Then wash twice with Hanks solution and centrifuge at 11000r/min for 10min each time.
  • Peptide probe fixation Couple the peptide probe with the sequence of amino acid residues as shown in SEQ ID NO.6 on the surface of a glass chip coated with bare gold, dissolve the peptide probe with pure water, and prepare the peptide with a concentration of 1 mmol/L Probe solution, apply the peptide probe solution on the surface of the chip, incubate it at a temperature of 4°C and a humidity of 40% for 24 hours, then wash it three times with 10x PBS, 1x PBS, and pure water respectively, and blow dry with nitrogen.
  • Tumor cell peptide probe capture Add 1x PBST to the lymphocytes obtained after sample processing to disperse to ensure no aggregation. Then use 1x PBST to dilute the cell suspension to 1 ⁇ 10 5 /mL, and pass through the coupling chamber at a flow rate of 5 ⁇ L/s. The peptide probe was attached to the chip surface for 500 seconds, and then 1x PBST was passed through the chip surface at a flow rate of 3 ⁇ L/s for 500 seconds to fully dissociate non-specific adsorption.
  • the magnetic beads bind to the cells and are characterized by cell staining.
  • Fluorescently labeled antibodies (different sites from the capture antibodies) are used to bind to the captured cells.
  • the captured cells are characterized by fluorescence microscopy to verify the positivity of the capture. Diagnosis of diffuse large B-cell lymphoma.
  • the polypeptide probe of the present disclosure has the characteristics of good selectivity, low immunogenicity, good biocompatibility, strong penetrability, easy excretion and clearance, etc., and has specificity and high efficiency in the detection of diffuse large B-cell lymphoma. sensitivity.
  • the polypeptide probe preparation method of the present disclosure can effectively increase the diversity of polypeptide probes by constructing a polypeptide probe library. This solves the problem of detection sensitivity in early diagnosis of diffuse large B-cell lymphoma.
  • the disclosed chip has the characteristics of high throughput, fast analysis speed, and low pollution, and has broad development prospects in the fields of clinical diagnosis and disease screening. Combining high-affinity peptide probes with chips can effectively improve the sensitivity and timeliness of detection.

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Abstract

Sonde polypeptidique, comprenant au moins deux résidus d'acides aminés hydrophobes, le nombre de résidus d'acides aminés hydrophobes représentant un quart à deux tiers du nombre des résidus d'acides aminés de la sonde polypeptidique.
PCT/CN2022/083202 2022-03-25 2022-03-25 Sonde polypeptidique, son procédé de préparation et son utilisation WO2023178704A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008013859A2 (fr) * 2006-07-28 2008-01-31 Adlyfe, Inc. Sondes peptidiques pour des diagnostics et des produits thérapeutiques
CN102643331A (zh) * 2012-04-25 2012-08-22 南方医科大学珠江医院 一种适用于肺癌分子影像诊断的小分子肽探针及其制备方法
CN103497235A (zh) * 2013-07-18 2014-01-08 广东药学院 一种小分子肽探针及其制备方法和应用
CN106632689A (zh) * 2016-12-23 2017-05-10 中国科学院深圳先进技术研究院 多肽探针、包含该探针的试剂盒及其应用
CN111855619A (zh) * 2020-07-09 2020-10-30 北京服装学院 表面等离子体共振传感芯片及其制备方法、传感设备
EP3796002A1 (fr) * 2006-07-14 2021-03-24 The Regents of The University of California Biomarqueurs du cancer et leurs procédés d'utilisation

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3796002A1 (fr) * 2006-07-14 2021-03-24 The Regents of The University of California Biomarqueurs du cancer et leurs procédés d'utilisation
WO2008013859A2 (fr) * 2006-07-28 2008-01-31 Adlyfe, Inc. Sondes peptidiques pour des diagnostics et des produits thérapeutiques
CN102643331A (zh) * 2012-04-25 2012-08-22 南方医科大学珠江医院 一种适用于肺癌分子影像诊断的小分子肽探针及其制备方法
CN103497235A (zh) * 2013-07-18 2014-01-08 广东药学院 一种小分子肽探针及其制备方法和应用
CN106632689A (zh) * 2016-12-23 2017-05-10 中国科学院深圳先进技术研究院 多肽探针、包含该探针的试剂盒及其应用
CN111855619A (zh) * 2020-07-09 2020-10-30 北京服装学院 表面等离子体共振传感芯片及其制备方法、传感设备

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